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Thrombolysis in acute myocardial infarctionBrügemann, Johannes

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Thrombolysis in Acute Myocardial Infarction

Factors Determining its Efficacy

Copyright 1994 by Johan Brügemann

ISBN 90-9007130-X

Printing Drukkerij Brügemann BV, Den Burg - TexelCover illustration Bas van der Meer, Zuidhorn (Gn)

RIJKSUNIVERSITEIT GRONINGEN

Thrombolysis in Acute Myocardial Infarction

Factors Determining its Efficacy

PROEFSCHRIFT

ter verkrijging van het doctoraat in de Geneeskundeaan de Rijksuniversiteit Groningen

op gezag van de Rector Magnificus Dr. S.K. Kuipersin het openbaar te verdedigen op woensdag 25 mei 1994

des namiddags te 4.00 uur

door

Johannes Brügemann

geboren 26 augustus 1955 te Den Burg - Texel

Promotores Prof.Dr. K.I. LieProf.Dr. M.R. HalieProf.Dr. F.W.A. Verheugt

Referent Dr. P.A. de Graeff

Promotiecommissie Prof.Dr. J.W. ten CateProf.Dr. W.D. ReitsmaProf.Dr. T. van der Werf

Financial support by the Netherlands Heart Foundation for the publication of this thesis isgratefully acknowledged.

Financial support by Astra Pharmaceutica BV, Boehringer Ingelheim BV, Bristol-MyersSquibb, Leo Pharmaceutical Producs BV, Lorex Synthélabo BV, Parke-Davis BV,Pharmacia Nederland BV for the publication of this thesis is gratefully acknowledged.

When examining a man for illness in his cordia, he has pain in his arms, in his breast, onthe site of his cardia ... it is death which approaches him (The Ebers papyrus, 3000 BC,cited by Tijssen, 1987).

voor Isabelle

ter nagedachtenis aan Ben van Gelder

Contents

Chapter I General introduction and aims of the thesis 1

1. The origin of myocardial infarction2. Management of myocardial infarction

2.1. Coagulation and fibrinolytic system2.2. Anticoagulants2.3. Plasminogen activators2.4. Thrombolytic therapy

2.4.1. Introduction of thrombolytic therapy, the first trials2.4.2. Towards the thrombolytic era, placebo controlled megatrials2.4.3. The thrombolytic era, comparative trials and adjuncts to therapy

3. Factors that determine the success of thrombolytic therapy3.1. Morphology and localization of the lesion3.2. Delay from onset of complaints to initiation of therapy3.3. Hematological factors

4. Aims of the thesis

Chapter II Time as a determinant for success of thrombolytic therapy 15

1. Introduction2. Time-loss between onset of symptoms and initiation of therapy

2.1. Delay by the patient2.2. Delay by the doctor and emergency crew2.3. Delay in transport to hospital2.4. Delay in the emergency room and/or CCU

3. Strategies to reduce the time from onset of symptoms to treatment3.1. Prehospital delay and in-hospital delay3.2. Prehospital thrombolysis

4. Strategies to accelerate reperfusion by different dosage schemes and new agents5. Conclusions

Chapter III Significance of hematological parameters in the outcomeof thrombolytic therapy 21

1. Introduction2. Systemic lytic state and anti-streptokinase antibodies (aSKa)

2.1. Determination of anti-streptokinase antibodies (aSKa)3. Role of plasminogen activator inhibitor (PAI)4. Impairment of plasminogen activation by lipoprotein(a) [Lp(a)]5. Proposed indicators of thrombolytic efficacy such as fibrinopeptide A (FPA)6. Conclusions and implications for thrombolytic therapy

Contents

Chapter IV Early and late reocclusion in patients with myocardial infarction 27

1. Introduction2. Occurrence and mechanism3. Factors determining reocclusion4. Traditional ways to prevent reocclusion5. New ways to prevent reocclusion6. Conclusions

Chapter V Summary and conclusions 33

References Chapter I-IV 35

Abbreviations and acronyms 53

Appendices:

appendix 1 55

Logistic Problems in Prehospital Thrombolysis.Eur Heart J 1989:10;122 (abstract)Eur Heart J 1992:13;787-8.

appendix 2 59

A Systemic Non-Lytic State and Local Thrombolytic Failure of Anistreplase(Anisoylated Plasminogen Streptokinase Activator Complex, APSAC) in AcuteMyocardial Infarction.J Am Coll Cardiol 1990;15:3A (abstract)Br Heart J 1990;64:355-8.

appendix 3 67

Rapid Enzyme Immunoassay of Anti-Streptokinase Antibodies in Human Plasma.Thromb Haemost 1991;65:1268 (abstract)Clinica Chimica Acta 1993;218:121-9.

Contents

appendix 4 79

Anti-Streptokinase Antibodies Inhibit Fibrinolytic Effects of Anistreplasein Acute Myocardial Infarction.Thromb Haemost 1991;65:1095 (abstract)Am J Cardiol 1993;72:462-4.

appendix 5 87

Anti-Streptokinase Antibodies are of Clinical Importance and can beMeasured Quantitatively in 0.5 hr Using a Simple Enzyme-LinkedImmunosorbent AssayBr Heart J 1994, in press (letter)

appendix 6 89

Outcome of Thrombolytic Therapy with Streptokinase Appears to beIndependent of Plasminogen Activator Inhibitor (PAI-1) levels.J Am Coll Cardiol 1992:19;179A (abstract)submitted

appendix 7 95

Lipoprotein(a) Levels in Myocardial Infarction Treated with Anistreplase:No Prediction of Efficacy but Inverse Correlation with PlasminogenActivation in Non-Patency.Thromb Res 1992;65:S98 (abstract)Eur Heart J 1992;13:27 (abstract)Int J Cardiol 1994, in press

appendix 8 105

Reocclusion Three Months after Successful Thrombolytic Treatment ofAcute Myocardial Infarction with Anisoylated PlasminogenStreptokinase Activating Complex (APSAC).Eur Heart J 1988;9:10 (abstract)Am J Cardiol 1990;65:1422-4.

Nederlandse samenvatting 115

Dankwoord 117

Introduction

Chapter I

General introduction and aims of the thesis

1. The origin of myocardial infarction

Already in ancient history it was noted that sudden pain in the chest could be aharbinger of death. The actual cause of death was not yet understood. First the circulationhad to be described which was done by Harvey in the 17th century. Medical sciencedeveloped slowly. Only at the beginning of the 20th century theories emerged on thepathophysiology of myocardial infarction (MI), which was defined as necrosis of heartmuscle tissue due to persisting ischemia.

In 1910, Russian pathologists described five patients with acute MI of whom threeshowed coronary thrombosis at autopsy (Obraztsov,1910). Subsequently, in 1912, Herrickwrote a publication on the syndrome of acute MI. He suggested that "hope for thedamaged myocardium lies in the direction of securing a supply of blood .. so as to restoreas far as possible its functional integrity" (Herrick,1912). So it was hypothesized for thefirst time that obstruction of the blood stream was the cause of subsequent necrosis anddysfunction of a part of the heart. The issue of thrombosis did not receive much attentionfor several decades. Clinicians and pathologists continued to argue about the questionwhether coronary thrombosis was a cause or a consequence of MI (Roberts,1972;Baroldi,1976; Silver,1980). The introduction of cardiac catheterization settled this dispute.Using this technique, a thrombus was shown in patients with symptoms and ECG signs ofMI (DeWood,1980). This confirmed the view that acute thrombotic occlusion was thecause of MI. In addition, Falk and Davies showed that focal arterial lesions, in particular afissured atherosclerotic plaque, were the origin of the thrombotic process (Falk,1983;Davies,1985). Adherence and aggregation of thrombi at the site of the culprit lesionpreceded the development of fibrin (Davies,1979). Currently, the process of plaque ruptureis more precisely understood (Richardson,1989; Chesebro,1991). This is depicted inFigure 1.

2. Management of myocardial infarction

Acute death in patients with MI is usually caused by extensive ischemia leading topump failure, ventricular arrhythmias, in particular ventricular fibrillation, and cardiacrupture. Initially, attention focused on the reduction of secondary complications. Duringthe last decade, however, emphasis in treatment of patients with MI has shifted tostrategies aimed at reducing the extent of necrosis. This was based on improvedunderstanding of the underlying mechanisms in evolving MI and supported bydevelopments in the field of antithrombotic and thrombolytic treatment. In the followingparagraphs major developments will be summarized.

2.1. Coagulation and fibrinolytic system

Clotting is a process that includes the conversion of soluble fibrinogen into fibrin by

1

Introduction

the action of thrombin. Thrombin is formed by proteolytic cleavage of the proenzymeprothrombin which is produced by activated factor X (Xa) in the presence of calcium.Activation of factor X may occur by either one of two separate pathways, the extrinsic orthe intrinsic pathway. In the extrinsic pathway, a tissue factor, released from damagedcells (tissue thromboplastin) activates factor X in the presence of factor VII (and calcium).In the intrinsic pathway, the contact of blood with a foreign surface such as collagen (or,in vitro, glass) activates factor XII. Cascadian activation of other coagulation proteinsfinally leads to activated factor X (Xa). Hemostatic platelet plugs in the injured vesselwall are stabilized by the fibrin network. Activated platelets release proaggregatorysubstances and catalyse the coagulation process. Thrombin, in its turn, is the most potentand physiologically important activator of platelets and is pivotal in the process of plateletrecruitment into thrombus formation after vascular injury (Harker,1992). The closeinteraction between platelet membrane receptors and the coagulation cascade isschematized inFigure 2.

In homeostasis, coagulation is in balance with fibrinolysis. Like coagulation, fibrinolysisis a complicated interplay between activators and inhibitors. In static blood, inhibitionoutweighs activity. If blood is allowed to clot in a test-tube and incubated at 37 C, theclot will remain solid for days or weeks (Fearnley,1961). The fibrinolytic system isdepicted inFigure 3.

2.2. Anticoagulants

Theoretically, there were several reasons to assume that anticoagulants might bebeneficial in patients with MI: a) anticoagulants might halt or slow the progression of thedevelopment of a thrombus, b) anticoagulants might be expected to inhibit the subsequentformation of mural thrombi, and c) anticoagulants might reduce the incidence of deepvenous thrombosis, and eventually subsequent pulmonary embolism, in immobilizedpatients. Antithrombotic treatment with heparin was propagated in the 1970s followingpublication of a number of randomized trials in the USA (Chalmers,1977). However, thediscussion continued and in 1984 one could read in Braunwalds’ textbook Heart Diseasethat "Despite several decades of evaluation, the results of the treatment of acute MI withanticoagulants are inconclusive" (Sobel,1984). Subsequently, the role of heparin inconjunction or adjunction to thrombolytic therapy has gained in importance due to thesupportive findings (MacMahon,1988; Yusuf,1988). Its intravenous use is currentlyadvocated in conjunction with recombinant tissue-type plasminogen activator (rt-PA),whereas its use might be delayed for several hours in those patients with MI who havebeen treated with streptokinase (SK) (Prins,1991). This will be discussed further in thechapters I and IV.

2.3. Plasminogen activators

In 1933 it was reported that filtrates of broth cultures of certain strains of hemolyticstreptococci contained a substance capable of inducing fibrinolysis in human plasma clots(Tillett 1933,1934). At first this substance was called streptococcal fibrinolysin.Christensen suggested that it be renamed into streptokinase (SK) because the streptococcal

3

Chapter I

product was an activator rather than a fibrinolysin. Actually, it activated a "lytic factor"present in plasma globulins, which Kaplan identified as plasminogen (Kaplan,1944).Another plasminogen activator found in human urine was called urokinase (Sobel,1952).Additional research on components of the fibrinolytic enzyme system was carried out byAstrup and Permin. They showed that animal tissue contained an agent which couldactivate plasminogen (Astrup,1947). This trypsin-like serine protease was t-PA. It was apoor plasminogen activator in the absence of fibrin but after binding to fibrin in a clot itactivated plasminogen several hundred-fold more efficient than in the circulation. Thischaracteristic was called fibrin-specificity. Investigators of the Genentech companysucceeded in the manufacturing of t-PA by means of recombinant DNA technology(Pennica,1983). rt-PA is known as alteplase or duteplase. Because of its specificity forfibrin and its nonallergenic structure, hope arose that bleeding and anaphylacticcomplications would decrease compared to those occurring after SK.

The knowledge, that SK-plasmin was a highly effective plasminogen activator guidedresearch to manufacture a thrombolytic agent that contained such an enzyme complex. Itwas intended to control its action by a specific temporary chemical protection of itscatalytic centre by inserting a p-anisoyl group. The complex would reactivate atphysiological pH following spontaneous deacylation. Such a compound was developed byBeecham pharmaceutical research and it was named anisoylated plasminogen streptokinaseactivator complex (APSAC or anistreplase) (Smith,1981).

As depicted inFigure 3, exogenous plasminogen activator converts plasminogen, whichis a single-chain glycoprotein consisting of 790 amino acids, into plasmin. Actually, theArg560-Val561 peptide bond in plasminogen is hydrolysed whereafter plasmin is formed.Lysine binding sites mediate its adherence to fibrin which is subsequently digested tosoluble degradation products. Plasmin is the key enzyme of the plasma fibrinolytic system,which is predominantly inhibited by the serine protease inhibitorα2-antiplasmin (Collen1976,1980).

2.4. Thrombolytic therapy

Administration of plasminogen activators is usually called thrombolytic therapy. Thisterm will also be used in the following paragraphs and chapters.

2.4.1. Introduction of thrombolytic therapy, the first trials

The fibrinolytic properties of SK in patients with MI were demonstrated for the firsttime in the late 1950s (Fletcher 1958,1959). The first controlled clinical trial with thepurified compound SK was conducted in Europe (European Working Party,1971). In thistrial patients with MI of less than 24 hours’ duration were randomly allocated to receiveintravenous SK (initial dose 250,000 U, maintenance dose 100,000 U hourly) or heparinfor 24 hours. After 24 hours and 6 weeks the mortality rate in patients treated with SKwere significantly lower (10.6% and 19.0%, respectively) compared to patients givenheparin (17.8% and 27.4%, respectively). However, these results were not generallyaccepted, reflecting doubts about the causal connection between coronary thrombosis andMI (Sherry,1989). During the early 1980s these doubts diminished because another trial

6

Introduction

again showed a significant reduction in mortality in patients with MI treated with i.v. SK(European Cooperative Study Group,1979) whereas other investigators showed rapidrecanalization of obstructed coronary vessels in patients with MI treated with intracoronarySK (Rentrop,1981). As intracoronary thrombolysis is not widely applicable because of itsdependence on catheterization facilities, intravenous administration was considered to bethe only realistic approach (Verstraete 1966,1985). Clinical investigation showed that 1.5million U SK, administered intravenously during 1 hour, appeared appropriate for patientswith MI (Schröder,1983). Subsequently, this dosage regimen was used in nearly all clinicalstudies. Among these was the landmark TIMI-trial (TIMI,1985). In this trial the efficacyof a 1-hour i.v. infusion of 1.5 million U of SK was compared with a 3-hour infusion of80 mg t-PA in patients with MI. The primary end point was angiographic grade 2 or 3patency at 90 minutes from start of the infusion, in patients who had grade 0 initially.Thus, patency was measured 30 minutes after infusion of SK and after 50 mg of the totaldose of 80 mg t-PA. Sixty percent of 99 patients assigned to t-PA had 90-minutes grade 2or 3 patency, as compared with only 40% of 115 patients assigned to SK (p <0.01).Because of this substantial, statistically significant difference in recanalization rate, phase Iof the trial was stopped by the TIMI policy advisory board. Since this trial t-PA therapywas considered to be more efficacious than treatment with SK in the USA. Recently, thisdifference in time to reperfusion between t-PA and SK has been confirmed (Ganz,1992).

In the early thrombolytic trials in patients with MI, mortality was chosen as the studyend point. Because of the inverse relation between low residual left ventricular ejectionfraction (LVEF) and post-MI mortality rate (Taylor,1980; Norris,1984), several subsequenttrials focussed on changes in this indicator of morbidity. In one of these studies it wasfound that patients with MI treated with SK had a significantly higher LVEF (57%)compared to those who were given placebo (54%) (ISAM,1986). Another smaller studyshowed corresponding results: SK recipients had a 18 ml smaller end-systolic volume,indicating decreased dilatation of the heart after MI treated with SK (White,1987). Theseinvestigators also found no differences between SK and rt-PA treatment with respect topreservation of LVEF (White,1989). These studies suggest that thrombolytic treatment inpatients with MI does not only reduce the mortality rate but also decreases morbidity asindicated by a higher residual LVEF.

2.4.2. Towards the thrombolytic era, placebo controlled megatrials

The "Schröder regimen" of SK administration was used in patients with MI in the firstItalian megatrial (GISSI-1,1986). In this trial, a 3-week mortality rate of 10.7% in the SKrecipients was found compared to 13% in the control patients (reduction in risk 18%;p=0.002). Treatment was safe, as the incidence of life threatening bleeding was only 0.2%.The first randomized placebo-controlled mortality trial with rt-PA in patients withsuspected MI was the Anglo-Scandinavian Study of Early Thrombolysis (ASSET). Patientsreceived a bolus of 10 mg, followed by an infusion of 50 mg in the 1st hour and then 20mg in each of the next 2 hours (or placebo). All patients received i.v. heparin. At 1 monththe overall case fatality rates were 7.2% in 2516 patients given 100 mg rt-PA and 9.8% in2495 patients given placebo, a relative reduction of 26%. Major bleeding was observed in1.4% of the patients treated with rt-PA versus 0.4% in those treated with placebo. The

7

Chapter I

incidence of stroke was similar: 1.1% in the rt-PA group and 1.0% in the placebo group(ASSET,1988).

The results of the APSAC intervention mortality study (AIMS,1988) were alsopresented in 1988. In this randomized, placebo-controlled, double-blind trial 1004 patientswith proven MI received 30 U of APSAC or placebo. Mortality after 30 days was 6.4% inpatients treated with APSAC and 12.2% in those treated with placebo, a 47% reduction ofrelative risk. Adverse events were few. Unexpectedly, 5 cerebrovascular bleedingsoccurred in the placebo group versus 2 in those patients who received APSAC. The fourththrombolytic megatrial, also published in 1988, was the ISIS-2 study. In this randomizedstudy 17,187 patients with suspected MI received SK, aspirin 160 mg daily, both, orneither (placebo). SK alone and aspirin alone each produced a highly significant reductionin 5-week vascular mortality. The combination of SK and aspirin was significantly betterthan either agent alone. Absolute mortality rates after treatment with SK plus aspirin andplacebo were 8% and 13.2%, respectively (ISIS-2,1988). Reductions in the odds of earlydeath in various trials are summarized inFigure 4.

2.4.3. The thrombolytic era, comparative trials and adjuncts to therapy

Thus, these thrombolytic drugs share the ability to reduce mortality in patients with MI,but they differ in several characteristics, especially speed of action, infusion time, fibrinspecificity, and also price. Suggested superiority in efficacy or safety of one drug to theother led to comparative trials. With this purpose, 20,891 and 41,299 patients with MIwere studied in GISSI-2 (and its international extension) and ISIS-3, respectively (GISSI-2,1990; The International Study Group,1990; ISIS-3,1992). In these megatrials, all patientswere given aspirin because this was shown to elicit an independent reduction of mortalityin the ISIS-2 study. No statistical difference in mortality rate between the thrombolyticagents was found in these trials. In ISIS-3, stroke, including intracranial bleeding, wasmore common in patients treated with APSAC or rt-PA compared to those receiving SKtherapy. Both GISSI-2 and ISIS-3 included a group of patients who were treated withtwice daily s.c. heparin, initiated 12 and 4 hours, respectively, after infusion of thethrombolytic agent to meet criticism against rt-PA monotherapy. This attempt was notsuccessful and the heparin controversy flared up (White,1990a). The latest thrombolyticmegatrial in patients with MI bears the acronym GUSTO (Global Utilization ofStreptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries)(GUSTO,1993). In GUSTO, 41,021 patients were randomized over 4 treatment arms: SK(1.5 million U over 60 min) with either s.c. or i.v. heparin, front-loaded rt-PA (15 mgbolus, 50 mg over 30 minutes and an additional 35 mg over the next 60 minutes) plus i.v.heparin, and a combination of rt-PA (≤ 90 mg) with SK (1.0 million U) plus i.v. heparin.All patients received aspirin. After combining the s.c. and i.v. heparin branch of patientswho received SK, 30-day mortality rates were 7.3, 6.3, and 7.0% for the SK, rt-PA and rt-PA/SK treatment arm, respectively. Subgroup analysis revealed that rt-PA therapy wassuperior to both of the SK regimens in patients with one (or more) of the followingcharacteristics: age under 75 years, anterior myocardial wall localization or duration ofchest pain shorter than 4 hours. A substudy recently showed that front-loaded

8

Chapter I

rt-PA therapy was associated with earlier patency and higher LVEF which might explainthe lower mortality (GUSTO Angiographic Investigators,1993). The findings support the"open-artery" hypothesis which means that more rapid and complete restoration ofcoronary flow through the infarct related artery will result in improved LVEF and lowermortality among patients with MI (Braunwald,1993).

3. Factors that determine the success of thrombolytic therapy

Apart from dosage, speed of administration and/or type of agent, several other factorsmay determine the success of thrombolytic therapy, especially a) morphology andlocalization of the culprit lesion, b) time delay to thrombolytic therapy, and c)hematological factors.

3.1. Morphology and localization of the lesion

One reason for failure of thrombolytic therapy may be that the composition of theobstruction is not susceptible to a thrombolytic agent. In other words, not a thrombus butsomething else is present, such as discharged atheromatous material from a cracked plaqueor an intimal flap obstructing the coronary lumen. These rare occurrences have been called"plaque disasters" (Falk,1991). Clots rich in platelets with few fibrin, and thereforeprobably less susceptible to fibrinolytic therapy, were also recognized as a cause oftreatment failure (Fuster,1988). Experiments in a rabbit model showed that such thrombi,compared to erythrocyte-rich clots, were intrinsically more resistant to thrombolysis(Jang,1989). Depth and length of the underlying plaque fissure were suggested to becontributing factors (Richardson,1989). Furthermore, occlusions in the anterior descendingartery were recanalized more frequently than those in the left circumflex or right coronaryartery and proximal occlusions showed higher opening rates than distal lesions afterintracoronary ostial thrombolysis (Tendera,1985).

3.2. Delay from onset of complaints to initiation of therapy

Duration of chest pain before seeking medical attention is of great predictive value forthe outcome of thrombolytic therapy. The sooner patients are treated in the hospital, thegreater the reduction in mortality compared to non-thrombolytic treatment. This wasclearly shown by the pooled results of the megatrials GISSI-1 and ISIS-2. The absolute(odds) reduction in mortality in hour 1, hours 2-3, hours 4-6, and hours 7-12, amounted6.5% (48.5%), 2.7% (25.4%), 2.5% (21.5%) and 1.3% (11.8%), respectively (Gersh,1993).Time as a determinant for success of thrombolytic therapy will be discussed in chapter II.

3.3. Hematological factors

It was already known in the 1950s from in vitro experiments that failure or delay of clotlysis was due to the binding of SK to inhibitory plasma components or antibodies, therebycreating a complex which rendered no plasminogen activation. The origin of theseantibodies in human plasma was sought in streptococcal infections which induced the

10

Introduction

immunosystem to create anti-streptococcal antibodies. It took a long time before otherblood constituents were found which played a role in the regulation of fibrinolysis, andpossibly in the failure of thrombolytic therapy. In 1983, the presence of a naturallyoccurring, fast-acting inhibitor of t-PA, plasminogen activator inhibitor (PAI), wasreported (Kruithof 1983,1984). The third factor that was identified as a possible factor thatmight impair fibrinolysis was lipoprotein(a) [Lp(a)]. After a striking homology was shownbetween the protein moiety apo(a) of Lp(a) and plasminogen (Berg,1963; McLean,1987;Eaton,1987), several authors suggested that Lp(a) can inhibit the binding of plasminogento immobilized fibrinogen or to specific binding sites of the endothelial cell (Scott,1989;Harpel,1989). So both molecules may compete for the same binding site as schematized inFigure 5. These in vitro findings (Edelberg,1989; Rouy 1991), however, have not yet beenconfirmed clinically.

Thus, the plasma constituents anti-streptokinase antibodies (aSKa), plasminogenactivator inhibitor (PAI) and lipoprotein(a) [Lp(a)], may play a role in the efficacy ofthrombolytic therapy. These factors will be discussed more extensively in chapter III.

11

Introduction

4. Aims of the thesis

In the following chapters the conclusions of our studies will be placed in a more generalcontext. Three topics are under discussion which involve most of the aforementionedfactors that determine the success of thrombolytic therapy. The first relates to delay fromonset of symptoms to initiation of thrombolytic therapy (Chapter II). Special attention willbe given to prehospital thrombolysis. Next, the significance of hematological parameters inthe outcome of therapy will be addressed with attention focused on endogenous inhibitorsof SK and plasminogen activation (Chapter III). Finally, the subject of early and latevessel reocclusion will be illuminated (Chapter IV). Following the summary andconclusions (Chapter V), the main studies, which have been published or are submitted forpublication, are added as appendices. These include data on: a) logistical problems inprehospital thrombolysis (no. 1), b) a characteristic condition of the coagulation systemreferred to as "the systemic lytic state" (no. 2), c) measurement and clinical role of anti-streptokinase antibodies (nos. 3, 4 and 5), d) the contribution of plasminogen activatorinhibitor (PAI) and lipoprotein(a) [Lp(a)] to failure of thrombolytic therapy (nos. 6 and 7),and e) the 3 months reocclusion rate after thrombolytic therapy with anistreplase (no. 8).

13

Time

Chapter II

Time as a determinant for success of thrombolytic therapy

1. Introduction

Time between onset of symptoms and initiation of treatment is a major determinant foroutcome of therapy in patients with MI. During the last decades attempts have been madeto shorten this period as much as possible. Already in the 1960s, reduction in prehospitalmortality was achieved by the introduction of ambulances equipped with a battery-operated DC defibrillator and a registrar or houseman, a so-called mobile intensive-careunit (Pantridge,1967; Mathewson,1985). Initially, attention was focused on the earlytreatment of life threatening arrhythmias. Subsequently, it was shown in animals that celldeath following coronary occlusion is a time-dependent wavefront-like progressivephenomenon (Reimer,1979). Reasons why valuable time was sometimes lost beforeinitiation of therapy were elucidated and strategies to circumvent this were developed.This became of major importance in the context of thrombolytic therapy, when time is aprimary determinant for success. This will be shown in this chapter.

2. Time-loss between onset of symptoms and initiation of therapy

2.1. Delay by the patient

The major contribution to the time-loss before initiation of treatment is the patients’delay. This delay was shown to have a skewed distribution with a median and mean of 2and 10 hours, respectively (Rawles,1988). Factors influencing the patients’ delay are age,education level and race. Older patients wait longer before calling for help, and blackswait longer than whites. Educated people are less likely to use the emergency system forsymptoms of chest pain (Schaeffer,1989; Weaver,1991; Kelly,1991). Reasons for delay bythe patient are, among others, the idea that the pain will subside, insufficient severity anddoubt about the cardiac origin of the pain (Ho,1988). In addition, variations in sensitivityto body sensations and emotions appear to play an important role in treatment seekingbehavior (Kenyon,1991).

2.2. Delay by the doctor and emergency crew

Doctor’s delay, both inside and outside the hospital, depends on the patients’ age, theduration of chest complaints and the site where the call came from. The median and meanvalue of the delay time in the settings of a) CCU, b) general ward, and c) home amount to10 to 16 minutes, 15 to 20 minutes, and 20 to 35 minutes, respectively. It had beensuggested that elements of the doctor’s behavior may be at a subconscious or instinctivelevel. The urgency of response is likely to depend on the perceived anxiety content of themessage (Rawles,1988).

Ambulance response times are short, for instance an average of 12 minutes in Cardiff(Wales, UK) and 7 minutes in Nottingham (UK) (McCabe,1991; Rowley,1992). In The

15

Chapter II

Netherlands these figures are comparable. This might be expected, because, according tothe Dutch law, an ambulance must be present within 15 minutes after dispatch(Bouten,1992). There is a striking difference in delay of hospital admission betweenpatients who choose to call a general practitioner compared to those who choose to call anemergency ambulance. If a general practitioner referred the patient to the hospital, mediandelay was 247 minutes, compared with 100 minutes when the patient called an ambulancedirectly (Rowley,1992). This difference remained unchanged between 1982-84 and 1989-90 (Gray,1993).

2.3. Delay in transport to hospital

Ambulance transportation time in most industrialized countries is of a short duration.Ambulances have priority in traffic and are well identified. In a medium sized townwithout regular traffic jams, a major gain in time in this link of the chain is not likely. Inrural areas, as well as in a metropole, the situation may be different, but even then the lossof time due to transport is limited. In the Nottingham registry, the median time from thepatient’s call for the ambulance to its hospital arrival was 29 minutes (Rowley,1992).

2.4. Delay in the emergency room and/or CCU

In a University hospital participating in the TIMI trial, patients waited for an average(±SD) of 20±18 minutes before the initial ECG was made following their arrival in theemergency department. After confirmation of MI, an additional 70±40 minutes elapsedbefore commencement of thrombolytic therapy (Sharkey,1989). This shows that eveninside the hospital valuable time is lost before thrombolysis is started.

3. Strategies to reduce the time from onset of symptoms to treatment

3.1. Prehospital and in-hospital delay

Reports on the effect of media campaigns showed that the delay time by the patientbefore presentation in an emergency room of a city hospital can be reduced, although atthe cost of a temporary increase in the number of patients with non-cardiac chest pain(Herlitz,1989; Blohm,1992). An effort in a rural community was, however, less successful(Moses,1991a). Short prehospital delay results in increased eligibility and likelihood ofreceiving thrombolytic therapy (Goldberg,1992). In an attempt to shorten transport time tohospital, some investigators even used a helicopter to transfer patients with suspected MI.Only in extraordinary situations time might be saved using this approach (Bellinger,1988).

It has been attempted to shorten the first phase of delay in the hospital by prehospital12-lead ECG recording and subsequent telephone transmittance to the emergencydepartment (Grim,1987; Karagounis,1990). This approach has not been generally adopted.Efficiency measures and initiation of thrombolytic therapy in the emergency room, insteadof the CCU, could lead to a substantial decrease of the delay from 70 to 34 minutes inpatients with MI (Verheugt,1989; MacCallum,1990; Moses,1991b). This was called the"fast-track" admission strategy (Pell,1992). Recently, the potential to save time in the

16

Time

emergency room was confirmed by the observation that the median time between arrival athospital to start of thrombolytic therapy, the so called "door-to-needle-time", was 31minutes for patients who received thrombolytic therapy in the emergency room versus 80minutes for those who were treated in the CCU (Birkhead,1992).

Finally, strategies can be designed to accelerate the diagnosis of MI in patients with anatypical history and/or a non-diagnostic ECG. In these situations enzymatic proof byassessment of creatine kinase isoforms or troponin can sometimes be helpful to establishthe diagnosis of MI quickly (Jaffe,1986; Abendschein,1988, Katus 1989,1991;Adams,1993). In addition, the plasma-myoglobin concentration may also be valuable in theestablishment of MI. As early as 1 hour after MI, serum concentrations exceeding thenormal range have been found. Its peak activity is after 4-12 hours (Stone,1983). By usinga quick myoglobin latex-agglutination test during the first 4-12 hours after onset ofsymptoms, it was possible to rule out MI within a few minutes (Endert,1987; Mair1991,1992). Early enzymatic confirmation of MI will eventually lead to reduction in timebefore thrombolytic therapy is administered.

3.2. Prehospital thrombolysis

Already at an early stage, investigators have tried to initiate thrombolysis in theprehospital setting (Koren,1985). In a small sample size they showed that patients with MIwho were treated at home showed a better ventricular performance compared to those whowere treated inside the hospital. Subsequent reports on prehospital thrombolysis came froma) the European myocardial infarction project (EMIP), b) the Grampian region earlyanistreplase trial investigators (GREAT), c) the reperfusion in acute infarction Rotterdamtrial (REPAIR), and d) the myocardial infarction triage and intervention study (MITI).

Patients participating in the EMIP trial who were randomly assigned to the prehospitaltreatment received anistreplase from a physician a median of 55 minutes earlier than thosewho were treated after arrival in the hospital. This was associated with a nonsignificant13% reduction in overall mortality at 30 days. However, death from cardiac causes wassignificantly less in the prehospital treated patients compared to the in-hospital treatedpatients (8.3 vs 9.8%; reduction in risk 16%; p=0.049) (The European MyocardialInfarction Project Group,1993). In GREAT, patients with suspected MI living in countrytowns and villages in Great Britain, received in a randomized design prehospital or in-hospital anistreplase therapy from the general practitioner. The median time anistreplasewas administered at home was 101 minutes after the onset of symptoms, whereas hospitaladministration occurred with a median delay of 240 minutes. Three months mortality ratefrom all causes of death was 8.0% and 15.5% for early and late receivers of anistreplase,respectively (p=0.04) (GREAT Group,1992). The 1-year mortality from all causes in homeand hospital treated patients was 10.4 and 21.6%, respectively (Rawles,1993). In theREPAIR trial 9052 patients were screened by the staff of the ambulance which did notinclude a doctor. Eligible patients underwent on-site ECG recording whereafter, on thebasis of the abnormalities, a computer-program suggested a treatment strategy(Kudenchuk,1991). Only 226 patients (2.5%) were treated with rt-PA. Prehospital treatedpatients saved 47 minutes compared with in-hospital treatment (Bouten,1992). The MITIinvestigators reported a 33 minutes decrease in the interval between symptom onset to

17

Chapter II

treatment. In the clinical phase, there were no significant differences in mortality, ejectionfraction or infarct size. A secondary analysis of time to treatment and outcome showedthat therapy initiated within 70 minutes of onset of symptoms was associated withsignificantly lower mortality (1.2% vs 8.7%), smaller infarct size (4.9% vs 11.2%) andhigher ejection fraction (53% vs 49%) than treatment initiated after a longer period(Weaver,1993a).

Based on the documented delay in each link of the chain from onset of chest pain toCCU admission, we designed the Groninger ambulance study. Our study was comparableto the GREAT trial with the addition of remote ECG confirmation of threatening MI. Wealso used anistreplase in preference to SK because this could be administered i.v. in 5minutes and therefore was considered convenient for domiciliary use.

Our study was discontinued after screening of 350 patients and inclusion of 7 patients(2%). As discussed in appendix 1, we were disappointed with the time consumingprocedure and the logistical problems associated with remote ECG assessment. Due tothese problems we did not gain but even lost time in the enrollment of patients. A silicon-rubber mat with electrodes mounted upon the patient’s chest, and a computer suggestingthe treatment strategy, as was performed in the REPAIR study, is probably quicker(Bouten,1992). Furthermore, we were discouraged by the low inclusion rate. This,however, was also low in the REPAIR and MITI trial, 226/9052 (2.5%) and 360/8863(4%), respectively (Bouten,1992; Weaver,1993a). Some characteristics and results of theaforementioned studies are summarized inTable 1.

4. Strategies to accelerate reperfusion by different dosage schemes and new agents

Different dosage schemes and new thrombolytic agents may reduce time betweeninitiation of thrombolytic treatment and time to reperfusion, thereby enhancing its ultimatesuccess. Whereas in the early days of thrombolytic treatment the duration of infusion wasvery long, currently short term strategies are under investigation. It became evident inseveral studies that the efficacy of 100 mg rt-PA, infused in 90 minutes, rather than in 3hours, was superior compared to the standard regimen. Ninety-minutes patency rates ashigh as 85% have been reported (Neuhaus,1989; Neuhaus,1992; Carney,1992; Wall,1992;GUSTO Angiographic Investigators,1993). This modified rt-PA regimen was called "front-loaded" because of an initial bolus of 15 mg instead of the earlier practiced 10 mg. Incontrast, rapid infusion of SK has been associated with hypotension (Lew,1985a), or a lessbeneficial outcome compared to rt-PA administration (Taylor,1993). Thus, optimal dosingof rt-PA may lead to a more rapid restoration of flow which eventually results in areduction of mortality and morbidity. This appears not to be applicable to treatment withSK.

Another way to enhance early reperfusion is the use of newer thrombolytic agents suchas pro-urokinase or staphylokinase. Pro-urokinase or recombinant single chain urokinase-type plasminogen activator (rscu-PA or saruplase), is a fibrin dependent thrombolyticagent (Gurewich,1984; Pannell,1986) whose use was associated with earlier reperfusion, ahigher patency rate, and fewer bleeding than treatment with SK (PRIMI,1989); thereocclusion rate was only 1.5% (Weaver,1993b). Staphylokinase, a small protein

18

Time

Table 1 Characteristics and results of five prehospital studies indicated by their acronym.

Patients(screened/enrolled)

Drug(active/reference)

Doctor(on thespot)

ECG(confir-med MI)

Time(gain inseconds)

Mortalityreduction(time)

EMIP -/5469 ani/P yes yes (S) 55 (M) no(1 mo)

GREAT -/311 ani/P yes no 139 (M) yes(3 mo)

REPAIR 9052/226 rt-PA/ref no yes (S) 47 (m) NA

MITI 8863/360 rt-PA/P no yes (R) 33 (M) no(dis)

GAS 350/7 ani/P no yes (R) -24 (m) NA

- = unknown; ani = anistreplase; rt-PA = alteplase; P = placebo; ref = historical referencepopulation of 220 patients; M = median, m = mean; S = on the spot; R = remote(hospital), mo = month(s), dis = discharge, NA = not applicable

References: EMIP (The European Myocardial Infarction Project Group,1993), GREAT(GREAT Group,1992), REPAIR (Bouten,1992), MITI (Weaver,1993a), GAS (GroningenAmbulance Study, appendix 1)

19

Chapter II

produced by certain strains of staphylococcus aureus, has been known since 1948 to havefibrinolytic properties (Lack,1948). In contrast to SK, staphylokinase appears to be moreefficient in platelet-rich thrombi and less immunogenic and allergenic (Collen, 1993a).Recombinant staphylokinase was recently given to 5 patients with MI of whom 4 showedreperfusion within 40 minutes (Collen,1993b). These promising results need furtherconfirmation.

5. Conclusions

Factors determining the time between symptoms of coronary occlusion and reperfusionof the occluded vessel have been extensively studied. This has led to a number ofstrategies all aimed at the reduction of this interval. The main emphasis has been put onprehospital thrombolytic therapy and measurements to increase in-hospital efficiency.

With respect to prehospital thrombolytic therapy problems have emerged related to a)ECG-confirmation of MI, b) the diagnostic and practical skills of the ambulance staff andc) the responsibility issue related to the absence of a physician. Using strict selectioncriteria only a minority of patients with symptoms of chest pain have been eligible outsidethe hospital. The reduction of the delay in time will depend on the early establishment ofthe diagnosis but, as was shown in our study, that was not always easy. Until now, areduction in mortality as a result of prehospital initiation of treatment was reported in onlyone study. Therefore, one must remain critical with respect to this approach especially in asituation where rapid transportation to the hospital is possible (Fox,1990; Wilcox,1990;Petch,1991; Gemmill,1993).

It is well known that valuable time can be lost after the patient’s arrival in the hospital.Therefore, it is essential that in-hospital procedures gain in efficiency in order to startthrombolysis as soon as possible after the diagnosis has been established, preferably in theemergency department. Furthermore, strategies should be introduced to speed up the timenecessary for confirmation of MI in patients with a atypical history and/or a non-diagnostic ECG. Further studies may tell us whether newer thrombolytic agents or reviseddosing schemes of currently available thrombolytic drugs can accelerate reperfusion,thereby further enhancing success.

Finally, one comment should be made which may seem paradoxical in the context ofthis chapter. So far, the maximum time limit for onset of symptoms to initiation ofthrombolytic therapy in patients with MI has been 6 hours. Recently a study has beenpublished which indicated that patients with symptoms for more than 6 but less than 12hours may also benefit from thrombolytic treatment (LATE,1993; Gil,1993), although thiswas not confirmed by another study (EMERAS,1993). Delayed successful thrombolytictherapy may be beneficial due to other mechanisms than by salvaging ischemicmyocardium such as a) improved healing of infarcted tissue, b) prevention of ventricularremodeling, c) perfusion of hibernating myocardium and d) increased electrical stability(Shah,1991; Kim,1993). Thus, although time is an essential determinant for success ofthrombolytic treatment, one should not apply currently used time limits too strictly.

20

aSKa, PAI and Lp(a)

Chapter III

Significance of coagulation parametersin the outcome of thrombolytic therapy

1. Introduction

In the early days of thrombolytic treatment SK was the only agent used. Therapy wasguided under the assumption that SK-inhibitory constituents in the patients’ plasma had tobe overruled before any local effect of therapy could be expected. Assessment of SK-inhibitory constituents in the patients’ plasma was done by use of the "predicted dose test"(Johnson,1957). On the basis of this method patients received an i.v. loading dose varyingfrom 35,000 to 1,500,000 U SK (Fletcher,1959). In those days, clinicians considered anti-streptokinase antibodies (aSKa) to be the origin of SK-resistance. Former humanstreptococcal infections were supposed to have elicited antibodies in the plasma whichcrossreacted with SK. In the 1960s, when thrombolytic therapy was administered on alimited scale, clinicians used a modification of the "predicted dose test", the "titratedinitial dose" test, to determine the loading dose of SK necessary to induce a systemic lyticstate. Applied dosages varied between 25,000 and 3,000,000 U (Verstraete,1966). Thiscorresponded with an interindividual variation in dosage of over a 100-fold range. Theloading dose was below 1,250,000 U SK in 97% of the patients. Subsequently, amaintenance dosage of 100,000 U SK per hour was given in order to keep fibrinogen andplasminogen low during several days of treatment.

SK-resistance was described in several studies in the 1970s. It was reported that 32 of236 blood donors (15.6%) would have required a loading dose of SK exceeding 250,000 Uto overcome resistance to treatment with SK (James,1973). In 312 patients in whom the"titrated initial dose" test was performed, it was found that 7% of them had a SK-resistance exceeding 250,000 U (Arnesen,1977). A recent streptococcal infection inpatients was associated with a significantly higher SK resistance. Patients, who had aantistreptolysin titre (AST) exceeding 625 U showed a greater SK resistance compared topatients who had an AST under 125 U (Aznar,1976). Very high levels of IgG to SK werenot found in a recent small study (Buchalter,1992). Thus, the reported incidence of aSKais subject to variability.

2. Systemic lytic state and anti-streptokinase antibodies (aSKa)

In the 1980s it was demonstrated that a systemic lytic state, defined as a certaindecrease in fibrinogen after thrombolytic therapy, was associated with success of SKtherapy (Rothbard,1985; Six,1987). On the other hand, a non-systemic lytic state inpatients who were treated with anistreplase was associated with non-patency(Marder,1987). In our study this finding was confirmed; all patients with MI showing asystemic non-lytic state, defined as fibrinogen in excess of 1 g/l, turned out to have non-patency of the infarct related vessel after treatment with anistreplase (appendix 2). It has

21

Chapter III

been suggested that aSKa might be responsible for the absence of a systemic lytic statebut at the time of this study aSKa could not be measured. Following completion of theassay (appendix 3), we found that a systemic non-lytic state in patients could be explainedby the presence of high levels of aSKa (appendix 4). However, the occurrence of asystemic lytic state after anistreplase administration was not absolutely predictive forsuccess of therapy. In fact, despite a lytic state, 17% (9/52 patients) showed a non-patentinfarct related vessel. It is therefore likely that other local or systemic antifibrinolyticfactors, such as Lp(a) and/or PAI, may also be involved.

In contrast to our data, another study, in which the pretreatment aSKa levels of 333patients were measured, reported no correlation between aSKa and patency afteranistreplase therapy (Fears,1992). These investigators used a radio-immuno assay (RIA),whereas we used an enzyme-linked immunosorbent assay (ELISA). Until now, these 2assay techniques have not been compared with each other.

2.1. Determination of anti-streptokinase antibodies (aSKa)

Variants of Johnson’s "predicted dose test", such as the "titrated initial dose test", the"streptokinase-resistenz-test" and the "streptokinase reactivity test" have been used fordetermination of the appropriate dosage in patients with thromboembolic disease(Deutsch,1960; Amery,1963). In 1983, a specific ELISA for quantitative assessment ofaSKa has been described (Leonardi,1983), but no reports on its use emerged. The aSKaassay using a RIA (Moran,1984) was used more often. However, this method is timeconsuming which disqualifies its application for immediate clinical practice purposes. Aquick aSKa level determination offers the opportunity to administer complementary oralternative thrombolytic therapy when titers are high. This strategy may thus renderclinical benefits in the first phase of MI (Sigwart,1985; appendix 5). Sofar, no studieshave been published which described pre-thrombolysis blood sampling, immediateassessment of aSKa and subsequently adaptation of the dosage of SK and/or adjunctive rt-PA infusion in case of a high aSKa titre. Because of its delayed assessment, aSKadetermination has never become popular. As a surrogate parameter, some cliniciansroutinely measured serum fibrinogen immediately after administration of SK in order toidentify patients in whom delayed or failed reperfusion is likely to be due to a high aSKatiter (Lew,1985b). As we illustrated in appendix 2, this appears to be a rational approachto detect failure of anistreplase (and probably SK). Now that a rapid and easy to performassay has been developed (appendix 3), its clinical benefit needs to be determined.

3. Role of plasminogen activator inhibitor (PAI)

Endogenous tissue-plasminogen activator (t-PA) is a serine protease which convertsplasminogen to plasmin. It is localized in endothelial cells and released into the blood inresponse to a variety of stimuli like thrombin formation, fibrin deposition, ischemia, stress,physical exercise or venous occlusion. The concentration of t-PA antigen in plasma isabout 3.8-4.5 ng/ml (Angleton,1989). Its activity is regulated by the inhibitor plasminogenactivator inhibitor (PAI) of which two forms are known: PAI type 1 (PAI-1) and PAI type2. The latter is found in pregnancy and not of relevance in MI. PAI-1, further referred to

22

aSKa, PAI and Lp(a)

as PAI, is one of the main inhibitors of endogenous fibrinolysis in blood. It neutralizesboth tissue-type PA and urinary-type plasminogen activator. PAI is present in endothelialcells, α-granules of platelets and in plasma (Erickson,1984; Sprengers,1987). PAIdetermines the amount of free t-PA that is available for actual plasminogen activation andendogenous fibrinolysis. Thus, PAI plays an important inhibitory role in endogenousfibrinolysis. It is one of the most highly regulated fibrinolytic components. Changes in itsactivity may dramatically disrupt the normal fibrinolytic balance. Determination of PAI ishampered because the molecule is very unstable (Loskutoff,1989). PAI antigen levels ofnormal human plasma may vary within the range 6.9 to 77 ng/ml. The same holds true forPAI activity levels which may vary from 1.9 to 12.4 U/ml (Nicoloso,1988). The antigenassay may detect more PAI than the activity assay suggesting that some of the PAI in thesample is latent or in complex with t-PA. PAI activity in plasma frequently increases aftersurgery or in response to MI. This indicates that PAI is an acute-phase reactant.

PAI levels might be clinically important in MI, as is illustrated by at least 4observations: a) Patients with coronary artery disease may have an impaired fibrinolyticactivity (Chakrabarti,1968), b) Reduced fibrinolytic activity was demonstrated in survivorsof MI who had no coronary artery disease at angiography (Verheugt,1987), c)Significantly elevated levels of PAI have been shown in survivors of MI compared to ahealthy subjects (Hamsten,1987), and d) Elevated PAI activity in the morning, incombination with almost undetectable free t-PA levels, corresponds with the documentedhigher incidence of MI during this part of the day (Muller,1985; Tofler,1987;Grimaudo,1988; Angleton,1989; Mulcahy,1991). The inhibitory potential of PAI mighteasily be exceeded by infusion of rt-PA in patients with MI as was illustrated by anabsence of correlation between the pre-existent PAI level and failure to achieve coronarypatency with rt-PA (Sane,1991). However, other authors showed an inverse relation of PAIlevels before rt-PA or urokinase administration with vessel patency (Barbash,1989;Sakamoto,1992). Failure of rt-PA therapy in MI has also been explained by excessivelyhigh local concentrations of PAI caused by activated platelets in the coronary obstruction(Kruithof,1986; Loskutoff,1988). Thus, no consensus exists concerning the significance ofplasma PAI levels in relation to the effect of thrombolytic therapy.

We did not find an increased PAI activity level in patients with a non-patent coronaryvessel compared to those with a patent vessel following SK therapy (appendix 6). Theseresults did not help to explain the superiority of a combination of non-fibrin and fibrinspecific thrombolytic agents in patients with MI compared to monotherapy (Califf,1991;Grines,1991). These findings, however, were not confirmed by the GUSTO trial(GUSTO,1993). Thus it may be concluded that the extent to which PAI may affect thesuccess rate of thrombolysis in patients with MI still needs to be determined.

4. Impairment of plasminogen activation by lipoprotein(a) [Lp(a)]

In clinical practice, high levels of lipoprotein(a) [Lp(a)] have been associated withcoronary artery disease, MI, coronary bypass vein graft stenosis, restenosis afterpercutaneous transluminal coronary angioplasty (PTCA) and cerebrovascular disease(Dahlen,1986; Zenker,1986; Hamsten,1987; Hoefler,1988; Hoff,1988; Seed,1990;

23

Chapter III

Hearn,1992). Lp(a) was identified as an independent risk factor for atherothromboticdisease (Merz,1989; MBewu,1990; Loscalzo,1990). Actually, its physiologic function isunknown but it is unlikely that the resemblance of apo(a) to plasminogen has nofunctional consequence in the fibrinolytic system (Brown,1987; Scott,1990; Rees,1991).Clinical studies on Lp(a), however, are hampered because the various Lp(a) assays arepoorly standardized. Dietary modification or drug therapy appeared to have little or noeffect on Lp(a) levels (Brewer,1990). In-vitro, Lp(a) appeared to enhance endothelial cellsynthesis of PAI without altering t-PA activity. This suggested that Lp(a) might support aspecific prothrombotic state (Etingin,1990). In contrast, other investigators reported a morecomplex interaction (Edelberg,1990).

In our study we found an inverse correlation between Lp(a) levels and plasminogendecrease, but only in patients with MI who had a non-patent coronary vessel atangiography after treatment with anistreplase (appendix 7). These results are in agreementwith the hypothesis that high Lp(a) levels may impair fibrinolysis. It must be said that thenumber of patients in our study was small and the relation between Lp(a) and plasminogendecrease was only of borderline significance. Thus, definite conclusions may not be drawn.Because of the possible interaction between Lp(a) and PAI, it appears prudent to measureboth parameters at the same time in further studies on factors which determine theoutcome of thrombolytic therapy.

5. Proposed indicators of thrombolytic efficacy such as fibrinopeptide A (FPA)

Fibrinopeptide A (FPA) is liberated from fibrinogen when it converts to fibrin by theaction of thrombin (Bettelheim,1956; Mosesson,1992). As a result, high FPA levels inplasma indicate thrombin activity and this reflects a status of hypercoagulability. Thepossibility of specific measurement by radio-immuno assay (RIA) made FPA a marker offibrin generation in vivo. Elevated plasma levels have been demonstrated in patients withthromboembolic disease like MI but such levels decrease promptly in response toadminstration of heparin (Mombelli,1984; Eisenberg,1985). Treatment of MI with SK orrt-PA was associated with increased plasma FPA levels (Eisenberg,1987; Rapold1989,1990). No firm relation between perfusion status and plasma FPA levels has beenreported following thrombolytic therapy. Whether thrombin/antithrombin III (TAT)complex levels may be used as an indicator for initial success of thrombolytic therapy washypothesized (Gulba,1991), but remains to be confirmed by other studies.

6. Conclusions and implications for thrombolytic therapy

When one considers coagulation parameters in relation to success of thrombolytictherapy with SK or anistreplase, only anti-streptokinase antibodies (aSKa) appear to affectits outcome in a direct manner. Former streptococcal infections and previous thrombolytictherapy with SK or anistreplase are responsible for elevated levels of aSKa. The antibodylevel was increased at the 3rd day after this treatment and levels remained elevated for upto 4.5 years (Blix,1961; Massel,1989; Jalihal,1990; Lynch,1991; Fears,1992; Elliott,1993;Lee,1993; Patel,1993; Buchalter,1993).

The development of a simple and rapid enzyme-linked immunosorbent assay now

24

aSKa, PAI and Lp(a)

allows a more routine quantification of aSKa. These antibodies, which prevent theoccurrence of a systemic lytic state, can also be identified indirectly by measurement ofthe fibrinogen level shortly after initiation of SK or anistreplase therapy. In case of a non-systemic lytic state, patency of the infarct related vessel was always absent. However,despite the occurrence of a systemic lytic state, success of thrombolytic therapy is notguaranteed. In these patients non-invasive parameters such as clinical condition, ECG ST-segment transformation and creatinine kinase levels, may offer clues with respect to infarctrelated vessel patency. These parameters, however, were not part of our studies. In ourview, patients with high plasma levels of aSKa are candidates for additional or alternativetreatment. This may include administration of a higher dosage of SK or administration ofa different thrombolytic agent. Consequently, an improved reperfusion rate may ensue.Mechanical intervention in patients who fail to respond to pharmacological intervention isalso optional but shall not be discussed here.

Besides efficacy, the presence of aSKa may also affect the safety of a repeatedtreatment with SK or anistreplase. Adverse events such as acute anaphylaxis, serumsickness, delayed hypersensitivity or vasculitis, which have been reported in 1.7 to 18% ofSK or anistreplase recipients (Alexopoulos,1984; McGrath,1985; Bucknall,1988), are likelyto occur more often following repeated treatment. Intra-dermal administration of a smalldosage SK may be used for identification of patients at risk for adverse events mediatedby IgE antibodies to SK (Dykewicz,1986). However, this test is not suitable for theassessment of IgG antibodies to SK. Only this type determines the efficacy of SK or itsderivate. AST titers are frequently raised without corresponding elevated specific IgGtiters (Elliott,1993). Thus, determining the plasma AST-level in the acute situation is notuseful. For clinical practice it is important to ask patients for any previous thrombolytictreatment. Although comparative trials to guide the choice for repeated treatment have notbeen performed, both urokinase or rt-PA can be used safely and probably effectively inpatients with reinfarction (White,1990b; White,1991).

Several authors have suggested that plasminogen activator inhibitor (PAI) levels mightbe relevant in MI. We studied its plasma level in patients with MI who received SK butfound no association with the outcome of therapy. In contrast, for Lp(a) some evidence forimpairment of clinical efficacy was demonstrated in non-patency. However, the relationbetween systemic levels of PAI and Lp(a) and the efficacy of thrombolytic therapyremains complex. This issue has to be studied further.

25

Reocclusion

Chapter IV

Early and late reocclusion in patients with myocardial infarction

1. Introduction

Following thrombolytic therapy, occluded infarct related coronary arteries can bereperfused as a result of plasminogen activation. Coronary angiography is the goldenstandard for its detection. The speed of reperfusion is, among other variables, dependenton the type of thrombolytic agent which is administered. Therapy with rt-PA dissolved aclot quicker compared to infusion of SK (TIMI,1985) although 3 to 4 hours after initiationof therapy reperfusion rates became similar (Ganz,1992; GUSTO AngiographicInvestigators,1993). This has been called the "catch-up" phenomenon of infarct vesselpatency. Very early reperfusion might be intermittent (Hackett,1987; Krucoff 1990,1993;Kwon,1991). In this phase it is not qualified as reocclusion. Persistent early reocclusion isoften accompanied by clinical symptoms and electrocardiographic signs. Since a relationbetween early reocclusion and increased in-hospital mortality has been demonstrated(Ohman,1990), its occurrence will be of clinical relevance.

2. Occurrence and mechanism

Quantification of the reocclusion rate will require repeated coronary angiography. Aftercombining the results of randomized trials in which rt-PA plus i.v. heparin was comparedwith non-fibrin specific thrombolytic agents, reocclusion rates, determined after severaldays or before discharge, were 13.5 and 8% (p=0.002), respectively (Granger,1992). Whenanistreplase was used, reocclusion after 24 hours was found in only 4% of the patients(Relik-van Wely,1991). This low figure is comparable to our results as no reocclusionoccurred between 1½ and 48 hours after therapy with anistreplase (appendix 8).Reocclusion 3 months after initial successful thrombolytic therapy with SK or anistreplasewas found in about 30 and 28% of the patients in the APRICOT study and our study,respectively (Meijer,1993; appendix 8). Patients in the APRICOT study with persistentpatency at coronary angiography after 3 months showed a slight increase in left ventricularejection fraction (LVEF), those with reocclusion did not. Their finding of an absence offurther LVEF deterioration despite reocclusion corresponds with ours. Presence, or evenenhanced, development of collateral blood flow might have been responsible for thestabilization of LVEF in these patients.

Early after reperfusion the stimulus for rethrombosis is strongest because of a) re-exposure of the cracked plaque, b) hypercoagulability and activation of platelets inducedby thrombolytic therapy (see below), and c) the presence of locally vasoactive substancesreleased by activated platelets. Thus, early reocclusion (within hours or days) may berelated to other mechanisms than reocclusion after weeks or months (late). In latereocclusion lesion remodelling due to endothelial growth may be of primary importance.In contrast to early reocclusion, late reocclusion often occurs silently (Ohman,1990;appendix 8). Actually, the precise course of events leading to late reocclusion is less wellunderstood than of those leading to early reocclusion.

27

Chapter IV

3. Factors determining reocclusion

Thrombolytic agents convert plasminogen to plasmin. Presence of plasmin in thecirculation leads to fibrinogenolysis, loss of activity coagulation factors V and VIII, andthe accumulation of fibrinogen degradation products. These effects impair bloodcoagulability. In addition, membrane glycoproteins at the platelet surface are degraded byplasmin, which may result in impaired adhesion and aggregation of platelets. Thus,initially, a state of hypocoagulability and platelet dysfunction will occur. However,abundance of free plasmin or the binding of anti-SK antibodies to the SK-plasminogencomplex located at the platelet surface, may trigger processes leading to platelet activationand hypercoagulability (Vaughan 1988,1991; Sherry,1992a).

Activated platelets release thromboxane A2. This peptide strongly supports plateletaggregation. During thrombolytic therapy with either rt-PA or SK, a striking increase ofthromboxane A2 metabolites in plasma and urine has been measured (Fitzgerald,1988;Kerins,1989). Therefore it is believed that marked platelet activation takes place followingthrombolytic therapy.

The paradoxical hypercoagulabile state in patients with MI after thrombolytic therapywth either SK or rt-PA is illustrated by the presence of clearly elevated fibrinopeptide A(FPA) levels in their plasma (Eisenberg 1986,1989; Owen,1988). As mentioned in the 5thparagraph of the former chapter, no relation between reperfusion and FPA levels has beendemonstrated. With regard to reocclusion, in patients treated with heparin in conjunction tothrombolytic therapy, an FPA threshold level of 50 ng/ml 24 hours followingthrombolysis, appeared to be a relative specific marker of subsequent vessel occlusion(86%), but lacked sensitivity (48%) (Rapold,1992).

When reperfusion of an occluded vessel has been achieved, the balance between pro-thrombotic and anti-thrombotic factors will determine the incidence of early reocclusion.Of importance are a) severity and geometry of the underlying coronary artery stenosis, b)presence of residual coronary arterial thrombosis, c) platelet adhesiveness and aggregationpotential, d) rheologic parameters like flow properties, shear stress, fibrinogen content and,possibly, e) fibrin(-ogen) degradation products which express anticoagulant activity, and f)anticoagulant and/or antiplatelet therapy (Latallo,1964; Marder,1969; Haverkate,1979;Harrison,1984; Gash,1986; Adams,1987; Moriarty,1988; Davies,1991; Hoffmannn,1993)

4. Traditional ways to prevent reocclusion

It has been suggested that the presence of a high-grade residual stenosis at coronaryangiography after successful thrombolysis may predict recurrent ischemia (Harrison,1984;Gash,1986). Subsequently, several well designed studies were performed to assess thehypothesized additional benefit of early coronary angioplasty (PTCA) after thrombolytictherapy because of MI (Topol and the TAMI Group,1987; TIMI,1988; Simoons,1988).These trials showed that thrombolyic therapy combined with immediate PTCA did notappear to be superior to early non-invasive treatment using i.v. rt-PA, heparin and acetyl-salicylic acid. In contrast, a complicated clinical course occurred more frequently andmortality may be higher due to an early invasive strategy. Recently, the APRICOT-investigators studied the relation between angiographic reocclusion at 3-months and

28

Reocclusion

residual stenosis after successful thrombolysis (Veen,1993). They showed that stenosisseverity exceeding 90% was a significant independent predictor for reocclusion. One mightobject that the APRICOT findings appears in contrast to the results of the TAMI group.However, the TAMI study focussed at recurrent ischemia before hospital discharge, not atreocclusion (Ellis,1989).

The severity of the underlying ruptured coronary lesion cannot be influenced bysystemic thrombolytic therapy, this in contrast to the mechanical approach by PTCA. Inskillful hands, this procedure, without previous thrombolytic therapy, was recently shownto be superior in patients with MI compared to i.v. SK therapy with respect to patencyrate, residual left ventricular function, and incidence of recurrent myocardial ischemia(Zijlstra,1993).

In the course of complete clot digestion by thrombolytic treatment, residual coronarythrombus is present. This material is a particularly long lasting thrombogenic substrate,even more thrombogenic than deeply injured arterial tissue (Badimon 1988,1991).Persistent exposure of thrombin may be a predisposing factor both to initial failure ofrecanalization and to early reocclusion.

The most widely used antithrombotic agent is heparin (Hirsh,1991). Following its use inpatients with MI, plasma FPA level decreased indicating its antithrombin effect(Gallino,1986). Heparin was given subcutaneously 12 hours after the thrombolytic agent inthe GISSI-2 study. This regimen was disapproved because the delay was qualified as toolong to prevent reperfused coronary arteries from reocclusion. Meeting the critics, in ISIS-3 subcutaneous administration of heparin was started 4 hours after thrombolytic therapy.However, as was indicated by only minimal extension of the activated partialthromboplastin time (aPTT), the degree of anticoagulation was definitely not in thetherapeutic range (Kroon,1992; Delanty,1992; Goldhaber,1992). It appeared that specificquestions like the usefulness of heparin and its optimal mode of administration, were moreadequately answered in small, but well designed and conducted trials. Such trialsdemonstrated that in patients with MI treated with rt-PA, i.v. heparin is of superiorefficacy compared to placebo or aspirin in obtaining patency of the infarct related vessel(Bleich,1990; Hsia,1990; de Bono,1992). Moreover, the quality of heparinization, asmeasured by the degree of prolongation of the aPTT, has been related with a morebeneficial outcome (Hsia,1992; Arnout,1992). Thus, when heparin is given in patients withMI, measurement and appropriate adjustment of the dosage guided by the aPTT are ofmajor importance. Currently, only in a minority of patients aPTTs two to three times thecontrol values were obtained during several days in various studies (Hsia,1992;Becker,1993).

An oral dose of 325 mg aspirin blunted the increase of thromboxane A2 metabolites inpatients with MI treated with rt-PA or SK (Fitzgerald,1988; Kerins,1989). This finding,among others, provided a rationale for adjunctive antiplatelet therapy to thrombolytictherapy (Stein,1989; Winters,1991). After performing a meta-analysis, it has been statedthat aspirin in the presence of heparin significantly reduced the incidence of coronaryreocclusion and recurrent ischemia after thrombolysis with either SK or rt-PA(Roux,1992). Criticizers, however, rebutted that the multiplicity of individual study designsdid not allow such a conclusion (Sherry,1992b; Ridker,1993).

With respect to late reocclusion and recurrent MI, it has been attempted to prevent this

29

Chapter IV

with coumadin treatment. However, the APRICOT-study showed that the reocclusion ratein patients who used either aspirin, coumadin or placebo was similar at angiography after3 months (Meijer,1993). Interestingly, in that study treatment with aspirin was associatedwith a significantly lower incidence of reinfarction compared to placebo.

5. New ways to prevent reocclusion

Given the overall poor quality of anticoagulation using i.v. heparin, one may attempt tospeed up aPTT or activated clotting time (ACT) measurements and perform these tests atthe bedside. Probably this might improve the efficacy of treatment (Ogilby,1989;Ansell,1991; Vacek,1991; Melandri,1993; Grill,1993).

Because residual anchored mural thrombus contains active thrombin, adsorbed to deeperlying layers of fibrin, it is poorly accessible to the large heparin-antithrombin III complex(Liu,1979; Bar-Shavit,1989; Hogg,1989; Weitz,1990). Thus, more effective antithrombinagents may be needed. These agents, named argatroban and hirudin, are currently studied(Fitzgerald,1989; Jang,1990; Clarke,1991; Gold,1993). Hirudin, a small peptide derivedfrom the pharyngeal glands of the leech was recently manufactured using a recombinanttechnique. It is the most potent and selective inhibitor of thrombin currently known.Unlike heparin, hirudin does not require any endogenous cofactor such as antithrombin IIIfor its anticoagulant effects (Hoet,1991; Deutsch,1993). In contrast to heparin, hirudinneutralizes fibrin-bound thrombin (Mirshahi,1989) and does not increase platelet adhesionto fibrin (Verstraete,1992). Two human pilot-studies showed hirudin, compared to heparin,to be as effective, or even more so, in preventing reocclusion after successfulthrombolysis, without any safety problems (Neuhaus,1993; Cannon,1993). Careful dose-ranging studies followed by large randomized controlled trials will be required to establishto what extent the ratio of anti-thrombotic efficacy to bleeding risk differs from that ofconventional therapy (Anonymous,1992).

When platelets are activated, the glycoprotein IIb/IIIa (GPIIb/IIIa) complex at theplatelets’ surface plays a prominent role in the binding of fibrinogen and other ligands(Coller,1990). Specific inhibition of this complex in vitro and in vivo was shown to bepossible with a monoclonal antibody named 7E3 [7E3-F(ab’)2]. After its administration,there was profound inhibition of platelet function, leading to acceleration of thrombolysiswith rt-PA and prevention of reocclusion (Coller,1985; Gold,1988). The clinical efficacyand safety of these antibodies as therapeutic agents are currently under investigation(Gold,1989; Kleiman,1993).

6. Conclusions

Mechanisms involved in early and late reocclusion are probably different. Earlyreocclusion appears related to factors such as hypercoagulability and platelet activation,which may paradoxically follow thrombolytic therapy. Exposure of the denuded crackedcoronary plaque or, in particular, residual mural thrombus, serves as a thrombogenicsubstrate. In addition, the extent of early reperfusion may also be important with regard toearly reocclusion: TIMI grade 2 flow (partial perfusion) may no longer be perceived asequal to TIMI grade 3 flow (complete perfusion) since patients with TIMI grade 2 flow

30

Reocclusion

have enzymatic, electrocardiographic and pump function indices of MI similar to those ofpatients with grade 0 or 1 flow (no or minimal perfusion) (Karagounis,1992;Anderson,1993).

Factors involved in late reocclusion have been identified as severity and surfacegeometry of the residual stenosis (Veen,1993). Rheologic factors may play an additionalrole. Late reocclusion, although occurring relatively frequently, is not necessarilyaccompanied by symptoms or deterioration of myocardial function.

Optimal reperfusion characterized by rapid (within 60-minutes), complete (TIMI grade 3flow) and sustained coronary recanalization with adequate myocardial tissue perfusion(absence of the "no-reflow" phenomenon) and absence of reocclusion, is only achieved in25% or less of the patients with MI treated with thrombolytic agents (Lincoff,1993).

Currently adjunctive and/or conjunctive therapy to thrombolysis with new antithrombinor platelet inhibiting agents appear a most promising area of which additional therapeuticbenefits could be expected in the near future.

31

Summary and Conclusions

Chapter V

Summary and conclusions

Insight in the mechanisms leading to acute myocardial infarction (MI) has resulted inthe administration of exogenous plasminogen activator, later called thrombolytic therapy.This treatment was associated with a significant reduction in mortality and morbidity.However, success has not always been achieved due to failure to achieve reperfusion oroccurrence of reocclusion. This thesis describes our studies on the factors which maydetermine the efficacy of thrombolytic therapy in patients with MI.

The adagium "time is muscle" led us to conduct a prehospital thrombolysis study withthe streptokinase-derivate anistreplase (appendix 1). This thrombolytic agent is easilyadministered as an intravenous infusion during 5 minutes in the prehospital setting. Welearned that this approach was associated with logistical problems. Remote ECGconfirmation of impending MI turned out to be the major time consuming procedure. Asthe driving time from the patient’s home to the hospital was less than the procedure athome, we prematurely discontinued this study. It was concluded that in medium sizedtowns like Groningen, prehospital initiation of thrombolytic therapy does not renderbenefits for patients with MI. Metropolises, with regular traffic jams, or distant rural sites,with a long transit time to the hospital, may offer better opportunities.

Already in the early 1960s, a relation was recognized between parameters of bloodcoagulation and the outcome of thrombolytic therapy. It was assumed that a certain degreeof fibrinogen breakdown following streptokinase (SK) administration, the so-calledsystemic lytic state, was a prerequisite for local coronary fibrinolysis. Our study confirmedthis hypothesis for the SK-derivate anistreplase (appendix 2). We showed that systemicfibrinogenolysis preceded and/or accompanied local lytic activity in most patients. Astrong relation was shown between a systemic non-lytic state 1.5 hour after initiation oftherapy and failure of therapy as defined as non-patency of the infarct related vesselduring angiography. So, measurement of fibrinogen, which is simple, rapid and cheap, inpatients who are treated with anistreplase and probably SK, offers the opportunity toidentify patients in whom thrombolytic therapy might not be successful.

We hypothesized that the reason why a systemic lytic state did not develop was thepresence and subsequent binding of antibodies to the SK component of anistreplase. Theseanti-streptokinase antibodies (aSKa) have probably been induced by previous streptococcalinfections. As currently available assays to determine aSKa levels are time consumingand/or unpractical, we developed a quick enzyme-linked immunosorbent assay, in order todetermine the aSKa level during the first hour of treatment (appendix 3). Measurement ofaSKa in plasma samples of patients with MI confirmed the hypothesized relation betweenhigh titers of antibodies and failure to achieve a systemic lytic state leading to non-patencyof the infarct related vessel (appendix 4). If high aSKa titers are found, there are twopossible alternatives: either increase the dosage of SK or select another thrombolytic agent(appendix 5).

Plasminogen Activator Inhibitor (PAI) is a recently identified component of thefibrinolytic system which may inhibit plasminogen activation and thus thrombolyticactivity. We measured both PAI and endogenous t-PA in patients following treatment with

33

Chapter V

SK because of MI. A paradoxical low level of both compounds in patients with a non-patent infarct related vessel was found compared to patients with a patent vessel(appendix 6).

Low density lipoprotein (LDL) is of major importance in the development ofcardiovascular disease. Lipoprotein(a) [Lp(a)] consists of a LDL part and a protein moiety,named apo(a). This apo(a) has a striking homology to plasminogen but does not have itsfunctional activity. As in vitro findings had shown that Lp(a) might impair fibrinolysis, wesought for an in-vivo effect of Lp(a) in thrombolysis. Inappendix 7 we describe aninverse correlation of Lp(a) levels with plasminogen decrease in patients with a non-patentinfarct related vessel. Thus, high Lp(a) levels, although not directly associated with a pooroutcome of anistreplase therapy, contribute to insufficient fibrinolysis in patients with anon-patent infarct related vessel.

The angiographic reocclusion rate at 48 hours and 3-months after anistreplase therapywas studied inappendix 8. No reocclusion occurred between 1½ and 48 hours. After 3months the reocclusion rate amounted 28% despite adequate anticoagulant therapy.Interestingly, neither overt reinfarction, nor a decrease of left ventricular ejection fractionwere observed in those patients with a late occluded vessel. Early persistent patencyappears to be of greater clinical value than late patency. This may be explained byrestriction of the cardiac area at risk in the acute phase through reperfusion and subsequentadaptation of the ventricle by collateral blood supply.

The following conclusions can be drawn from these studies:

- Initiation of thrombolytic therapy in the prehospital setting is cumbersome and not thetreatment of choice in medium sized towns and/or when transit time to hospital is short,provided that the in-hospital triage is quick.

- Fibrinogen assessment 1 hour after initiation of treatment with streptokinase oranistreplase is a simple and quick method for identification of patients who might benefitfrom supplementary (thrombolytic) therapy.

- The presence of anti-streptokinase antibodies may determine the absence of a systemiclytic state 1 hour after streptokinase or anistreplase administration. An simple enzyme-linked immunosorbent assay for measurement of these antibodies has been developedwhich allows rapid detection. Its value, however, needs further clinical validation.

- The relation between lipoprotein(a) levels and insufficient thrombolysis in clinicalpractice is complex. It is likely that lipoprotein(a) is acting in concert with otherfibrinolysis inhibiting factors such as anti-streptokinase antibodies and plasminogenactivator inhibitor. The relative contribution of each of these factors needs furtherquantification.

- Late reocclusion, within 3 months after initial coronary patency, occurs relativelyfrequent, despite adequate anticoagulant therapy, and is not necessarily accompanied bysymptoms or deterioration of left ventricular function.

34

References

References

Abendschein D, Seacord LM, Nohara R, Sobel BE, Jaffe AS. Prompt detection of myocardial injury by assay o fcreatine kinase isoforms in initial plasma samples. Clin Cardiol 1988;11:661-4.

Adams PC, Fuster V, Badimon L, Badimon JJ, Chesebro JH. Platelet/vessel wall interactions, rheologic factorsand thrombogenic substrate in acute coronary syndromes: preventive strategies. Am J Cardiol 1987;60:9G-16G.

Adams JE, Bodor GS, Dávila-Román VG, et al. Cardiac troponin I, a marker with high specificity for cardiacinjury. Circulation 1993;88:101-6.

AIMS Trial Study Group . Effect of intravenous APSAC on mortality after acute myocardial infarction:preliminary report of a placebo controlled clinical trial. Lancet 1988;i:545-9.

Alexopoulos D, Raine AE, Cobbe SM. Serum-sickness complicating intravenous streptokinase therapy in acutemyocardial infarction. Eur Heart J 1984;5:1010-2.

Amery A , Maes H, Vermijlen J, Verstraete M. The streptokinase-reactivity test. Thromb Diath Haemorrh1963;9:175-88.

Anderson JL, Karagounis LA, Becker BC, Sorensen SG, Menlove RL, for the TEAM-3 Investigators. TIMIperfusion grade 3 but not grade 2 results in improved outcome after thrombolysis for myocardial infarction:Ventriculographic, enzymatic, and electrocardiographic evidence from the TEAM-3 study. Circulation1993;87:1829-39.

Angleton P, Chandler WL, Schmer G. Diurnal variation of tissue-type plasminogen activator and its rapidinhibitor (PAI-1). Circulation 1989;79:101-6.

Anonymous editorial. Hirudins: return of the leech? Lancet 1992;340:579-80.

Ansell J, Tiarks C, Hirsh J, McGehee W, Adler D, Weibert R. Measurement of the activated partialthromboplastin time from a capillary (fingerstick) sample of whole blood. A new method for monitoringheparin therapy. Am J Clin Pathol 1991;95:222-7.

Arnout J , Simoons M, de Bono D, Rapold HJ, Collen D, Verstraete M. Correlation between level ofheparinization and patency of the infarct-related coronary artery after treatment of acute myocardial infarctionwith alteplase (rt-PA). J Am Coll Cardiol 1992;20:513-9.

Astrup T , Permin PM. Fibrinolysis in animal organism. Nature 1947;159:681-2.

Aznar J, Delgado F, Estellés A. Streptokinase resistance test in patients with streptococcal infection and/or highantistreptolysin titers. Scand J Haematol 1976;16:141-143.

Arnesen H, Rygh M, Ly B, Jakobsen E. Streptokinase resistance in medical patients in Oslo. Scand J Haematol1977;18:348-52.

Badimon L, Lassila R, Badimon J, Vallabhajosula S, Chesebro JH, Fuster V. Residual thrombus is morethrombogenic than severely damaged vessel wall. Circulation 1988;78:II-119.

Badimon L, Badimon JJ, Lassila R, Heras M, Chesebro JH, Fuster V. Thrombin regulation of plateletinteraction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcinemodel: effects of hirudins, heparin, and calcium chelation. Blood 1991;78:423-34.

35

References

Badimon L, Badimon JJ, Lassila R, Merino A, Chesebro JH, Fuster V. Re-thrombosis on an evolving thrombusis mediated by thrombus-bound thrombin that is not inhibited by systemic heparin. Thromb Haemost1991;65:760.

Barbash GI, Hod H, Roth A, et al. Correlation of baseline plasminogen activator inhibitor activity with patencyof the infarct artery after thrombolytic therapy in acute myocardial infarction. Am J Cardiol 1989;64:1231-35.

Baroldi G . Coronary thrombosis: facts and beliefs. Am Heart J 1976;91:683-8.

Bar-Shavit R, Eldor A, Vlodavsky I. Binding of thrombin to subendothelial extracellular matrix. Protection andexpression of functional properties. J Clin Invest 1989;84:1096-1104.

Becker RC, Corrao JM, Ball SP, Gore JM. A comparison of heparin strategies after thrombolytic therapy. AmHeart J 1993;126:750-2.

Bellinger RL , Califf RM, Mark DB, et al. Helicopter transport of patients during acute myocardial infarction A mJ Cardiol 1988;61:718-22.

Bettelheim FR. The clotting of fibrinogen: II. Fractionation of peptide material liberated. Biochim Biophys Acta1956;19:121-9.

Berg K. A new serum type system in man: the Lp system. Acta Pathol Microbiol Scan 1963;59(suppl):369-82.

Birkhead JS on behalf of the joint audit committee of the British Cardiac Society and a cardiology committee ofthe Royal College of Physicians of London. Time delays in provision of thrombolytic treatment in six districthospitals. Br Med J 1992;305:445-8.

Bleich SD, Nichols TC, Schumacher RR, Cooke DH, Tate DA, Teichman SL. Effect of heparin on coronaryarterial patency after thrombolysis with tissue plasminogen activator in acute myocardial infarction. Am JCardiol 1990;66:1412-7.

Blix S. Antibodies after streptokinase treatment, and their importance for repeated fibrinolytic therapy. Acta MedScandinav 1961;170:201-9

Blohm M , Herlitz J, Hartford M, et al. Consequences of a media campaign focusing on delay in acutemyocardial infarction. Am J Cardiol 1992;69:411-3.

Bouten MJM , Simoons ML, Hartman JAM, van Miltenburg AJM, van der Does E, Pool J. Prehospitalthrombolysis with alteplase (rt-PA) in acute myocardial infarction. Eur Heart J 1992;13:925-31.

Braunwald E. The open-artery theory is alive and well-again. N Engl J Med 1993;329:1650-2.

Brewer HB Jr. Effectiveness of diet and drugs in the treatment of patients with elevated Lp(a) levels. In ScanuAM, ed. Lipoprotein(a). New York: Academic Press, 1990: 211-20.

Brown MS, Golstein JL. Teaching old dogmas new tricks. Nature 1987;330:113-4.

Buchalter MB , Suntharalingam G, Jennings I, et al. Streptokinase resistance: when might streptokinaseadministration be ineffective? Br Heart J 1992;68:449-53.

Buchalter MB . Are streptokinase antibodies clinically important. Br Heart J 1993;70:101-2.

Bucknall C, Darley C, Flax J, Vincent R, Chamberlain D. Vasculitis complicating treatment with intravenousanisoylated plasminogen streptokinase activator complex in acute myocardial infarction. Br Heart J 1988;59:9-

36

References

11.

Califf RM , Topol EJ, Stack RS, et al. for the TAMI Study Group. Evaluation of combination thrombolytictherapy and timing of cardiac catheterization in acute myocardial infarction. Results of thrombolysis andangioplasty in myocardial infarction - phase 5 randomized trial. Circulation 1991;83:1543-6.

Cannon CP, McCabe CH, Henry TD, et al. for the TIMI 5 Investigators. Hirudin reduces reocclusioncompared to heparin following thrombolysis in acute myocardial infarction: results of the TIMI 5 trial. J AmColl Cardiol 1993;21:136A (abstr.).

Carney RJ, Murphy GA, Brandt TR, et al. for the RAAMI Study Investigators. Randomized angiographic trial ofrecombinant tissue-type plasminogen activator (alteplase) in myocardial infarction. J Am Coll Cardiol

1992;20:17-23.

Chakrabarti R , Hocking ED, Fearnley GR, Mann RD, Attwell TN, Jackson D. Fibrinolytic activity andcoronary artery disease. Lancet 1968;i:987-90.

Chalmers TC, Matta RJ, Smith H Jr, Kunzler AM. Evidence favouring the use of anticoagulants in the hospitalphase of acute myocardial infarction. N Engl J Med 1977;297:1091-6.

Chesebro JH, Zoldhelyi P, Fuster V. Pathogenesis of thrombosis in unstable angina. Am J Cardiol 1991;68:2B-10B.

Collen D. Identification and some properties of a new fast reacting plasmin inhibitor in human plasma. Eur JBiochem 1976;69:209-16.

Collen D. On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture. Thromb Haemost1980;43:77-89.

Collen D, De Cock F, Stassen J-M. Comparative immunogenicity and thrombolytic properties toward arterial a n dvenous thrombi of streptokinase and staphylokinase in baboons. Circulation 1993;87:996-1006 (a).

Collen D, Van de Werf F. Coronary thrombolysis with recombinant staphylokinase in patients with evolvingmyocardial infarction. Circulation 1993;87:1850-3 (b).

Coller BS, Scudder LE. Inhibition of dog platelet function by in vivo infusion of F(ab’)2 fragments of amonoclonal antibody. Blood 1986;66:1456-9.

Coller BS. Platelets and thrombolytic therapy. N Engl J Med 1990;322:33-42.

Clarke RJ, Mayo G, FitzGerald GA, Fitzgerald DJ. Combined administration of aspirin and a specific thrombininhibitor in man. Circulation 1991;83:1510-8.

Dahlen GH, Guyton JR, Attar M, Farmer JA, Kautz JA, Gotto AM. Association of levels of lipoprotein Lp(a),plasma lipids and other lipoproteins with coronary artery disease documented by angiography. Circulation1986;74:758-65.

Davies MJ, Fulton WFM, Robertson WB. The relation of coronary thrombosis to ischaemic myocardialnecrosis. J Pathol 1979;172:99-110.

Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction, sudden ischaemic death, a n dcrescendo angina. Br Heart J 1985;53:363-73.

Davies SW, Marchant B, Lyons JP, et al. Irregular coronary lesion morphology after thrombolysis predicts early

37

References

clinical instability. J Am Coll Cardiol 1991;18:669-74.

de Bono DP, et al. for the European Cooperative Study Group. Effect of early intravenous heparin on coronarypatency, infarct size, and bleeding complications after alteplase thrombolysis: a result of a randomized doubleblind European Cooperative Study Group trial. Br Heart J 1992;67:122-8.

Delanty N, Fitzgerald DJ. Subcutaneous heparin during coronary thrombolysis. Too little, too late. Circulation1992;86:1636-8.

Deutsch E, Fischer M, Marschner I, Kock M. Die Wirkung intravenös applizierter Streptokinase auf Fibrinolyseund Blutgerinnung. Thromb Diath Haemorrh 1960;4:482-506.

Deutsch E, Rao AK, Colman RW. Selective thrombin inhibitors: The next generation of anticoagulants. J AmColl Cardiol 1993;22:1089-92.

DeWood MA, Spores J, Notske R, Mouser LT, Burroughs R, Golden MS, Lang HT. Prevalence of totalcoronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med 1980;303:897-902.

Dykewicz MS, McGrath KG, Davison R, Kaplan KJ, Patterson R. Identification of patients at risk foranaphylaxis due to streptokinase. Arch Intern Med 1986;146:305-7.

Eaton DL, Fless GM, Kohr WJ, et al. Partial amino acid sequence of apolipoprotein(a) shows that it ishomologous to plasminogen. Proc Natl Acad Sci USA 1987;84:3224-8.

Edelberg JM, Gonzalez-Gronow M, Pizzo SV. Lipoprotein a inhibits streptokinase-mediated activation ofhuman plasminogen. Biochemistry 1989;28:2370-4.

Edelberg JM, Reilly CF, Pizzo SV. The inhibition of tissue type plasminogen activator by plasminogen activatorinhibitor-1. The effects of fibrinogen, heparin, vitronectin and lipoprotein(a). J Biol Chem 1991;266:7488-93.

Eisenberg PR, Sherman LA, Schectman K, Sobel BE, Perez J, Sobel BE, Jaffe AS. Fibrinopeptide A: a markerof acute coronary thrombosis. Circulation 1985;71:912-8.

Eisenberg PR, Sherman L, Rich M, et al. Importance of continued activation of thrombin reflected byfibrinopeptide A to the efficacy of thrombolysis. J Am Coll Cardiol 1986;7:1255-62.

Eisenberg PR, Sherman LA, Jaffe AS. Paradoxic elevation of fibrinopeptide A after streptokinase: evidence forcontinued thrombosis despite intense fibrinolysis. J Am Coll Cardiol 1987;10:527-9.

Eisenberg PR, Miletich JP. Induction of marked thrombin activity by pharmacologic concentrations ofplasminogen activators in nonanticoagulated whole blood. Thromb Res 1989;55:635-43.

Elliot JM , Cross DB, Cederholm-Williams SA, White HD. Neutralizing antibodies to streptokinase four yearsafter intravenous thrombolytic therapy. Am J Cardiol 1993;71:640-5.

Ellis SG, Topol EJ, George BS, et al. Recurrent ischemia without warning. Analysis of risk factors for in-hospital ischemic events following successful thrombolysis with intravenous tissue plasminogen activator.Circulation 1989;80:1159-65.

Endert E, Delemarre BJM, Sanders GTB. De latex-agglutinatietest voor myoglobine bij het acute hartinfarct. NedTijdschr Geneeskd 1987;131:1309-11.

Erickson LA , Ginsberg MH, Loskuthoff DJ. Detection and partial characterization of an inhibitor of

38

References

plasminogen activator in human platelets. J Clin Invest 1984;74:1465-72.

EMERAS (Estudio Multicéntrico Estreptoquinasa Republicas de América del Sur) Collaborative Group.Randomized trial of late thrombolysis in patients with suspected acute myocardial infarction. Lancet1993;342:767-72.

Etingin OR , Hajjar DP, Hajjar KA, Harpel PC, Nachman RL. Lipoprotein(a) regulates plasminogen activatorinhibitor expression in endothelial cells. A potential mechanism in thrombogenesis. J Biol Chem1991;266:2459-65.

European Cooperative Study Group. Streptokinase in acute myocardial infarction. N Engl J Med1979;301:797-802.

European Working Party . Streptokinase in acute myocardial infarction. Br Med J 1971;3:325-31.

The European Myocardial Infarction Project Group . Prehospital thrombolytic therapy in patients withsuspected acute myocardial infarction. N Engl J Med 1993;329:383-9.

Falk E. Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis: characteristics ofcoronary atherosclerotic plaques underlying fatal occlusive thrombi. Br Heart 1983;50:127-34.

Falk E. Coronary thrombosis: pathogenesis and clinical manifestations. Am J Cardiol 1991;68:28B-35B.

Fearnley GR. A concept of natural fibrinolysis. Lancet 1961;i:992-4.

Fears R, Hearn J, Standring R, Anderson JL, Marder VJ. Lack of influence of pretreatment antistreptokinaseantibody on efficacy in a multicenter patency comparison of intravenous streptokinase and anistreplase in acute

myocardial infarction. Am Heart J 1992;124:305-14.

Fears R, Ferres H, Glasgow E, et al. Monitoring of streptokinase resistance titre in acute myocardial infarctionpatients up to 30 months after giving streptokinase or anistreplase and related studies to measure specificantistreptokinase IgG. Br Heart J 1992;68:167-70.

Fitzgerald DJ, Catella F, Roy L, FitzGerald GA. Marked platelet activation in vivo after intravenousstreptokinase in patients with acute myocardial infarction. Circulation 1988;77:142-50.

Fitzgerald DJ, Fitzgerald GA. Role of thrombin and thromboxane A2 in reocclusion following coronarythrombolysis with tissue-type plasminogen activator. Proc Natl Acad Sci USA 1989;86:7585-9.

Fletcher AP, Alkjaersig N, Smyrniotis FE, Sherry S. Treatment of patients suffering from early, myocardialinfarction with massive and prolonged streptokinase therapy. Trans Assoc Am Physicians 1958;71:287-96.

Fletcher AP, Alkjaersig N, Sherry S. The maintenance of a sustained thrombolytic state in man. I. Induction a n deffects. J Clin Invest 1959;38:1096-1110.

Fletcher AP, Sherry S, Alkjaersig N. The maintenance of a sustained thrombolytic state in man. II. Clinicalobservations on patients with myocardial infarction and other thrombo-embolic disorders. J Clin Invest1959;38:1111-9.

Fox KAA . Thrombolysis and the general practitioner. Practicable only under certain circumstances. Br Med J1990;300:867-8.

Fuster V, Badimon L, Cohen M, Ambrose JA, Badimon JJ, Chesebro J. Insights into the pathogenesis of acuteischemic syndromes. Circulation 1988;77:1213-20.

39

References

Ganz W, Shah PK. Temporal distribution of treatment to reperfusion times in patients with acute myocardialinfarction: The effect of tissue plasminogen activator vs. streptokinase. J Am Coll Cardiol 1992;19:361A.

Gash A, Spann JF, Sherry S, et al. Factors influencing reocclusion after coronary thrombolysis for acutemyocardial infarction. Am J Cardiol 1986;57:175-7.

Gallino A , Haeberli A, Hess T, Mombelli G. Fibrin formation and platelet aggregation in patients with acutemyocardial infarction: effects of intravenous and subcutaneous low-dose heparin. Am Heart J 1986;12:285-90.

Gemmill JD, Lifson WK, Rae AP, Hillis WS, Dunn FG. Assessment by general practitioners of suitability ofthrombolysis in patients with suspected acute myocardial infarction. Br Heart J 1993;70:503-6.

Gil V , Antunes A, Ventosa A, Morais J, Skene A, Seabra-Gomes R, on behalf of the Portuguese CooperativeGroup on Ventricular Function of the LATE Study. Late thrombolysis with alteplase improves left ventricularejection fraction at 1 month after myocardial infarction - a double blind, placebo controlled study. J Am CollCardiol 1993;21:300A.

Gersh BJ, Anderson JL. Thrombolysis and myocardial salvage. Results of clinical trials and the animalparadigm, paradoxic or predictable? Circulation 1993;88:296-306.

Gold HK , Coller BS, Yasuda T, et al. Rapid and sustained coronary artery recanalization with combined bolusinjection of recombinant tissue-type plasminogen activator and monoclonal antiplatelet GPIIb/IIIa antibody in a

canine preparation. Circulation 1988;77:670-7.

Gold HK , Gimple L, Yasuda T, et al. Phase I human trial of the potent antiplatelet agent 7E3-F(ab’)2, amonoclonal antibody to the GPIIb/IIIa receptor. Circulation 1989;80 (Suppl II):II-267.

Gold HK , Torres FW, Garabedian HD, et al. Evidence for a rebound coagulation phenomenon after cessation ofa 4-hour infusion of a specific thrombin inhibitor in patients with unstable angina pectoris. J Am Coll Cardiol1993;21:1039-47.

Goldberg RJ, Gurwitz J, Yarzebski J, et al. Patient delay and receipt of thrombolytic therapy among patientswith acute myocardial infarction from a community-wide perspective. Am J Cardiol 1992;70:421-5.

Goldhaber SZ. Conjunctive heparin therapy. Circulation 1992;86:1639-41.

GREAT Group . Feasibility, safety, and efficacy of domiciliary thrombolysis by general practitioners: Grampianregion early anistreplase trial. Br Med J 1992;305:548-53.

Granger CB, Califf RM, Topol EJ. Thrombolytic therapy for acute myocardial infarction. A review. Drugs1992;44:293-325.

Gray D, Keating NA, Murdock J, Skene AM, Hampton JR. Impact of hospital thrombolysis policy on out-of-hospital response to suspected myocardial infarction. Lancet 1993;341:654-7.

Grill HP , Spero JE, Granato JE. Comparison of activated partial thromboplastin time to activated clotting timefor adequacy of heparin anticoagulation just before percutaneous transluminal coronary angioplasty. Am JCardiol 1993;71:1219-20.

Grim P , Feldman T, Martin M, Donovan R, Nevins V, Childers RW. Cellular telephone transmission of 12- leadelectrocardiograms from ambulance to hospital. Am J Cardiol 1987;60:715-20.

Grimaudo V , Hauert J, Bachmann F, Kruithof EKO. Diurnal variation of the fibrinolytic system. ThrombHaemostas 1988;59:495-9.

40

References

Grines CL, Nissen SE, Booth DC, et al. and the Kentucky Acute Myocardial Infarction Trial (KAMIT) group. Aprospective, randomized trial comparing combination half-dose tissue-type plasminogen activator and

streptokinase with full-dose tissue-type plasminogen activator. Circulation 1991;84:540-9.

Gruppo Italiano per lo Studio Della Streptochinasi Nell’Infarto Miocardico . GISSI-1: Effectiveness ofintravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986;i:397-401.

Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico . GISSI-2: A factorial randomizedtrial of alteplase versus streptokinase and heparin versus no heparin among 12.490 patients with acutemyocardial infarction. Lancet 1990;336:65-71.

Gulba DC, Barthels M, Westerhoff-Bleck M, et al. Increased thrombin levels during thrombolytic therapy inacute myocardial infarction. Relevance for the success of therapy. Circulation 1991;83:937-44.

Gurewich V, Pannell R, Louie S, Kelley P, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by azymogenprecursor form of urokinase (pro-urokinase), a study in vitro and in two animal species. J Clin Invest1984;73:1731-9.

The GUSTO Angiographic Investigators. The effects of tissue plasminogen activator, streptokinase, or both oncoronary-artery patency, ventricular function, and survival after acute myocardial infarction. N Engl J Med1993;329:1615-22.

The GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acutemyocardial infarction. N Engl J Med 1993;329:673-82.

Hackett D, Davies G, Chierchia S, Maseri A. Intermittent coronary occlusion in acute myocardial infarction. NEngl J Med 1987;317:1055-9.

Hamsten A, Wiman B, de Faire U, et al. Increased plasma levels of a rapid inhibitor of tissue plasminogenactivator in young survivors of myocardial infarction. N Engl J Med 1985;313:1557-63.

Hamsten A, de Faire U, Walldius G, et al. Plasminogen activator inhibitor in plasma: risk factor for recurrentmyocardial infarction. Lancet 1987;ii:3-9.

Harker LA , Mann KG. Thrombosis and fibrinolysis. In: Fuster V, Verstraete M, ed. Thrombosis incardiovascular disorders. Philadelphia: Saunders, 1992:1-16.

Harpel PC, Gordon BR, Parker TS. Plasmin catalyzes binding of lipoprotein(a) to immobilized fibrinogen andfibrin. Proc Natl Acad Sci USA 1989;86;3847-51.

Harrison DG , Ferguson DW, Collins SM, et al. Rethrombosis after reperfusion with streptokinase: importance o fgeometry of residual lesions. Circulation 1984;69:991-9.

Haverkate F, Timan G, Nieuwenhuizen W. Anticlotting properties of fragments from human fibrinogen andfibrin. Eur J Clin Invest 1979;9:253-5.

Hearn JA, Donohue BC, Ba’albaki H, et al. Usefulness of serum lipoprotein(a) as a predictor of restenosis afterpercutaneous transluminal coronary angioplasty. Am J Cardiol 1992;69:736-9.

Herrick JB . Clinical features of sudden obstruction of the coronary arteries. JAMA 1912;59:2015-20.

Herlitz J , Hartford M, Blohm M, et al. Effect of a media campaign on delay times and ambulance use insuspected acute myocardial infarction. Am J Cardiol 1989;64:90-3.

41

References

Hirsh J . Heparin. N Engl J Med 1991;324:1565-74.

Ho MT , Eisenberg MS, Litwin PE, et al. Reasons chest pain patients delay or do not call 911. Circulation1988;78:II-187.

Hoefler G, Harnoncourt F, Paschke E, et al. Lipoprotein Lp(a). A risk factor for myocardial infarction.Arteriosclerosis 1988;8:398-401.

Hoet B, Close P, Vermylen J, Verstraete M. Hirudo medicinalis and hirudin. In: Poller L, ed. Recent advances i nblood coagulation. Edinburgh: Churchill Livingstone, 1991:223-44.

Hoff HF , Beck GJ, Skibinski CI, et al. Serum Lp(a) level as a predictor of vein graft stenosis after coronaryartery bypass surgery in patients. Circulation 1988;77:1238-44.

Hoffmann JJML , Bonnier JJRM, Melman PG, Bartholomeus I. Blood viscosity during thrombolytic therapy withanistreplase in acute myocardial infarction. Am J Cardiol 1993;71:14-8.

Hogg PJ, Jackson CM. Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III:Implications for heparin efficacy. Proc Natl Acad Sci USA 1989;86:3619-23.

Hsia J, Kleiman N, Aguirre F, Chaitman BR, Roberts R, Ross AM, for the HART Investigators. Heparin-induced prolongation of partial thromboplastin time after thrombolysis: relation to coronary artery patency. JAm Coll Cardiol 1992;20:31-5.

Hsia J, Hamilton WP, Kleiman N, Roberts R, Chaitman BR, Ross AM, for the Heparin-Aspirin Reperfusion Trial(HART) Investigators. A comparison between heparin and low- dose aspirin as adjunctive therapy with

tissue plasminogen activator for acute myocardial infarction. N Engl J Med 1990;323:1433-7.

ISAM Study Group . A Prospective trial of intravenous streptokinase in acute myocardial infarction (ISAM).Mortality, morbidity, and infarct size at 21 days. N Engl J Med 1986;314:1465-71.

International Study Group . In-hospital mortality and clinical course of 20.891 patients with suspected acutemyocardial infarction randomized between alteplase and streptokinase with or without heparin. Lancet1990;336:71-5.

Jaffe AS, Serota H, Grace A, Sobel BE. Diagnostic changes in plasma creatine kinase isoforms early after theonset of acute myocardial infarction. Circulation 1986;74:105-9.

Jalihal S, Morris GK. Antistreptokinase titers after intravenous streptokinase. Lancet 1990;i:184-5.

James DCO. Anti-streptokinase levels in various hospital patient groups. Postgrad Med J 1973;49(suppl):26-9.

Jang I-K , Gold HK, Ziskind AA, et al. Differential sensitivity of erythrocyte-rich and platelet-rich arterialthrombi to lysis with recombinant tissue-type plasminogen activator. A possible explanation for resistance tocoronary thrombolysis. Circulation 1989;79:920-8.

Jang I-K , Gold HK, Ziskind AA, Leinbach RC, Fallon JT, Collen D. Prevention of platelet-rich arterialthrombosis by selective thrombin inhibition. Circulation 1990;81:219-24.

Johnson AJ, Fletcher AP, McCarty WR, Tillett WS. The intravascular use of streptokinase. Ann N Y Acad Sci1957;68:201-6.

Kaplan MH . Nature and role of lytic factor in hemolytic streptococcal fibrinolysis. Proc Soc Exp Biol Med1944;57:40-3.

42

References

Karagounis L, Ipsen SK, Jessop MR, et al. Impact of field-transmitted electrocardiography on time to in-hospital thrombolytic therapy in acute myocardial infarction. Am J Cardiol 1990;66:786-91.

Karagounis L, Sorensen SG, Menlove RL, Moreno F, Anderson JL, for the TEAM-2 Investigators. Doesthrombolysis in myocardial infarction (TIMI) perfusion grade 2 represent a mostly patent artery or a mostlyoccluded artery. Enzymatic and electrocardiographic evidence from the TEAM-2 study. J Am Coll Cardiol1992;19:1-10.

Katus HA , Remppis A, Looser S, Hallermeier K, Scheffold T, Kübler W. Enzyme linked immuno assay ofcardiac troponin T for the detection of acute myocardial infarction in patients. J Mol Cell Cardiol1989;21:1349-53.

Katus HA , Remppis A, Neumann FJ, et al. Diagnostic efficacy of troponin T measurements in acute myocardialinfarction. Circulation 1991;83:902-12.

Kelly SF, Flood R, Atkinson HP, et al. Racial differences in delay time during the prehospital phase of acutemyocardial infarction. Circulation 1991;84:II-334.

Kenyon LW , Ketterer MW, Gheorghiade M, Goldstein S. Psychological factors related to prehospital delayduring acute myocardial infarction. Circulation 1991;84:1969-76.

Kerins DM , Roy L, FitzGerald GA, Fitzgerald DJ. Platelet and vascular function during coronary thrombolysiswith tissue-type plasminogen activator. Circulation 1989;80:1718-25.

Kim CB , Braunwald E. Potential benefits of late reperfusion of infarcted myocardium. The open arteryhypothesis. Circulation 1993;88:2426-36.

Kleiman NS, Ohman EM, Califf RM, et al. Profound inhibition of platelet aggregation with monoclonalantibody 7E3 Fab after thrombolytic therapy. Results of the thrombolysis and angioplasty in myocardialinfarction (TAMI) 8 pilot study. J Am Coll Cardiol 1993;22:381-9.

Koren G, Weiss AT, Ben-David AT, Hasin Y, Luria AH, Gotsman MS. Prevention of myocardial damage inacute myocardial ischemia by earlier treatment with intravenous streptokinase. N Engl J Med 1985;313:1384-9.

Kroon C , ten Hove WR, de Boer A, et al. Highly variable anticoagulant response after subcutaneousadministration of high-dose (12.500 IU) heparin in patients with myocardial infarction and healthy volunteers.Circulation 1992;86:1370-5.

Krucoff MW , Wagner NB, Pope JE, et al. The portable programmable microprocessor-driven real-time 12-leadelectrocardiographic monitor: a preliminary report of a new device for the noninvasive detection of successfulreperfusion or silent coronary reocclusion. Am J Cardiol 1990;65:143-8.

Krucoff MW , Croll MA, Pope JE, et al. Continuous 12-lead ST-segment recovery analysis in the TAMI 7study. Performance of a noninvasive method for real-time detection of failed myocardial reperfusion.Circulation 1993;88:437-46.

Kruithof EKO , Tran-Thang C, Bachman F. Studies on the release of plasminogen activator inhibitor fromhuman platelets. Thromb Haemost 1986;55:201-5.

Kruithof EKO , Ransijn A, Bachmann F. Inhibition of tissue plasminogen activator by human plasma. In:Progress in Fibrinolysis, vol 6. Davidson JF, Bachmann F, Bouvier CA, Kruithof EKO (eds). Edingburgh:Churchill-Livingstone, 365-9, 1983.

Kruithof EKO , Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen

43

References

activators in human plasma. Blood 1984;64:907-13.

Kudenchuk PJ, Ho MT, Weaver WD, et al. for the MITI Project Investigators. Accuracy of computer-interpreted electrocardiography in selecting patients for thrombolytic therapy. J Am Coll Cardiol1991;17:1486-91.

Kwon K-i , Freedman B, Wilcox I, et al. The unstable ST segment early after thrombolysis for acute infarctionand its usefulness as a marker of recurrent coronary occlusion. Am J Cardiol 1991;67:109-15.

Lack CH . Staphylokinase, an activator of plasma protease. Nature 1948;161:559-60.

Latallo ZS, Budzynski AZ, Lipinski B, Kowalski E. Inhibition of thrombin and fibrin polymerization, twoactivities derived from plasmin-digested fibrinogen. Nature 1964;203:1184-5.

LATE Study Group . Late assessment of thrombolytic efficacy (LATE) study with alteplase 6-24 hours afteronset of acute myocardial infarction. Lancet 1993;342:759-66.

Lee HS, Cross S, Davidson R, Reid T, Jennings K. Raised levels of antistreptokinase antibody andneutralization titers from 4 days to 54 months after administration of streptokinase or anistreplase. Eur Heart J1993;14:84-9.

Leonardi MS, Gazzarra D, Fava C, Focà A, Mastroeni P. Enzyme-linked immunosorbent assay (ELISA) forstreptokinase antibodies. Diagn Immunol 1983;1:64-7.

Lew AS, Laramee P, Cercek B, Rodriguez L, Shah PK, Ganz W. The effects of the rate of intravenous infusionof streptokinase and the duration of symptoms on the time interval to reperfusion in patients with acutemyocardial infarction. Circulation 1985;72:1053-8 (a).

Lew AS, Laramee P, Ganz W. Reply to Sigwart, et al. J Am Coll Cardiol 1985;5:1500 (b).

Lincoff AM , Topol EJ. Illusion of reperfusion. Does anyone achieve optimal reperfusion during acutemyocardial infarction? Circulation 1993;88:1361-74.

Liu CY , Nossel HL, Kaplan KL. The binding of thrombin by fibrin. J Biol Chem 1979;254:10421-5.

Loscalzo J. Lipoprotein(a). A unique risk factor for atherothrombotic disease. Arteriosclerosis 1990;10:672-9.

Loskutoff DJ . Type 1 plasminogen activator inhibitor and its potential influence on thrombolytic therapy. SeminThromb Hemost 1988;14:100-9.

Loskutoff DJ , Sawdey M, Mimuro J. Type 1 plasminogen activator inhibitor. Prog Hemost Thromb 1989;9:87-115.

Lynch M , Littler WA, Pentecost BL, Stockley RA. Immunoglobin response to intravenous streptokinase in acutemyocardial infarction. Br Heart J 1991;66:139-142.

MacCallum AG , Stafford PJ, Jones C, Vincent R, Perez-Avila C, Chamberlain DA. Reduction in hospital time tothrombolytic therapy by audit of policy guidelines. Eur Heart J 1990;11 (Suppl F):48-52.

MacMahon S, Collins R, Knight C, Yusuf S, Peto R. Reduction in major morbidity and mortality by heparin inacute myocardial infarction. Circulation 1988;78:II-98.

Mair J , Smidt J, Artner-Dworzak E, Lechleitner P, Dienstl F, Puschendorf B. Rapid diagnosis of myocardialinfarction by immunoturbidimetric myoglobin measurement. Lancet 1991;337:1343-4.

44

References

Mair J , Artner-Dworzak E, Lechleitner P, et al. Early diagnosis of acute myocardial infarction by a newlydeveloped rapid immunoturbidimetric assay for myoglobin. Br Heart J 1992;68:462-8.

Marder VJ , Kinsella PA, Brown J. Fibrinogen concentration and coronary artery reperfusion after intravenousanisoylated plasminogen streptokinase activator complex or intracoronary streptokinase therapy. Drugs1987;33 (Suppl. 3):237-41.

Marder VJJ , Shulman NR. High molecular weight derivates of human fibrinogen produced by plasmin, II.Mechanisms of their anticoagulant activity. J Biol Chem 1969;244:2120-4.

Massel D, Turpie AGG, Gill JB, Cairns A, Russett J. Development of neutralizing antibodies after 1.5 millionunits of streptokinase in the treatment of acute myocardial infarction. Circulation 1989;80:II-350.

Mathewson ZM, McCloskey BG, Evans AE, Russell CJ, Wilson C. Mobile coronary care and communitymortality from myocardial infarction, Lancet 1985;i:441-4.

MBewu AD, Durrington PN. Lipoprotein(a): structure, properties and possible involvement in thrombogenesisand atherogenesis. Atherosclerosis 1990;85:1-14.

McCabe M, Weston C. Ambulance response times. Br Med J 1991;302:181.

McGrath KG , Zeffren B, Alexander J, Kaplan K, Patterson R. Allergic reactions to streptokinase consistent w i thantigen-antibody complex-mediated damage. J Allergy Clin Immunol 1985;76:453-7.

McLean JW, Tomilnson JE, Kuang W-J, et al. cDNA sequence of human apolipoprotein(a) is homologous toplasminogen. Nature 1987;330:132-7.

Melandri G , Branzi A, Traini AM, Semprini F, Cervi V, Magnani B. On the value of the activated clotting t i m efor monitoring heparin therapy in acute coronary syndromes. Am J Cardiol 1993;71:469-71.

Meijer A , Verheugt FWA, Werter CJPJ, Lie KI, van der Pol JMJ, van Eenige MJ. Aspirin versus coumadin inthe prevention of reocclusion and recurrent ischemia after successful thrombolysis: A prospective placebo-controlled angiographic study. Results of the APRICOT Study. Circulation 1993;87:1524-30.

Meijer A . Clinical aspects of reocclusion after coronary thrombolysis, Thesis. Amsterdam: VUBoekhandel/Uitgeverij bv, 1993.

Merz B. Lp(a) joins other serum cholesterol lipoproteins as risk determinant. JAMA 1989;261:2013-4.

Mirshahi M , Soria J, Soria C, et al. Evaluation of the inhibition by heparin and hirudin of coagulation activationduring rt-PA induced thrombolysis. Blood 1989;74:1025-30.

Mombelli G , Imhof V, Haeberli A, Straub PW. Effect of heparin on plasma fibrinopeptide A in patients withacute myocardial infarction. Circulation 1984;69:684-89.

Moran DM , Standring R, Lavender EA, Harris GS. Assessment of anti-streptokinase antibody levels in humansera using a microradioimmunoassay procedure. Thromb Haemost 1984;52:281-7.

Moriarty AJ , Hughes R, Nelson SD, Belnave K. Streptokinase and reduced plasma viscosity: a second benefit.Eur J Haematol 1988;41:25-36

Moses HW, Engelking N, Taylor GJ, et al. Effect of a two-year public education campaign on reducingresponse time of patients with symptoms of acute myocardial infarction. Am J Cardiol 1991;68:249-51 (a).

45

References

Moses HW, Bartolozzi JJ, Koester DL, et al. Reducing delay in the emergency room in administration ofthrombolytic therapy for myocardial infarction associated with ST-elevation. Am J Cardiol 1991;68:251-3 (b).

Mosesson MW. The roles of fibrinogen and fibrin in hemostasis and thrombosis. Seminars in Hematology1992;29:177-88.

Mulcahy D, Purcell H, Fox K. Should we get up in the morning? Observations on circadian variations incardiac events. Br Heart J 1991;65:299-301.

Muller JE , Stone PH, Turi ZG, et al. and the MILIS Study Group. Circadian variation in the frequency of onsetof myocardial infarction. N Engl J Med 1985;313:1315-22.

Neuhaus K-L, Feuerer W, Jeep-Tebbe S, Niederer W, Vogt A, Tebbe U. Improved thrombolysis with amodified dose regimen of recombinant tissue-type plasminogen activator. J Am Coll Cardiol 1989;14:1566-9.

Neuhaus K-L, Von Essen R, Tebbe U, et al. Improved thrombolysis in acute myocardial infarction with front-loaded administration of alteplase: Results of the rt-PA-APSAC patency study (TAPS). J Am Coll Cardiol1992;19:885-91.

Neuhaus K-L, Von Essen R, Tebbe U, et. al. Hirudin and thrombolysis with front-loaded alteplase in acutemyocardial infarction; results of a pilot study. J Am Coll Cardiol 1993;21:418A (abstr.).

Nicoloso G, Hauert J, Kruithof EKO, Van Melle G, Bachmann F. Fibrinolysis in normal subjects: Comparisonbetween plasminogen activator inhibitor and other components of the fibrinolytic system. Thromb Haemost1988;59:299-303.

Norris RM , Barnaby PF, Brandt PWT, et al. Prognosis after recovery from first acute myocardial infarction:determinants of reinfarction and sudden death. Am J Cardiol 1984;53:408-13.

Obraztsov VP, Stazhesko ND. Simptomatiologii II. Diagnostike Tromboza Venechinikh Arteril Cerdtsa (on thesymptomatology and diagnosis of coronary thrombosis). In: Vorobeva VA, Konchalovski MO, eds. Trudipervogo sesda rossushkihkh terapevtov (works of the first congress of Russian Therapists). ComradeshipTypography of AE Mamontov 1910:26-43.

Ogilby JD, Kopelman HA, Klein LW, Agarwal JB. Adequate heparinization during PTCA: assessment usingactivated clotting times. Cathet Cardiovasc Diag 1989;18:206-9.

Ohman EM, Califf RM, Topol EJ, et al. for the TAMI Study Group. Consequences of reocclusion aftersuccessful reperfusion therapy in acute myocardial infarction. Circulation 1990;82:781-91.

Owen J, Friedman KD, Grossman BA, Wilkins C, Berke AD, Powers ER. Thrombolytic therapy with tissueplasminogen activator or streptokinase induces transient thrombin activity. Blood 1988;77:616-20.

Pannell R, Gurewich V. Pro-urokinase: A study of its stability in plasma and of a mechanism for its selectivefibrinolytic effect. Blood 1986;67:1215-23.

Pantridge JF, Geddes JS. A mobile intensive-care unit in the management of myocardial infarction. Lancet1967;ii:271-3.

Patel S, Jalihal S, Dutka DP, Morris GK. Streptokinase neutralisation titers up to 866 days after intravenousstreptokinase for acute myocardial infarction. Br Heart J 1993;70:119-21.

Pell ACH, Miller HC, Robertson CE, Fox KAA. Effect of "fast track" admission for acute myocardialinfarction on delay to thrombolysis. Br Med J 1992;304:83-7.

46

References

Pennica D, Holmes WE, Kohr WJ, et al. Cloning and expression of human tissue-type plasminogen activatorcDNA in E. Coli. Nature 1983;301:214-21.

Petch MC. Coronary thrombolytic treatment at home. An aspirin and quick admission to a coronary care unit isprobably a better bet. Br Med J 1991;302:1287-8.

PRIMI Trial Study Group . Randomized double-blind trial of recombinant pro-urokinase against streptokinase i nacute myocardial infarction. Lancet 1989;i:863-8.

Prins MH , Hirsh J. Heparin as an adjunctive treatment after thrombolytic therapy for acute myocardialinfarction. Am J Cardiol 1991:67:3A-11A.

Rapold HJ, Kuemmerli H, Weiss M, Baur H, Haeberli A. Monitoring of fibrin generation during thrombolytictherapy of acute myocardial infarction with recombinant tissue-type plasminogen activator. Circulation1989;79:980-9.

Rapold HJ. Promotion of thrombin activity by thrombolytic therapy without simultaneous anticoagulation.Lancet 1990;335:481-2.

Rapold HJ, de Bono D, Arnold AER, et al. for the European Cooperative Study Group. Plasma fibrinopeptide Alevels in patients with acute myocardial infarction treated with alteplase. Correlation with concomitant

heparin, coronary artery patency, and recurrent ischaemia. Circulation 1992;85:928-34.

Rawles JM, Haites NE. Patient and general practitioner delay in acute myocardial infarction. Br Med J1988;296:882-4.

Rawles J on behalf of the GREAT Group. Halving of mortality at 1 year by domiciliary thrombolysis in theGrampian region early anistreplase trial (GREAT). J Am Coll Cardiol 1994;23:1-5.

Rees A. Lipoprotein(a): a possible link between lipoprotein metabolism and thrombosis. Br Heart J 1991;65:2-3.

Relik-van Wely L , Visser RF, van der Pol JMJ, et al. Angiographically assessed coronary arterial patency andreocclusion in patients with acute myocardial infarction treated with anistreplase: Results of the anistreplasereocclusion multicenter study (ARMS). Am J Cardiol 1991;68:296-300.

Rentrop P, Blanke H, Karsch KR, Kaiser H, Köstering H, Leitz K. Selective intracoronary thrombolysis inacute myocardial infarction and unstable angina pectoris. Circulation 1981;63:307-17.

Reimer KA , Jennings RB. The "wavefront phenomenon" of myocardial ischemic cell death. II. Transmuralprogression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral flow.Lab Invest 1979;40:633-44.

Richardson PD, Davies MJ, Born GVR. Influence of plaque configuration and stress distribution on fissuring ofcoronary atherosclerotic plaques. Lancet 1989;ii:941-4.

Richardson SG, Allen DC, Morton P, Murtagh JG, Scott ME, O’Keeffe DB. Pathological changes afterintravenous streptokinase treatment in eight patients with acute myocardial infarction. Br Heart J1989;61:390-5.

Ridker PM , Hebert PR, Fuster V, Hennekens CH. Are both aspirin and heparin justified as adjuncts tothrombolytic therapy for myocardial infarction. Lancet 1993;341:1574-7.

Roberts WC, Buja LM. The frequency and significance of coronary arterial thrombi and observations in fatalacute myocardial infarction: a study of 107 necroscopy patients. Am J Med 1972;52:425-43.

47

References

Rothbard RL , Fitzpatrick PG, Francis CW, Caton DM, Hood WB, Marder VJ. Relationship of the lytic state tosuccessful reperfusion with standard- and low-dose intracoronary streptokinase. Circulation 1985;71:562-70.

Roux S, Christeller S, Lüdin E. Effects of aspirin on coronary reocclusion and recurrent ischemia afterthrombolysis: a meta-analysis. J Am Coll Cardiol 1992;19:671-7.

Rouy D, Grailhe P, Nigon F, Chapman J, Anglés-Cano E. Lipoprotein(a) impairs generation of plasmin byfibrin-bound tissue-type plasminogen activator, in vitro studies in a plasma milieu. Arteriosclerosis andThrombosis 1991;11:629-38.

Rowley JM, Mounser P, Harrison EA, Skene AM, Hampton JR. Management of myocardial infarction:implications for current policy derived from the Nottingham Heart Attack Register. Br Heart J 1992;67:255-62.

Sakamoto T, Yasue H, Ogawa H, Misumi I, Masuda T. Association of patency of the infarct-related coronaryartery with plasma levels of plasminogen activator inhibitor activity in acute myocardial infarction. Am JCardiol 1992;70:271-6.

Sane D, Stump D, Topol E, et al. Correlation between baseline plasminogen activator inhibitor levels andclinical outcome during therapy with tissue-type plasminogen activator for acute myocardial infarction.Thromb Haemost 1991;65:275-9.

Schaeffer SM, Eisenberg MS, Ho MT, Weaver D, Larsen MP. The relationship of education to use of 911 forsymptoms of myocardial infarction. Circulation 1989;80:II-637.

Schröder R, Biamco G, v Leitner E-R, et al. Intravenous short-term infusion of streptokinase in acutemyocardial infarction. Circulation 1983;67:536-48.

Scott J. Lipoprotein(a), thrombotic and atherogenic. Br Med J 1990;303:663-4.

Second International Study of Infarct Survival Collaborative Group . Randomized trial of intravenousstreptokinase, oral aspirin, both, or neither among 17.187 cases of suspected acute myocardial infarction:ISIS-2. Lancet 1988;ii:349-60.

Seed M, Hoppichler F, Reaveley D, et al. Relation of serum lipoprotein(a) concentration and apolipoprotein(a)phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med1990;322:1494-9.

Shah PK. The role of thrombolytic therapy in patients with acute myocardial infarction presenting later than sixhours after the onset of symptoms. Am J Cardiol 1991;68:72C-77C.

Sharkey SW, Brunette DD, Ruiz E, et al. An analysis of time delays preceding thrombolysis for acutemyocardial infarction. JAMA 1989;262:3171-4.

Sherry S. The origin of thrombolytic therapy. J Am Coll Cardiol 1989;14:1085-92.

Sherry S. Fibrinolysis, thrombosis and hemostasis. Pennsylvania: Lea & Febiger, 1992 (a).

Sherry S, Marder VJ. Thrombolytic therapy: reocclusion rates with adjunctive aspirin and its relation to heparintherapy. J Am Coll Cardiol 1992;19:671-7 (b).

Sigwart U, Grbic M, Bachmann F. Measurement of antistreptokinase antibodies. J Am Coll Cardiol1985;5:1500.

Silver MD , Baroldi G, Mariani F. The relationship between acute occlusive coronary thrombi and myocardial

48

References

infarction studied in 100 consecutive patients. Circulation 1980;61:219-27.

Simoons ML, Betriu A, Col J, et al. for the European Cooperative Study Group for recombinant tissue-typeplasminogen activator (rt-PA). Thrombolysis with tissue plasminogen activator in acute myocardial infarction:no additional benefit from immediate percutaneous coronary angioplasty. Lancet 1988;i:197-203.

Six AJ, Brommer EJP, Müller EJ, Kerkhoff HF. Activation of the fibrinolytic system during intracoronarystreptokinase administration. J Am Coll Cardiol 1987;9:189-96.

Smith RAG, Dupe RJ, English PD, Green J. Fibrinolysis with acyl-enzymes: a new approach to thrombolytictherapy. Nature 1981;290:505-8.

Sobel BE, Braunwald E. The management of acute myocardial infarction. In: Braunwald E, ed. Heart disease.Philadelphia: Saunders, 1984:1301-33.

Sobel GW, et al. Urokinase, an activator of profibrinolysin extracted from urine. Am J Physiol 1952;171:768- ..

Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987;69:381-7.

Stein B, Fuster V, Halperin JL, Chesebro JH. Antithrombotic therapy in cardiac disease. An emerging approachbased on pathogenesis and risk. Circulation 1989;80:1501-13.

Stone MJ, Willerson JT. Myoglobulinemia in myocardial infarction. Int J Cardiol 1983;4:49-52.

Taylor GJ , Humphries JO, Mellits ED, et al. Predictors of clinical course, coronary anatomy and leftventricular function after recovery from acute myocardial infarction. Circulation 1980;62:960-70.

Taylor GJ , Moses HW, Koester D, et al. A difference between front-loaded streptokinase and standard-doserecombinant tissue-type plasminogen activator in preserving left ventricular function after acute myocardialinfarction (the central Illinois thrombolytic therapy study). Am J Cardiol 1993;72:1010-4.

Tendera MP, Campbell WB, Tennant SN, Ray WA. Factors influencing probability of reperfusion withintracoronary ostial infusion of thrombolytic agent in patients with acute myocardial infarction. Circulation1985;71:124-8.

Third International Study of Infarct Survival Collaborative Group . ISIS-3: a randomized comparison ofstreptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin aloneamong 41.299 cases of suspected acute myocardial infarction. Lancet 1992;339:753-70.

Tillett WS , Garner RL. The fibrinolytic activity of hemolytic streptococci. J Exp Med 1933;58:485-502.

Tillett WS , Edwards LB, Garner RL. Fibrinolytic activity of hemolytic streptococci. The development ofresistance to fibrinolysis following acute hemolytic streptococcus infections. J Clin Invest 1934;13:47-78.

Tijssen JGP, Simoons ML, de Feijter PJ, Hugenholtz PG, Lubsen J. Unstable angina pectoris. Eur Heart J1987;8 (Suppl H):3-15.

TIMI Study Group . The thrombolysis in myocardial infarction (TIMI) trial: phase I findings. N Engl J Med1985;312:932-6.

TIMI Research Group . Immediate vs delayed catheterization and angioplasty following thrombolytic therapy foracute myocardial infarction, TIMI IIA results. JAMA 1988;260:2849-58.

Tofler GH , Brezinski D, Schager AI, et al. Concurrent morning increase in platelet aggregability and the risk of

49

References

myocardial infarction and sudden cardiac death. N Engl J Med 1987;316:1514-18.

Topol EJ, Califf RM, George BS, et al. and the Thrombolysis and Angioplasty in Myocardial Infarction StudyGroup. A randomized trial of immediate versus delayed elective angioplasty after intravenous tissueplasminogen activator in acute myocardial infarction. N Engl J Med 1987;317:581-8.

Vacek JL, Hibiya K, Rosamond TL, Kramer PH, Beauchamp GD. Validation of a bedside method of activatedpartial thromboplastin time measurement with clinical range guidelines. Am J Cardiol 1991;68:557-9.

Vaughan DE, Kirstenbaum JM, Loscalzo J. Streptokinase induced, antibody-mediated platelet aggregation: apotential cause of clot propagation in vivo. J Am Coll Cardiol 1988;11:1343-8.

Vaughan DE, Van Houtte E, Declerck PJ, Collen D. Streptokinase-induced platelet aggregation. Prevalence andmechanism. Circulation 1991;84:84-91.

Veen G, Meyer A, Verheugt FWA, et al. Culprit lesion morphology and stenosis severity in the prediction ofreocclusion after coronary thrombolysis: angiographic results of the APRICOT study. J Am Coll Cardiol1993;22:1755-62.

Verheugt FWA, ten Cate JW, Sturk A, et al. Tissue plasminogen activator activity and inhibition in acutemyocardial infarction and angiographically normal coronary arteries. Am J Cardiol 1987;59:1075-9.

Verheugt FWA, Funke Kupper AJ, Sterkman LGW, Meijer A, Peels CH, Roos JP. Emergency room infusions o fintravenous streptokinase in acute myocardial infarction: feasibility, safety and hemodynamic consequences. A mHeart J 1989;117:1018-21.

Verstraete M, Vermylen J, Amery A, Vermylen C. Thrombolytic therapy with streptokinase using a standarddosage scheme. Br Med J 1966;1:454-6.

Verstraete M. Intravenous administration of a thrombolytic treatment is the only realistic therapeutic approach inevolving myocardial infarction. Eur Heart J 1985;6:586-93.

Verstraete M. Novelties in antithrombotic and thrombolytic therapy. In: Fuster V, Verstraete M, ed.Thrombosis in cardiovascular disorders. Philadelphia: Saunders, 1992:529-43.

Wall TC , Califf RM, George BS, et al. for the TAMI-7 Study Group. Accelerated plasminogen activator doseregimens for coronary thrombolysis. J Am Coll Cardiol 1992;19:482-9.

Weaver WD, Litwin PE, Martin JS, et al. Effect of age on the use of thrombolytic therapy and mortality inacute myocardial infarction. J Am Coll Cardiol 1991;18:657-62.

Weaver DW, Cerqueira M, Hallstrom AP, et al. for the myocardial infarction triage and intervention projectgroup. Prehospital-initiated vs hospital-initiated thrombolytic therapy -The myocardial infarction triage andintervention (MITI) trial. JAMA 1993;270:1211-6 (a).

Weaver WD, Hartmann JK, Reddy PS, Anderson J, for the Pro-UK Study Group. Prourokinase achieves rapidand sustained patency for treatment of acute myocardial infarction. J Am Coll Cardiol 1993;21:397A (b).

Weitz JI , Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition byheparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J ClinInvest 1990;86:385-91.

White HD , Norris RM, Brown MA, et al. Effect of intravenous streptokinase on left ventricular function andearly survival after acute myocardial infarction. N Engl J Med 1987;317:850-5.

50

References

White HD , Rivers JT, Maslowski AH, et al. Effect of intravenous streptokinase as compared with that of tissueplasminogen activator on left ventricular function after first myocardial infarction. N Engl J Med1989;320:817-21.

White HD . GISSI-2 and the heparin controversy. Lancet 1990;336:297-8 (a).

White HD , Cross DB, Williams BF, Norris RM. Safety and efficacy of repeat thrombolytic treatment after acutemyocardial infarction. Br Heart J 1990;64:177-81 (b).

White HD . Thrombolytic treatment for recurrent myocardial infarction. Avoid repeating streptokinase oranistreplase. Br Med J 1991;302:429-30.

Wilcox RG, Lippe von der G, Olsson CG, Jensen G, Skene AM, Hampton JR. Trial of tissue plasminogenactivator for mortality reduction in acute myocardial infarction. Anglo-Scandinavian Study of EarlyThrombolysis (ASSET). Lancet 1988;ii:525-30.

Wilcox RG. Thrombolysis and the general practitioner. Useful after careful evaluation of patients. Br Med J1990;300:869-70.

Winters KJ , Santoro SA, Miletich JP, Eisenberg PR. Relative importance of thrombin compared to plasmin-mediated platelet activation in response to plasminogen activation with streptokinase. Circulation1991;84:1552-60.

Yusuf S, Wittes J, Friedman L. Overview of results of randomized clinical trials in heart disease. JAMA1988;260:2088-93.

Zenker G, Költringer P, Boné G, Niederkorn K, Pfeiffer K, Jürgens G. Lipoprotein Lp(a) as a strong indicatorfor cerebrovascular disease. Stroke 1986;17:942-5.

Zijlstra F , de Boer MJ, Hoorntje JCA, Reiffers S, Reiber JHC, Suryapranata H. A comparison of immediatecoronary angioplasty with intravenous streptokinase in acute myocardial infarction. N Engl J Med1993;328:680-4.

51

Abbreviations and acronyms

Abbreviations and acronyms

APSAC Anisoylated Plasminogen Streptokinase Activator Complex (anistreplase)[Eminase™]

aPTT activated partial thromboplastin timeaSKa anti-streptokinase antibodiesAST antistreptolysin titreCCU coronary care unitECG electrocardiogram/electrocardiographicECSG European Cooperative Study GroupELISA enzyme-linked immunosorbent assayEMIP European Myocardial Infarction ProjectFPA fibrinopeptide AGISSI Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto MiocardicoGREAT Grampian Region Early Anistreplase TrialGUSTO Global Utilization of Streptokinase and Tissue Plasminogen Activator for

Occluded Coronary ArteriesISIS International Study of Infarct Survivali.v. intravenous(ly)Lp(a) lipoprotein(a)LVEF left ventricular ejection fractionMI myocardial infarctionMITI Myocardial Infarction Triage and InterventionPAI plasminogen activator inhibitorPTCA percutaneous transluminal coronary angioplastyREPAIR REPerfusion in Acute Infarction RotterdamRIA radio-immuno assayrt-PA recombinant tissue-type plasminogen activator (alteplase) [Actilyse™]s.c. subcutaneous(ly)SK streptokinase [Kabikinase™/Streptase™]TIMI Thrombolysis In Myocardial InfarctionTAMI Thrombolysis and Angioplasty in Myocardial InfarctionTAT thrombin-antithrombin III complexU Unit(s)vs versus

53

appendix 1

Logistic Problems in Prehospital Thrombolysis

Johan Brügemann, Jan van der Meer, Pieter A. de Graeff, Bert H. Takens and Kong I. Lie

Department of Cardiology, Thoraxcenter, University Hospital Groningen, The Netherlands

Eur Heart J 1989:10;122 (abstract)Eur Heart J 1992:13;787-8.

Abstract

In this study we compared efficacy and safety of prehospital with in-hospital thrombolytictreatment with anistreplase in patients with acute myocardial infarction (AMI). Three-hundred and fifty patients with chest pain were screened for eligibility by the municipalambulance staff and/or the general practitioner. Patients were included in absence ofcontraindications and if the telephone-transmitted ECG showed AMI. In a 6 months 16patients (5%) were eligible, but only seven were randomized. Age over 70 years, durationof chest pain for longer than 4 h and logistic problems were the major factors responsiblefor the low inclusion rate. The mean time spent at home with and without the ECGprocedure amounted 38±14 and 14±8 minutes, respectively (p< 0.001). These resultsdemonstrate that in a medium sized town prehospital delivery of intravenous thrombolytictherapy by paramedics and/or the general practitioner is not feasible, leads to unnecessarytime delay and may therefore yield no clinical benefits.

Introduction

Thrombolytic therapy in patients with acute myocardial infarction (AMI) reducescardiovascular morbidity and mortality and efficacy is increased by early administration(1). Often time is lost before therapy is initiated (2), but if the selection of patients wasperformed by a physician in a mobile care unit, administration of prehospital thrombolytictherapy might shorten the ischemic period (3). On the basis of these data, we designed astudy to determine the efficacy and safety of therapy at home with anistreplase in patientswith AMI. Because the ambulance staff in The Netherlands does not usually include aphysician, we sought support of the general practitioner (GP) and the paramedicalambulance staff for selection purposes and initiation of thrombolytic treatment. In thispaper we describe our experience with prehospital thrombolysis.

Methods

The study was performed in Groningen, The Netherlands, a town of 200.000inhabitants, from July 1988 to January 1989. Prior to the study, the ambulance staff wasextensively educated and trained and the GPs were asked for their participation.

If there was suspicion of AMI on the basis of a telephone call by a patient to the GP,

55

appendix 1

an ambulance, equipped with an ECG device (Marquette Electronics) and the studymedication, was sent to the spot. Subsequently, the patient was subjected to a list of 20questions which was preferentially checked by the GP or otherwise by a member of theambulance staff. Inclusion criteria for the study were age less than 70 years, chest pain ofover 20 minutes duration, but less than 4 h, unresponsiveness to sublingual nitroglycerin,and absence of contraindications for thrombolytic therapy. If the patient was eligible, a 12-lead ECG was made and transmitted by telephone for review to the hospital. ECG ST-segment elevation of over 0.1 mV in more than one of the standard leads or over 0.2 mVin more than two of the precordial leads was required before permission was given toproceed. Treatment was started with 100 mg prednisolone and 100 mg lignocaine.Subsequently, either 30 U of anistreplase (Eminase, Trade Mark of the Beecham Groupplc) or placebo was given intravenously (i.v.) over 4-5 min in a double blind randomizedfashion. After admission to one of the three local participating hospitals, the second vial ofthe study medication, containing either placebo or anistreplase, was infused. Thus, eachrandomized patient received anistreplase. Infusion of heparin was started 4-6 h afteradmission and continued for 48 h when coronary angiography was performed. The studywas approved by the ethical committee, provided that no more than 20 min were lost dueto inclusion procedures.

Results for continuous variables are presented as mean ± standard deviation. Student’s 2sample t-test was used to assess differences between the pre- and in-hospital treatedpatients.

Results

During 6 month period 350 patients were screened. The mean ambulance travel time tothe patient’s house after a telephone call was 10±7 min.

In only 24 cases (7%) was an ECG transmitted and an AMI was diagnosed in 16 (5%)of these recordings. The most important reasons for ineligibility were age over 70 years(over a third of the patients), chest pain of more than 4 h and maximal delay at homeexceeded. Following diagnosis of AMI, nine of the 16 eligible patients were notrandomized because of: telephone communication problems (four times); inability toachieve i.v. access (three times); chaotic situation (once); in the last case the reason isunknown. Consequently, no more than seven patients (2%), with a mean age of 57±7years, who had complaints for 125±55 minutes, received the study medication. Patency ofthe affected vessel was found in six of these seven patients (86%).

For the 24 patients in whom an ECG was registered at home, the time from arrival ofthe ambulance to completion of the procedure, including review of the ECG, was 38±14minutes. For the other patients, who were not eligible for the study, the time spent athome was l4±8 minutes, which was significantly shorter (p< 0.001). Transportation of apatient to the emergency room lasted 9±5 minutes, irrespective of inclusion.

Three patients experienced ventricular fibrillation prior to infusion of the studymedication. Minor bleeding was seen in two and hypotension in one patient. One patientshowed a mild allergic reaction. None of the patients died.

Discussion

56

appendix 1

In the prehospital studies published so far, a considerable reduction of the time prior tothrombolytic treatment of patients with AMI has been reported: 40-45 minutes (4-7), 60minutes (8,9) and 73 minutes (10). Results concerning clinical benefits of this earlyintervention are ambiguous. In an earlier study, a higher left ventricular ejection fractionwas shown in those patients who were treated less than 1.5 h after the onset of pain (3).However, this result was not confirmed in larger, more recent studies (4,7,8). Only onestudy showed a decrease of mortality in the prehospital treated patients (5). Nearly allprehospital thrombolysis protocols prescribed an ECG which was assessed on the spot bya physician (3,5,7,8), or, as in the REPAIR study, by a computer (6).

In our study, thrombolytic therapy at home was administered by the GP and paramedicsfollowing remote ECG assessment. In the field only 16 out of 350 patients (5%) had AMIand seven patients (2%) were treated. Because of these small numbers, the efficacy ofprehospital versus in-hospital treatment cannot be compared. The low percentage ofpatients eligible for prehospital thrombolysis is in accordance with two similarly designedstudies in which 107 out of 2472 patients (4%) (10) and 3 out of 85 patients (3.5%) (11)were eligible.

Due to the ECG procedure, eligible patients spent on average 24 minutes more at home.This time was well in excess of the mean transportation time of 9 minutes to the hospital.

Our study demonstrates that prehospital delivery of i.v. thrombolytic therapy byparamedics and/or GP is not feasible, leads to unnecessary time delay and may thereforerender no clinical advantages. This may, in part, be due to the size of our town. Incontrast to a metropolis, we do not experience regular traffic jams and the travel time to ahospital is short. Moreover, due to the low number of eligible patients, each paramedicdealt with too few patients with AMI to maintain competence of skills, a problem that wasalso noted by others (11). In our view, the conditions in Groningen are representative formany European medium-sized towns. In these circumstances, patients with chest pain mustbe delivered to hospital immediately, which is also the current opinion of the British HeartFoundation Working Group (12).

References

1. The GISSI Study Group. Effectiveness of intravenous thrombolytic treatment inacute myocardial infarction. Lancet 1986;i:397-402.

2. Sharkey SW, Brunette DD, Ruiz E, et al. An analysis of time delays precedingthrombolysis for acute myocardial infarction. JAMA 1989;262:3171-4.

3. Koren G, Weiss AT, Ben-David AT, Hasin Y, Luria AH, Gotsman MS. Preventionof myocardial damage in acute myocardial ischemia by earlier treatment withintravenous streptokinase. N Engl J Med 1985;313:1384-89.

4. The Thrombolysis Early in Acute Heart Attack Trial Study Group. Very earlythrombolytic therapy in suspected acute myocardial infarction. Am J Cardiol1990;65:401-7.

57

appendix 1

5. Barbash GI, Roth A, Hod H et al. Improved survival but not left ventricular functionwith early and prehospital treatment with tissue plasminogen activator in acutemyocardial infarction. Am J Cardiol 1990;66:261-6.

6. Bouten MJM, Simoons ML, Pool J, Hartman JAM, van Zijl AJM, REPAIR studyGroup. Prehospital thrombolysis by ambulance paramedics. Eur Heart J 1989;10:123(Abstr).

7. Schofer J, Büttner J, Geng G et al. Prehospital thrombolysis in acute myocardialinfarction. Am J Cardiol 1990;66:1429-33.

8. Castaigne AD, Hervé C, Duval-Moulin AM et al. Prehospital use of APSAC: resultsof a placebo-controlled study. Am J Cardiol 1989;64:30A-33A.

9. European Myocardial Infarction Project (EMIP) Sub-committee. Potential timesaving with pre-hospital intervention in acute myocardial infarction. Eur Heart J1988;9:118-24.

10. Weaver WD, Eisenberg MS, Martin JS et al. Myocardial infarction triage andintervention project-phase 1: patient characteristics and feasibility of prehospitalinitiation of thrombolytic therapy. J Am Coll Cardiol 1990;15:925-31.

11. Gibler WB, Kereiakes DJ, Dean EN et al. Prehospital diagnosis and treatment ofacute myocardial infarction: A North-South perspective. Am Heart J 1991;1:1-11.

12. British Heart Foundation Working Group. Role of the general practitioner inmanaging patients with myocardial infarction: impact of thrombolytic treatment. BrMed J 1989;299:555-7.

58

appendix 2

A Systemic Non-lytic State and Local Thrombolytic Failure ofAnistreplase (Anisoylated Plasminogen Streptokinase ActivatorComplex, APSAC) in Acute Myocardial Infarction

Johan Brügemann, Jan van der Meer (*), Bert H. Takens, Hans L. Hillege and Kong I. Lie

Departments of Cardiology and Haematology (*), University of Groningen, TheNetherlands

J Am Coll Cardiol 1990;15:3A (abstract)Br Heart J 1990;64:355-8.

Abstract

The relation between coronary thrombolysis and coagulation variables after administrationof anistreplase (anisoylated plasminogen streptokinase activator complex, APSAC) wasstudied in patients with an acute myocardial infarction. Fifty-eight consecutive patientswith acute myocardial infarction were given 30 U of anistreplase intravenously within 4hours of the onset of symptoms. A fall in the plasma concentration fibrinogen level to <1.0 g/l, within 90 minutes after administration of anistreplase was considered to reflect asystemic lytic state. Coronary angiography was performed 48 hours after thrombolytictreatment. The overall patency rate was 74% (43/58). Patency rates were significantlydifferent in patients with a systemic lytic [83% (43/52)] and a systemic non-lytic state [0%(0/6)]. The absence of a systemic lytic state after anistreplase administration seemed to behighly predictive of the failure of coronary thrombolysis. Coagulation studies showedevidence of inhibition of anistreplase induced fibrinolytic activity which may explain thefailure of thrombolytic treatment in patients with evidence of a systemic non-lytic state.

Introduction

Thrombolytic drugs reduced mortality in patients with acute myocardial infarctiontreated within 6, 12 or even 24 hours after onset of symptoms (1-3). When treatment wasstarted within the first 4-6 hours after the onset of chest pain, reperfusion was shown inmost of the infarct related coronary arteries. However, in up to 30-40% of the patients noreperfusion could be achieved (4). Failure of thrombolytic treatment has been reportedirrespective of the drug used (4). The configuration of coronary obstruction may be animportant determinant of the success of treatment (5), but inhibition of drug activity hasnever been ruled out.

In general, streptokinase and anistreplase (anisoylated plasminogen streptokinaseactivator complex, APSAC) caused comparable changes in hematological variables such asfibrinogen, plasminogen, andα2-antiplasmin during the first 24 hours after they weregiven (6). These changes were ascribed to systemic effects. Some patients, however,

59

appendix 2

showed no substantial decrease in plasma-fibrinogen after anistreplase or streptokinaseadministration (7). This suggests resistance to these drugs. It has been suggested that asystemic lytic state, defined as a low plasma concentration of fibrinogen after thrombolytictreatment, is a prerequisite for local thrombolytic efficacy (8).

To investigate the possibility of drug resistance as an explanation for failure ofthrombolytic treatment, we performed a retrospective study to assess the relation betweenthe systemic fibrinolytic effects and the local efficacy of anistreplase in patients with acutemyocardial infarction.

Patients and methods

Patients: We studied 58 consecutive patients (47 men, 11 women), mean age 57 years(range 34-71), who presented within 4 hours of the onset of chest pain. Selection criteriafor thrombolytic treatment included the presence of characteristic symptoms of myocardialinfarction and ST-segment elevation of at least 0.1 mV in one or more of the standardleads or at least 0.2 mV in two or more of the precordial leads in a 12-leadelectrocardiogram and the presence of symptoms unresponsive to sublingual glyceryltrinitrate. We excluded patients with contraindications for thrombolytic treatment and thosewho had been treated with streptokinase or anistreplase within the previous 6 months.

Study Protocol: Patients were treated with 30 U of anistreplase (Eminase™,SmithKline Beecham) administered intravenously in 4-5 minutes. Infusion with heparin(30.000 U in 24 hours) was started 4-6 hours after thrombolytic treatment and wascontinued until an adequate level of anticoagulation had been achieved with oralacenocoumarol, which was started after 48-72 hours. To assess patency of the infarctrelated artery, coronary angiography was performed 48 hours (range 36-60) after theadministration of anistreplase in all patients. In the first 30 consecutive patients patencywas also assessed after 90 minutes (range 1 to 3 hours). Patency was documentedaccording to the score used in the thrombolysis in myocardial infarction (TIMI) trial (9).Scores of grade 0 or 1 indicated occlusion of the infarct related vessel and grades 2 and 3patency.

Coagulation analyses:Coagulation and fibrinolytic variables were studied immediatelybefore and 90 minutes and 48 hours after anistreplase administration. Venous bloodsamples were collected on ice in a 1/10 volume 3.05% trisodium citrate for measurementsof fibrinogen, plasminogen,α2-antiplasmin, reptilase time and euglobulin clot lysis time.Assays were performed immediately or plasma was stored at -80oC for analysis later.Fibrinogen was measured according to the method of Clauss (10). Plasminogen andα2-antiplasmin assays were performed with a synthetic chromogenic substrate (Kabi)according to the method of Friberger et al (11). Reptilase time was determined by themethod of Soria et al. (12) and euglobulin clot lysis times by the method of Buckell (13).The assay for fibrinogen/fibrin degradation products was carried out on serum collected atthe times mentioned above with a latex agglutination kit (Wellcome) according to themethod of Pitcher (14).

A systemic lytic state was defined as a decrease of the plasma concentration offibrinogen to below 1.0 g/l, measured 1.5 hours after the administration of anistreplase.

Statistical analysis: Plasma concentrations of fibrinogen, plasminogen, andα2-

60

appendix 2

antiplasmin were expressed as mean (SD). Statistical comparisons between patientsshowing a systemic lytic state and a systemic non-lytic state were performed by means ofthe Student’s t-test for independent samples. Comparisons within the groups were madewith the paired Student’s t-test.

Measurements of reptilase time, euglobulin clot lysis time, and fibrinogen/fibrindegradation products were expressed as median (range). Patient groups were compared bythe Mann-Whitney U/Wilcoxon rank sum test. Differences within the groups were testedby the Wilcoxon matched paired signed ranks test. We used Fisher’s exact test to comparethe result of treatment in terms of patency and the presence of a systemic lytic state. Atwo-tailed p-value of <0.05 was regarded as statistically significant.

Results

Coagulation data: Fifty-eight patients were retrospectively classified into two groups.Fifty-two showed a systemic fibrinolytic state and in six patients plasma fibrinogen did notdecrease below 1.0 g/l. Initial values of fibrinogen, plasminogen,α2-antiplasmin, reptilasetime, euglobulin clot lysis time, and fibrinogen/fibrin degradation products were similar inthe two groups (Table I).

After 90 minutes, fibrinogen, plasminogen, andα2-antiplasmin concentrations weresignificantly reduced in both the lytic and the non-lytic groups. Mean plasmaconcentrations of fibrinogen in the lytic and the non-lytic groups were 0.0 g/l and 2.3 g/l(normal range 1.7-3.5); of plasminogen 11% and 57% (normal range 70-130); andα2-antiplasmin 4% and 35% (normal range 90-130) respectively. These differences werestatistically significant. Individual values for fibrinogen in the six non-lytic patients beforeand 90 minutes after treatment with anistreplase were: 3.3 vs 2.7; 2.7 vs 2.3; 2.4 vs 1.1;3.0 vs 2.2; 2.4 vs 1.8, and 3.7 vs 3.6 g/l respectively. The reptilase time was considerablyprolonged in the lytic group from 19 to 109 seconds, but did not change in the non-lyticgroup (19 vs 24 seconds). Euglobulin clot lysis time was shortened from >120 before to<10 minutes after the administration of anistreplase in both groups (normal value >120minutes). Serum concentrations of fibrinogen/fibrin degradation products remained withinnormal ranges (<8 µg/ml) in the non-lytic group, whereas they were considerablyincreased in the lytic group (median value >256 µg/ml).

These changes declined after 48 hours. At that time mean plasma concentrations offibrinogen, plasminogen, andα2-antiplasmin were still significantly lower in the lyticgroup, and, with the exception of fibrinogen, below the normal ranges. Values for reptilasetime, euglobulin clot lysis time, and fibrinogen/fibrin degradation products were normal oralmost normal.

Patency: Ninety minutes after anistreplase administration angiography showed patencyin 20 (67%) of 30 patients. No early reocclusion occurred in these patients. Overallpatency at 48 hours was achieved in 43 (74%) of 58 patients. The patency rate was 83%(43/52) in the patients showing a systemic lytic state and 0% (0/6) in those showing anon-systemic lytic state (Table II). The relation between systemic non-lytic state and non-patency of the infarct related vessel was statistically significant (p <0.001).

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appendix 2

Table I Coagulation variables of all patients stratified according to fibrinolytic state

variable lytic non-lytic p-value@

fibrinogen (g/l)before1.5 h after48 hrs after

3.1 (0.96)0.0 (0.15)*

2.5 (0.68)*

2.9 (0.48)2.3 (0.78)#

4.2 (0.84)

NS<0.01<0.01

plasminogen (%)before1.5 h after48 hrs after

97 (18)11 (13)*

55 (13)*

104 (6)57 (9)*

78 (12)#

NS0.01<0.01

α2-antiplasmin (%)before1.5 h after48 hrs after

93 (14)4 (5)*

80 (16)*

90 (12)35 (2)#

99 (8)

NS<0.01<0.01

reptilase time (sec)before1.5 h after48 hrs after

19 (10-27)109 (44-201)*

19 (15-23)

19 (18-20)24 (18-31)20 (19-21)

NS<0.01NS

euglobulin clotlysis time (sec)

before1.5 h after48 hrs after

>120 (>120)<10 (<10)*

>120 (95->120)

>120 (>120)<10 (<10-15)#

>120 (>120)

NSNSNS

fibrinogen/fibrindegradationproducts (µg/ml)

before1.5 h after48 hrs after

<8 (<8)>256 (<8->256)*

36 (<8->256)#

<8 (<8)<8 (<8)<8 (<8)

NS<0.01<0.05

* p <0.01 vs baseline, # p <0.05 vs baseline, @ p value for between group comparison.

Fibrinogen, plasminogen andα2-antiplasmin expressed as mean (SD). Values of reptilasetime, euglobulin clot lysis time and fibrinogen/fibrin degradation products as median(range).

62

appendix 2

Table II Relation of coagulation variables to patency of infarct related vessel 48 hoursafter treatment with anistreplase

lytic state non-lytic state p-value

patency 43 0

0.00012non-patency 9 6

total 52 6

63

appendix 2

Discussion

The predictive value of a systemic lytic state for the efficacy of thrombolytic treatmentwith streptokinase, urokinase, or anistreplase was the subject of several previous studies.White et al. did not find that systemic hematological markers of fibrinolysis were helpfulin explaining the success or failure of intracoronary thrombolysis (15). In contrast,Rothbard et al. showed a close relation between a systemic lytic state and reperfusion ofthe infarct related vessel (8). Burket et al. stated that a systemic lytic state, rather thanbeing considered an adverse effect of treatment, might serve as a reasonable clinical goalwhen thrombolysis is attempted (16). Lew et al. showed that high residual fibrinogenconcentrations identified patients in whom thrombolytic treatment was relativelyineffective (17). It is difficult to compare the results of these studies. In the first threestudies streptokinase or urokinase was given, whereas in Lew et al’s study streptokinasewas administered intravenously. The dosages of streptokinase varied widely as did theinterval between onset of chest pain and thrombolytic treatment. Finally, a systemic lyticstate was defined differently in these studies -as a reduction in fibrinogen of at least 50%(15), at least 10% (8) or to below 0.5 g/l (17), and Burket et al. did not define a cut offpoint (16). Marder et al. studied 106 patients treated with streptokinase or anistreplase (7).A systemic lytic state was defined as a fall of >20% in plasma fibrinogen concentration.In 4 of the 58 patients treated with 30 U of anistreplase a systemic non- lytic state wasfound and none of these patients achieved reperfusion. None the less, no statisticallysignificant relation between a systemic non-lytic state and failure of reperfusion wasfound. In the remaining 48 patients treated with a low dose of intracoronary streptokinasethere was also no statistical correlation. Despite the presence of a systemic non-lytic statereperfusion occurred in 10 patients. This discrepancy was partly explained by localthrombolytic effects of intracoronary streptokinase.

In our patients we regarded a systemic lytic state as being likely if the concentration ofplasma fibrinogen was <1.0 g/l 90 minutes after the administration of anistreplase. Thisvalue was chosen because it is commonly accepted as the hemostatic concentration offibrinogen (18). In the lytic group there was an almost complete depletion of fibrinogen,plasminogen, andα2-antiplasmin, associated with a short euglobulin clot lysis time,considerably prolonged reptilase time, and high concentration of fibrinogen/fibrindegradation products. In both the lytic and the non-lytic patients there was a comparableshortening of the euglobulin clot lysis time. Euglobulin clot lysis time reflects thefibrinolytic activity of plasma after inhibitors have been removed. Apparently, thefibrinolytic system was activated by anistreplase in all patients. The moderate decrease inplasminogen andα2-antiplasmin in the non-lytic group also accorded with activation ofthe fibrinolytic system. Because neither reptilase time nor the concentration offibrinogen/fibrin degradation products changed, whereas the fibrinogen concentrationdecreased but remained within normal ranges, it seems likely that inhibition wasresponsible for the limited expression of fibrinolytic activity.

None of the patients in the non-lytic group showed reperfusion of the infarct relatedvessel. Thus fibrinolytic inhibition seems to be restricted not only in terms of systemiceffects but also for local thrombolytic failure. Initial plasma concentrations ofα2-antiplasmin plasma were similar in both groups. Therefore, it is unlikely that this potent

64

appendix 2

inhibitor is responsible for the supposed fibrinolytic inhibition. Anti-streptokinaseantibodies from earlier treatment with streptokinase or anistreplase can be excludedbecause none of the patients had previously received one of these drugs. There may havebeen naturally occurring anti-streptokinase antibodies (19), but they were not sought in ourpatients.

A systemic non-lytic state 90 minutes after the administration of anistreplase in aproportion of patients with myocardial infarction predicted failure of thrombolysis. Theabsence of systemic and local fibrinolytic activity was probably due to fibrinolyticinhibitors. These compounds are currently under investigation. The reported findings arerelevant not only to explain the mechanism of thrombolytic failure but may also haveimplications for clinical practice. A simple and rapid laboratory test to detect thrombolyticfailure of anistreplase would lead to the option of additional treatment.

Acknowledgment

We thank Beecham Research Laboratories, The Netherlands, for providing us with thethrombolytic drug (anistreplase, Eminase™).

References

1. AIMS Trial Study Group. Effect of intravenous APSAC on mortality after acutemyocardial infarction: preliminary report of a placebo controlled clinical trial. Lancet1988;i:545-9.

2. The GISSI Study Group. Effectiveness of intravenous thrombolytic treatment inacute myocardial infarction. Lancet 1986;i:397-402.

3. ISIS-2 Collaborative Group. Randomized trial of intravenous streptokinase, oralaspirin, both, or neither among 17187 cases of suspected acute myocardial infarction:ISIS-2. Lancet;ii:349-60.

4. Marder VJ, Sherry S. Thrombolytic therapy: current status (first of two parts). NEngl J Med 1988;318:1512-20.

5. Davies MJ. Successful and unsuccessful coronary thrombolysis. Br Heart J1989;61:381-4.

6. Monassier JP, Hanssen M. Hematological effects of anisoylated plasminogenstreptokinase activator complex and streptokinase in patients with acute myocardialinfarction, interim report of the IRS II study. Drugs 1987;33 (suppl.3):247-52.

7. Marder VJ, Kinsella PA, Brown MJ. Fibrinogen concentration and coronary arteryreperfusion after intravenous anisoylated plasminogen streptokinase activatorcomplex or intracoronary streptokinase therapy. Drugs 1987;33 (suppl.3):237-41.

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8. Rothbard RL, Fitzpatrick PG, Francis CW, Caton DM, Hood WB, Marder VJ.Relationship of the lytic state to successful reperfusion with standard- and low-doseintracoronary streptokinase. Circulation 1985;71:562-70.

9. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial: phase Ifindings. N Engl J Med 1985;312:932-6.

10. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung desFibrinogens. Acta Haematol 1957;17:237-46.

11. Friberger P, Knös M, Gustavsson S, Aurell L, Claeson G. Methods for determinationof plasmin, antiplasmin and plasminogen by means of substrate S2251. Haemostasis1978;7:138-45.

12. Soria J, Soria C, Yver J, Samama M. Temps de reptilase, étude de la polymerisationde la fibrin en présence de reptilase. Coagulation 1969;173:2.

13. Buckell M. The effect of citrate on euglobulin methods of estimating fibrinolyticactivity. J Clin Pathol 1958;11:403-5.

14. Pitcher PM. The detection of fibrinogen degradation products (FDP) in serum andurine. Canad J Med Tech 1972;34:166-78.

15. White CW, Schwartz JL, Ferguson DW, et al. Systemic markers of fibrinolysis afterunsuccessful intracoronary streptokinase thrombolysis for acute myocardialinfarction. Am J Cardiol 1984;54:712-7.

16. Burket MW, Smith MR, Walsh TE, Brewster PS, Fraker TD. Relation ofeffectiveness of intracoronary thrombolysis in acute myocardial infarction tosystemic thrombolytic state. Am J Cardiol 1985;56:441-4.

17. Lew AS, Cercek B, Hod H, Shah PK, Ganz W. Usefulness of residual plasmafibrinogen after intravenous streptokinase for predicting delay or failure ofreperfusion in acute myocardial infarction. Am J Cardiol 1986;58:680-5.

18. Colman RW, Hirsh J, Marder VJ, Salzman EW. Hemostasis and Thrombosis. 2nded. Philadelphia: Lippincott, 1987:922.

19. Moran DM, Standring R, Lavender EA, Harris GS. Assessment of anti-streptokinaseantibody levels in human sera using a microradioimmunoassay procedure. ThrombHaemostas 1985;52:281-7.

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Rapid Enzyme Immunoassay of Anti-Streptokinase Antibodies inHuman Plasma

Victor J.J. Bom, Johan Brügemann (*), Wim van der Schaaf, Roelie T. van Wijk and Janvan der Meer

Department of Haemostasis, Thrombosis & Rheology and Department of Cardiology (*),University Hospital of Groningen, Groningen, The Netherlands

Thromb Haemost 1991;65:1268 (abstract)Clinica Chimica Acta 1993;218:121-9.

Abstract

A simple enzyme immunoassay for determination of anti-streptokinase antibodies (aSKa)in plasma is described. Commercially available reagents have been used for the assay,which is calibrated with a reference preparation of aSKa containing 100 AU/ml. The assayis specific and reproducible with a variation coefficient of 4.8%. In healthy individuals abroad range of values between 4 and 291 AU/ml was observed with a large differencebetween the mean and median value (55 and 27 AU/ml, respectively). Data from a studyon 21 patients with myocardial infarction treated with the streptokinase derivativeanistreplase suggest that a high titre of aSKa before treatment is associated with failure ofthrombolytic therapy. The assay procedure can be shortened to 0.5 h to screen patients fora high aSKa level. This assay allows a more routine assessment of aSKa in the clinic.

Introduction

Streptokinase (SK), a protein produced by group C ß-hemolytic streptococci, is nowroutinely used in thrombolytic treatment of myocardial infarction (1-4). The mechanism bywhich SK restores patency in occluded vessels is based on formation of a complexbetween SK and circulating plasminogen that acts as an activator of both free andthrombus-bound plasminogen, resulting in dissolution of the thrombus by formed plasmin(5,6). An acylated form of the complex, p-anisoyl plasminogen streptokinase activatorcomplex (APSAC, anistreplase, Eminase™) has been introduced as a second generationthrombolytic agent.

Though early administration of SK or APSAC significantly reduces the mortality due tomyocardial infarction, reperfusion studies have shown that 20-40% of patients fail torespond, for reasons that have remained largely unclarified (3,4,7,8). One interesting butstill controversial explanation is that high levels of circulating antibodies to SK mayreduce the efficacy of the thrombolytic therapy (8-11).

Circulating anti-SK antibodies (aSKa) are found in most people. They are probablyinduced by Streptococcal infections (11,12) which might also explain the considerable

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variation in aSKa levels observed previously (2,8,13). In general these levels have beenmeasured by a functional assay, the SK-resistance (SKR) test (1,9-11) which is based onneutralization of SK activity in a clot-lysis system. Though this assay can be performedrapidly, it demands appreciable technical skill to obtain reproducible results and also itsspecificity is limited. Alternatively, aSKa has been measured by radioimmunoassay (8,13)which requires a long assay period, radioactive reagents and expensive instruments,thereby hampering its routine application in clinical laboratories. A non-radioactiveimmuno assay has also been described, however the assay was only poorly characterized(12).

In this report we describe in detail a rapid and simple enzyme immunoassay of aSKa(14,15), which has been used to assess aSKa levels in healthy individuals and patientswith myocardial infarction treated with anistreplase.

Materials and Methods

Materials

Microtiterplates of high protein-binding capacity were obtained from Nunc,Copenhagen, Denmark. Bovine serum albumin (grade V) was obtained from SigmaChemical Co., St Louis, MO, USA. Streptokinase was purchased from Behringwerke,Marburg, Germany (Streptase™: 100,000 IU; 133-174 mg or 750,000 IU; 150-160 mg)and from KabiVitrum, Stockholm, Sweden (Kabikinase™: 100,000 IU, 26-27 mg) whoalso supplied human fibrinogen. Eminase™ (anistreplase, APSAC) is a registeredtrademark of Beecham Pharmaceuticals, Great Burgh, Epsom, England. Human thrombinwas purchased from the Central Laboratory of the Red Cross Blood Transfusion Service,Amsterdam, The Netherlands. Peroxidase-conjugated rabbit antibodies to human IgG(P214) and tablets of ortho-phenylenediamine dihydrochloride (OPD) were obtained fromDakopatts, Glostrup, Denmark. Tween 20 was purchased from Pierce Chemical Co.,Rockford, IL. Other chemicals were of high purity and obtained from Merck, Darmstadt,Germany or BioRad Laboratories, Richmond, VA.

Plasmas were prepared from venous blood samples anticoagulated with 1:10 volume ofa 0.109 M solution of trisodium citrate, pH 6.0. After centrifugation at 2200 x g, thesupernatants were rendered platelet free by subsequent centrifugation at 16,000 x g(Eppendorf centrifuge 5415-C). Aliquots were stored at -80°C. Blood samples wereobtained from healthy individuals and patients referred to the emergency ward of ourhospital because of acute myocardial infarction. Normal plasma pools were made ofplasma samples of 50-59 healthy hospital staff members and one of these pools wasassigned as the reference preparation of aSKa.

Enzyme immunoassay of aSKa

The assay was performed similarly to standard ELISA procedures. First the wells of amicrotiterplate were coated with 100 µl of a 10 IU/ml solution of SK (routinelyStreptase™: 100,000 IU, 133-174 mg) dissolved in PBS (0.15 M NaCl, 0.01 M sodiumphosphate pH 7.2) during 1 hr at 37°C. Afterwards the solution was aspirated and the

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wells were washed and incubated with PBS+ (PBS supplemented with 0.35 M NaCl, 0.002M EDTA, 1 g/l of bovine serum albumin and 1 ml/l of Tween 20) during 5 min at roomtemperature to block remaining binding sites, and washed again, before 100 µl of plasmasample dilutions in PBS+ were incubated in duplicate for 2 hr.

Thereafter non-bound material was removed and the wells were washed 3 times withPBS+. The amount of bound aSKa was determined by incubation with 100 µl of a solutionof peroxidase-conjugated anti-human IgG diluted 1:3000 in PBS+ at room temperature for1 hr.

After washing again, colour was developed by incubation with 100 µl of OPD/H2O2

reagent (8 mg OPD in 12 ml 0.065 M sodium phosphate, 0.035 M citric acid (pH 5.0),supplemented with 5 µl of a 30% solution of H2O2) for 5 min. The reaction was stoppedby adding 150 µl of 3M H2SO4 and the absorbance read at 492 nm using a microplatereader.

The results were calculated using a standard dilution curve of reference plasma, whichwas plotted against the absorbance on a double-logarithmic scale. The reference plasmahad an assigned value of 100 AU/ml. Plasma samples were tested in at least 2 dilutions inPBS+, routinely 1:100 and 1:200.

In the rapid "cito" procedure a pre-coated microtiterplate (5 IU of SK per well; keepingquality ≥1wk at 4°C) is used. This plate is incubated with 3 sample dilutions (1:100,1:200, 1:400) together with 4 reference plasma dilutions (1:50, 1:100, 1:200, 1:500) foronly 10 min in the first stage and with conjugate 1:500 for only 5 min in the second stageof the assay.

Streptokinase resistance (SKR) test

This assay was performed as described by Moran et al. (13). In short, 20 µl of a SKdilution in 0.9% NaCl was added to 160 µl test plasma and the mixture was vortexedimmediately. Then 20 µl of a thrombin solution (50 IU/ml) were added and the mixturewas vortexed again. Clot formation occurred within 30 sec and lysis was established after10 min. The SKR titre is defined as the lowest plasma concentration of SK that results incomplete lysis of the clot within 10 min and is expressed in IU/ml of SK (16).

Patency assessment

Patency of the infarct related vessel was assessed by coronary angiography performed48 hr after thrombolytic therapy. According to the thrombolysis in myocardial infarction(TIMI) trial (17) scores of grade 0 or 1 indicated occlusion and scores of grade 2 and 3indicated patency.

Statistical analysis

Correlation analysis was done by Spearman rank correlation test and a p-value < 0.01was considered to indicate significance.

Results

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Characterisation of the enzyme immunoassay of aSKa

The enzyme immunoassay of aSKa was performed using a typical ELISA incubationscheme, starting with immobilized streptokinase to capture the analyte, in casu aSKa, andwith enzyme-labeled rabbit anti-human IgG antibodies for the final analysis. Thecalibration curve with reference plasma containing 100 AU/ml is sigmoidal on a double-logarithmic scale as shown in Figure 1. Parallel dilution curves were obtained with 2 otherpooled plasmas containing 46 and 115 AU/ml of aSKa, respectively, and with mostindividual plasmas. Some individual plasmas showed non-parallelism at dilutions below1:100 (i.e. at 1:50 to 1:10). This is probably due to interferences which are commonlyencountered in immunoassays at low dilutions (18). Therefore, plasma samples wereroutinely tested at dilutions of 1:100 and 1:200 and the corresponding results (defined asdiffering less than 15%) were averaged. At these dilutions the detection limit of the assayis 1 AU of aSKa per ml of plasma.

Most experimental conditions of the assay were not very critical, i.e. SK could be usedat a 5-fold higher concentration or after coating overnight at 4°C in stead of 1 hour at37°C; also the amount of conjugate could be varied 2- to 3-fold with only minoradaptation of the color development time. The final conditions as described underMaterials and Methods have been chosen based on the highest, most reproducible signal-to-noise ratio at the lowest costs. Under these conditions the inter-assay coefficient ofvariation for a sample containing 44 AU/ml of aSKa was determined to be 4.8% (n=6).

For rapid application, i.e. for patients who present with chest pain andelectrocardiographic characteristics indicating acute myocardial infarction, the assay can beshortened to 0.5 hour to give quantitative values of aSKa in the range of 40 to 400 AU/ml(see Materials and Methods). The variation coefficient of this rapid "cito" assay for asample containing 137 AU/ml of aSKa was found to be 1.7% (n = 6).

The specificity of the assay was established in five different ways. Firstly,preincubations of several plasmas with an excess of SK showed a reduction of themeasured aSKa level of at least 50-fold. Secondly, the use of a SK preparation of a 7.5-fold higher specific activity or of SK obtained from another manufacturer did not affectthe results. Thirdly, when SK as coating ligand was replaced by other proteins like bovineserum albumin and human fibrinogen, the analytes were unreactive in the assay (thoughthe background was slightly higher). Fourthly, mixtures of plasmas with high and lowaSKa titres in ratios of 3:1, 1:1 and 1:3 all gave nearly expected values: for nine mixturesthe mean deviation was 1 AU/ml (range -7 to 7 AU/ml). Finally, in 3 patients having ahigh aSKa level, this level decreased by on the average 92% within 1.5 hr after treatmentwith the SK-derivative anistreplase (19) [see below].

Distribution of aSKa levels in a human population

The concentration of aSKa was determined in the plasmas of 63 healthy volunteers aged18-59 years. A broad range of values was observed between the extremes of 4 and 291AU/ml. The distribution, given as a histogram in Figure 2, was evidently not normal with

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a mean and a median value of 55 and 27 AU/ml, respectively. This was mainly due tosome rather high plasma levels of aSKa: in 10% of the individuals aSKa levels were 5-fold higher than the median value.

Correlation between aSKa level and SKR titre

The aSKa level was compared with the SKR titre in 57 different plasmas as shown inFigure 3. There was a statistically significant correlation: r=0.6975, p<0.001. However,large individual discrepancies were noticed, i.e., a SKR titre of 24 IU/ml was found inplasmas containing 20-180 AU/ml of aSKa. Also three different normal plasma poolsdiffered in their ratio of SKR titre to aSKa level, ranging from 0.30 to 0.78 (mean 0.48).

ASKa levels in patients with myocardial infarction treated with anistreplase

In a retrospective study we analyzed the plasma levels of aSKa in 21 patients withmyocardial infarction before onset of the thrombolytic therapy. The results together withthe angiographical findings with respect to patency or non-patency of the infarct-relatedvessel are shown in Figure 4. Three patients (14%) were found to have an aSKa levelabove 135 AU/ml, so 5-fold higher than the median normal value, and these patients didnot respond to the thrombolytic treatment with anistreplase. The mean aSKa level in thesepatients dropped from 321 AU/ml to 25 AU/ml within 1.5 hr after administration of theanistreplase (results not shown, see ref. 19), which is most likely explained by a strongreactivity to the SK-derivative. Patency was demonstrated in the remaining patients, allhaving a lower aSKa level.

Discussion

The enzyme immunoassay of aSKa reported here is very simple and rapid to perform inany laboratory familiar with standard ELISA techniques. All reagents are commerciallyavailable and most of the assay conditions are not very critical. The "cito" procedure israpid enough (0.5 hr) to be valuable for clinical practice. The assay seems specific and isaccurate as indicated by variation coefficients of 4.8 and 1.7% for the standard and "cito"assay, respectively. In our opinion this assay is superior to the radioimmunoassaydescribed by Moran et al. (13), not only because of its rapidity and elimination ofradioactive materials, but also because incubation of plasma with free SK, resulting in arapidly degradable SK-plasminogen complex to which aSKa have to bind, is avoided bythe use of immobilized SK; also the use of protein A, which does not react with allsubclasses of immunoglobulin G, is eliminated.

The results of the aSKa assay have been expressed in an arbitrary unit (AU/ml), relativeto the aSKa concentration in a home-made pooled normal reference plasma. As threedifferent plasma pools (each made of at least 50 individual plasmas) were found to varyconsiderably in their aSKa content (between 46 and 115 AU/ml), it is obvious that aninternational standard would be necessary to compare results between differentlaboratories. In the SKR test, a pooled plasma, containing 100 AU/ml of aSKa, has onaverage an inhibitory potential of 48 IU/ml of SK.

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The aSKa level in plasma was found to be significantly correlated to the SKR titre,though a wide scatter in individual ratios was observed. This is similar to what has beenobserved by others using different assay techniques to measure aSKa (7,13). Also a 2.6-fold variation of the SKR/aSKa ratio (0.30-0.78) was observed between three differentnormal plasma pools. A lower SK neutralizing capacity than expected from the aSKa levelmight be explained by the presence of low-affinity antibodies (which do not fully exerttheir effect within 10 min. in the SKR test) and by the presence of weakly- or non-neutralizing antibodies. Besides, interfering plasma factors may have affected (some) SKRtitres and hence the ratios.

A useful application of the enzyme immunoassay of aSKa might be the screening ofpatients with myocardial infarction who will be treated with SK or anistreplase. In aretrospective study we analyzed 21 patients and found plasma levels of aSKa above 135AU/ml (5-fold higher than the median normal value) in 3 (14%) of them; this frequency ofhigh aSKa levels in the patients was in good agreement with that (10%) among healthyindividuals. All three patients with high plasma aSKa levels failed to respond tothrombolytic treatment with anistreplase. In these patients we also observed a decrease ofthe mean aSKa level of 321 AU/ml to 25 AU/ml within 1.5 hour after treatment, which islikely to be due to reactivity of aSKa to the SK-derivative. The combined data stronglysuggest that aSKa had neutralised anistreplase, at least partly. With a presumed plasmavolume of 3000 ml, these patients would have a mean plasma SK neutralization capacityof 462,000 IU, which constitutes 31% of the administered dose of anistreplase (6).

Our findings support the hypothesis that high concentrations of aSKa can reduce theefficacy of anistreplase in vivo. However, Hoffmann et al. (8) did not find a correlationbetween a high aSKa level and failure of anistreplase therapy in a group of 32 patients.Whether this discrepancy is related to the radio-immunoassay they used to measure aSKa(vide supra) needs further investigation. The availability of the reported rapid "cito" aSKaassay would be relevant for clinical practice if high aSKa levels in patients are predictivefor the outcome of anistreplase or SK therapy. In such patients either a higher dose or another thrombolytic agent like tissue-type plasminogen activator or urokinase might bepreferred, primarily or additionally.

In conclusion, the simplicity, rapidity and reliability of the enzyme immunoassay ofaSKa as well as its possible relevance for clinical practice make this assay valuable forclinical laboratories.

Acknowledgements

We are grateful to W.M. Smid, MD for his valuable contributions to both the figuresand the statistical analysis and to Mrs. P. Wetterauw and Mrs. H. Sterenberg-Frieling forthe final preparation of the manuscript.

References

1. Deutsch E, Fischer M. Die wirkung intravenös applizierter streptokinase auffibrinolyse und blutgerinnung. Thromb Diath Haemorrh 1960;4:482-506.

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2. Verstraete M, Vermylen J, Amery A, Vermylen C. Thrombolytic therapy withstreptokinase using a standard dosage scheme. Br Med J 1966;1:454-456.

3. Rentrop KP. Thrombolytic therapy in patients with acute myocardial infarction.Circulation 1985;71:627-631.

4. Marder VJ. Sherry S. Thrombolytic therapy: current status. First of two parts. NEngl J Med 1988;318:1512-1520.

5. McClintock DK, Bell PH. Mechanism of activation of human plasminogen bystreptokinase. Biochem Biophys Res Commun 1971;43:694-702.

6. Robbins KC. Fibrinolytic therapy: biochemical mechanisms. Sem Thromb Haemost1991;17:1-6.

7. Brügemann J, Meer J van der, Takens BH, Hillege H, Lie KI. A systemic non-lyticstate and local thrombolytic failure of anistreplase (anisoylated plasminogenstreptokinase activator complex, APSAC) in acute myocardial infarction. Br Heart J1990;64:355-358.

8. Hoffmann JJML, Fears R, Bonnier JJRM, Standring R, Ferres H, Swart JBRM de.Significance of antibodies to streptokinase in coronary thrombolytic therapy withstreptokinase or APSAC. Fibrinolysis 1988;2:203-210.

9. Rothbard RL, Fitzpatrick PG, Francis CW, Caton DM, Hood WB jr, Marder VJ.Relationship of the lytic state to successful reperfusion with standard- and low-doseintracoronary streptokinase. Circulation 1985;71:562-570.

10. Lew AS, Neer T, Rodriguez L, Geft IL, Shah PK, Ganz W. Clinical failure ofstreptokinase due to an unsuspected high titre of antistreptokinase antibody. J AmColl Cardiol 1984;4:183-185.

11. Jalihal S, Morris GK. Antistreptokinase titres after intravenous streptokinase. Lancet1990;335:184-185.

12. McGrath K, Patterson R. Immunology of streptokinase in human subjects. Clin ExpImmunol 1985;62:421-426

13. Moran DM, Standring R, Lavender EA, Harris GS. Assessment of anti-streptokinaseantibody levels in human sera using a microradioimmunoassay procedure. ThrombHaemost 1985;52:281-287.

14. Vatassery GT, Quach HT, Smith WE, Benson BA, Eckfeldt JH. A sensitive assay oftransthyretin (prealbumin) in human cerebrospinal fluid in nanogram amounts byELISA. Clin Chim Acta 1991;197:19-26.

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15. Haimoto H, Kurobe N, Hosoda S, Kato K. Sensitive enzyme immunoassay forhuman aldolase B. Clin Chim Acta 1989; 181:27-36.

16. Bangham DR, Walton PL. The international standard for Streptokinase-Streptodornase. Bull WHO 1965;33:235-242.

17. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial. Phase Ifindings. N Engl J Med 1985;313:932-936.

18. Weber TH, Käpyaho KI, Tanner P. Endogenous interference in immunoassays inclinical chemistry. A review. Scand J Clin Lab Invest 1990;50(suppl 201):77-82.

19. Brügemann J, Meer J van der, Bom VJJ, Schaaf W van der, Lie KI. Anti-streptokinase antibodies before and absence of a systemic lytic state afterthrombolytic therapy with anistreplase predict failure of treatment in patients with amyocardial infarction. Thromb Haemost 1991;65:1095A.

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Anti-Streptokinase Antibodies Inhibit Fibrinolytic Effects ofAnistreplase in Acute Myocardial Infarction

Johan Brügemann, Jan van der Meer (*), Victor J.J. Bom (*), Wim van der Schaaf (*),Pieter A. de Graeff, and Kong I. Lie

Departments of Cardiology and Haematology (*), Division of Haemostasis, Thrombosis &Rheology, University Hospital Groningen, The Netherlands

Thromb Haemost 1991;65:1095 (abstract)Am J Cardiol 1993;72:462-4.

Abstract

Twenty-one patients with acute myocardial infarction (MI) were treated with anistreplase(Eminase™) within 4 hours after onset of symptoms. Coagulation parameters and the IgGantibody level to streptokinase (aSKa) were determined in pretreatment blood samples andafter 1.5 and 48 hours. Coronary angiography was performed after 48 hours. A systemicnon-lytic state was characterized by plasma fibrinogen in excess of 1.0 g/liter 1.5 hoursafter therapy. A high pretreatment aSKa level was found in 3 of the patients (mean 321Arbitrary Units/ml). In these patients no systemic lytic state was induced and coronaryangiography showed non-patency of the infarct related vessel. When aSKa levels werewithin the normal range (mean 55 Arbitrary Units/ml), a systemic lytic state was alwaysobtained and a high patency rate was seen (16/18 patients). Binding of aSKa to thestreptokinase component of anistreplase was suggested by a temporary decline of its levelfollowing anistreplase administration. Thus, the pretreatment IgG aSKa level is of majorimportance to achieve a systemic lytic state and subsequent local thrombolysis whenanistreplase and probably streptokinase, is used in patients with MI.

Introduction

Streptococcal fibrinolysin, later renamed streptokinase (SK), has been used widely inpatients with acute myocardial infarction since publication of the GISSI (Gruppo Italianoper lo Studio della Streptochinasi nell’Infarto Miocardio) trial (1). However, this therapy isnot always successful. Thirty to 40% failure of reperfusion has been reported (2). Thismay, among other causes, be due to systemic inactivation of the drug by immunoglobulinG antibodies to streptokinase (aSKa). These antibodies may be present as a result ofprevious infections with streptococci, or previous treatment with SK (3-5). Plasma ofpatients who had been treated with SK showed resistance to the action of a repeat dose inthe course of 1 to 2 months after initial therapy (6). In the late 1950s, it was thought thatlocal coronary thrombolysis was the result of a systemic lytic state. Two decades later, thispoint of view was advocated again by Rothbard et al. (7). They showed that a systemic

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lytic state, as characterized by a fibrinogen decrease of >10% after SK administration, wasrelated to the success of therapy. Recently, we described a strong relation between asystemic non-lytic state (fibrinogen >1 g/liter) and local thrombolytic failure of the SK-containing thrombolytic drug anistreplase in acute myocardial infarction (8). It washypothesized that a systemic lytic state was not achieved, because of the presence of ahigh aSKa level. To verify this hypothesis, in the present study we determined the aSKalevel in plasma of patients who had been treated with anistreplase.

Methods

In 21 patients (17 men, 4 women; mean age 56 years, range 34-70) withelectrocardiographically documented acute myocardial infarction, an intravenous bolusinjection of 30 U anistreplase (Eminase™, SmithKline Beecham) was administered≤4hours after the onset of chest pain. Adequate anticoagulation with intravenous heparin(30.000 U/24 hours) was begun 1-4 hours after thrombolytic treatment and continued for48 hours. Subsequently, coronary angiography was performed, and patency of the infarctrelated artery was documented according to the score used in the Thrombolysis inMyocardial Infarction trial (9). Grade 0 or 1 was considered to reflect occlusion, whereasgrade 2 or 3 was considered to indicate patency. Venous blood samples were obtainedbefore treatment, and after 1.5 and 48 hours, and placed on ice in 1/10 volume 3.05%trisodium citrate for measurement of fibrinogen, plasminogen,α2-antiplasmin and aSKa.Fibrinogen was determined according to the method of Clauss (10); plasminogen andα2-antiplasmin were determined according to the method of Friberger (11). A decrease in thefibrinogen level to <1.0 g/liter 1.5 hours after administration of anistreplase wasconsidered to reflect a systemic lytic state. Human aSKa was determined using a newlydeveloped enzyme-linked immunosorbent assay (12). The assay was performed similar tostandard enzyme-linked immunosorbent assay procedures using SK-coated microtiterplates.Plasma sample dilutions were incubated in duplicate for 2 hours, and then the nonboundmaterial was removed. The amount of bound aSKa was quantified spectrophotometricallyafter 1-hour incubation with a peroxydase conjugated to anti-human immunoglobulin G.Results were calculated using a standard dilution curve of reference plasma with anassigned value of 100 Arbitrary Units/milliliter (AU/ml). In addition to plasma samples ofpatients, blood samples were obtained from healthy hospital staff members as a referenceto determine aSKa levels in the population.

Results

After administration of anistreplase, levels of fibrinogen, plasminogen andα2-antiplasmin decreased by a variable extent. The patient group was dichotomized accordingto the fibrinogen level 1.5 hours after treatment, resulting in 18 patients beingcharacterized with a lytic state and 3 with a non-lytic state. Sixteen of 18 lytic patients(89%) had a patent infarct related artery and aSKa levels before treatment in the range of1 to 79 AU/ml (mean 42). The remaining 2 lytic patients had a non-patent coronary arteryand aSKa levels before treatment of 51 and 54 AU/ml, respectively. In the 3 patients witha non-lytic state, the infarct-related vessel was found to be non-patent; the plasma of these

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patients before treatment showed high aSKa levels (171, 343 and 450 AU/ml, respectively[mean 321 AU/ml]). Values before, and 1.5 and 48 hours after treatment are summarizedin Table I and depicted in Figure I. One and a half hours after treatment with anistreplase,a decrease in the aSKa level was found in both groups of patients. The mean and medianaSKa plasma levels in 63 healthy hospital staff members were 55 and 27 AU/ml, res-pectively, with a broad range of between 4 and 291 AU/ml. Thus, the aSKa levels in thesystemic lytic patients corresponded to the mean value in healthy subjects.

Discussion

Since the late 1950s, the plasma of patients with thromboembolic disease was assayedfor its content of SK inhibitory constituents (6); depending on the measured level, patientsreceived a varying dose of SK. The concept of SK inactivation by aSKa was revived in1984 when Lew et al. described clinical failure of SK in a patient with acute myocardialinfarction due to a high titer of aSKa (13). That patient had only a minimal decrease inserum plasminogen and no decrease in serum fibrinogen after therapy; thus, in contrast tomost other patients, neither a systemic lytic state nor local coronary thrombolysis occurred.Although the definition of a systemic lytic state varies among investigators, it generallycorresponds to a certain degree of fibrinogen decrease after administration of SK. It wasshown previously that a systemic lytic state is associated with successful therapy (7,8,14).In a Dutch study, it was observed that the degree of systemic lysis correlated inverselywith the preexisting aSKa level, but a causal relation between a high preexisting aSKalevel and unsuccessful thrombolytic therapy with SK or anistreplase, was not found (15).

In our study, we found a high aSKa level before treatment in 3 of 21 patients (14%)with acute myocardial infarction. These patients had relatively high levels of fibrinogen,plasminogen andα2-antiplasmin after administration of anistreplase, and coronaryangiography showed non-patency of the infarct related vessel. When aSKa levels were inthe normal range, a systemic lytic state was always obtained; a high level of aSKaapparently prevented the induction of such a state and subsequently, local thrombolysis.The binding of aSKa by the SK component of anistreplase was clearly shown by thetemporary reduction in their levels after its administration. This corresponds to the virtualdisappearance of aSKa due to treatment with SK, as was shown recently (16,17).Unsuccessful therapy may be avoided by the administration of a higher dosage of SK oranistreplase, or administering supplementary tissue-type plasminogen activator; assessmentof the aSKa level before treatment in patients with acute myocardial infarction enables theidentification of patients in whom this strategy should be considered. Several investigatorsused the radioimmunoassay of Moran et al. (18) to quantify the aSKa level. We developedan enzyme-linked immunosorbent assay for aSKa, which was originally performed in 3hours (12). Simplification and shortening of the assay has made it possible to perform thisquantification in 1 hour. A quicker procedure is currently being investigated so thatroutine clinical application would be more worth while. It is concluded that high aSKalevels may result in the failure of SK-containing thrombolytic agents, because a systemiclytic state is not induced; however, as was shown in 2 patientsTable I Patients dichotomised according to the fibrinogen level 1.5 hours after treatmentwith anistreplase. Mean values are expressed. Systemic lytic status represents fibrinogen

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<1.0 g/L, and non-lytic state is characterized by higher values. Corresponding values ofcoagulation parameters, aSKa level, and number of patients with a patent and a non-patentvessels are shown.

coagulation status lytic (n=18) non-lytic (n=3)

time (hours) 0 1.5 48 0 1.5 48

fibrinogen (g/l) 3.3 0.0 2.8 3.2 2.7 4.4

plasminogen (%) 100 15 58 102 59 76

α2-antiplasmin (%) 94 4 84 96 37 102

aSKa (AU/ml) 42 16 25 321 25 88

patency (n) - - 16 - - 0

non-patency (n) - - 2 - - 3

aSKa = anti-streptokinase antibody, 0 = before treatment, AU/ml = ArbitraryUnits/milliliter

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with normal aSKa levels in whom a systemic lytic state was induced, even this does notalways guarantee success.

References

1. The Gruppo Italiano per lo Studio della Streptochinasi nell’Infarto Miocardio(GISSI) Study Group. Effectiveness of intravenous thrombolytic treatment in acutemyocardial infarction. Lancet 1986;i:397-401.

2. Marder VJ, Sherry S. Thrombolytic therapy: current status (first of two parts). NEngl J Med 1988;318:1512-1520.

3. Hirsh J, O’Sullivan F, Martin M. Evaluation of a standard dosage schedule withstreptokinase. Blood 1970;35:341-349.

4. Aznar J, Delgado F, Estellés A. Streptokinase resistance test in patients withstreptococcal infection and/or high antistreptolysin titers. Scand J Haematol1976;16:141-143.

5. Jalihal S, Morris GK. Antistreptokinase titres after intravenous streptokinase. Lancet1990;i:184-185.

6. Fletcher AP, Alkjaersig N, Sherry S. The maintenance of a sustained thrombolyticstate in man. I. Induction and effects. J Clin Invest 1959;38:1096-1110.

7. Rothbard RL, Fitzpatrick PG, Francis CW, Caton DM, Hood WB, Marder VJ.Relationship of the lytic state to successful reperfusion with standard- and low-doseintracoronary streptokinase. Circulation 1985;71:562-570.

8. Brügemann J, van der Meer J, Takens LH, Hillege H, Lie KI. A systemic non-lyticstate and local thrombolytic failure of anistreplase (anisoylated plasminogenstreptokinase activator complex, APSAC) in acute myocardial infarction. Br Heart J1990;64:355-358.

9. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial: phase Ifindings. N Engl J Med 1985;312:932-936.

10. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung desFibrinogens. Acta Haematol 1957;17:237-246.

11. Friberger P, Knös M, Gustavsson S, Aurell L, Claeson G. Methods for determinationof plasmin, antiplasmin and plasminogen by means of substrate S2251. Haemostasis1978;7:138-145.

12. Bom VJJ, van der Schaaf W, van Wijk RT, Brügemann J, van der Meer J. Solid

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phase enzyme immuno assay of anti-streptokinase antibodies in human plasma.Thromb Haemost 1991;65:1268 (abstr. 2070).

13. Lew AS, Neer T, RodriguezL, Geft IL, Shah PK, Ganz W. Clinical failure ofstreptokinase due to unsuspected high titer of antistreptokinase antibody. J Am CollCardiol 1984;4:183-185.

14. Lew AS, Cercek B, Hod H, Shah PK, Ganz W. Usefulness of residual plasmafibrinogen after intravenous streptokinase for predicting delay or failure ofreperfusion in acute myocardial infarction. Am J Cardiol 1986;58:680-685.

15. Hoffmann JJML, Fears R, Bonnier JJRM, Standring R, Ferres H, De Swart BRM.Significance of antibodies to streptokinase in coronary thrombolytic therapy withstreptokinase or APSAC. Fibrinolysis 1988;2:203-210.

16. Lynch M, Littler WA, Pentecost BL, Stockley RA. Immunoglobin response tointravenous streptokinase in acute myocardial infarction. Br Heart J 1991;66:139-142.

17. Fears R, Ferres H, Glasgow E, Standring R, Hogg KJ, Gemmill JD, Burns JMA, RaeAP, Dunn FG, Hillis WS. Monitoring of streptokinase resistance titre in acutemyocardial infarction patients up to 30 months after giving streptokinase oranistreplase and related studies to measure specific antistreptokinase IgG. Br Heart J1992;68:167-170.

18. Moran DM, Standring R, Lavender EA, Harris GS. Assessment of anti-streptokinaseantibody levels in human sera using a microradioimmunoassay procedure. ThrombHaemost 1984;52:281-287.

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Anti-streptokinase antibodies are of clinical importance and can bemeasured quantitatively in 0.5 hr using a simple enzyme-linkedimmunosorbent assay

Johan Brügemann, Jan van der Meer (*), Victor J.J. Bom (*), Kong I. Lie

Department of Cardiology and Haematology (*), Division of Haemostasis, Thrombosis &Rheology, University Hospital Groningen, The Netherlands

Br Heart J 1994, in press (letter)

Sir, -Buchalter (1) and Patel et al. (2) stated that the relation between anti-streptokinaseantibodies and lytic efficacy of streptokinase or its derivate anistreplase in patients withacute myocardial infarction is unknown. Furthermore, the authors appeared not to beaware of a rapid assay for anti-streptokinase antibodies. However, these issues have beendescribed recently. Firstly, a strong relation between a systemic non-lytic state andangiographic non-patency of the infarct related vessel in patients with myocardialinfarction has been reported in this Journal (3) and elsewhere (4). Secondly, high levels ofanti-streptokinase antibodies before thrombolytic therapy with anistreplase have beenshown to lead to failure in achieving a systemic lytic state and subsequently, infarctrelated vessel patency (5). Finally, following the development of a simple enzyme-linkedimmunosorbent assay, the level of these antibodies can be measured in half an hour (6).As this method is quick and can be performed easily, its use might have beneficial clinicalconsequences since additional or alternative thrombolytic therapy, potentially leading toearly coronary artery reperfusion, could be applied.

It has been advocated that anti-streptokinase antibodies should be searched for routinelyin patients undergoing thrombolytic therapy in myocardial infarction (7). However, thecommonly used radio-immunoassay is time consuming and unpractical and therefore notsuitable to guide clinical strategies in critically ill patients. In order to identify patients inwhom delayed or failed reperfusion is likely to occur due to the presence of anti-streptokinase antibodies, some clinicians routinely measure serum fibrinogen immediatelyafter administration of streptokinase (8). Whereas this method is still useful, a quick andeasy measurement of anti-streptokinase antibodies can now be performed for screeningpurposes and/or to guide clinical treatment.

References

1. Buchalter MB. Are streptokinase antibodies clinically important. Br Heart J1993;70:101-2.

2. Patel S, Jalihal S, Dutka DP, Morris GK. Streptokinase neutralisation titers up to 866days after intravenous streptokinase for acute myocardial infarction. Br Heart J

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1993;70:119-21.

3. Brügemann J, van der Meer J, Takens LH, Hillege H, Lie KI. A systemic non-lyticstate and local thrombolytic failure of anistreplase (anisoylated plasminogenstreptokinase activator complex, APSAC) in acute myocardial infarction. Br Heart J1990;64:355-8.

4. Lew AS, Neer T, Rodriguez L, Geft IL, Shah PK, Ganz W. Clinical failure ofstreptokinase due to unsuspected high titer of antistreptokinase antibody. J Am CollCardiol 1984;4:183-5.

5. Brügemann J, van der Meer J, Bom VJJ, van der Schaaf W, de Graeff PA, Lie KI.Anti-streptokinase antibodies inhibit fibrinolytic effects of anistreplase in acutemyocardial infarction. Am J Cardiol 1993;72:462-4.

6. Bom VJJ, Brügemann J, van der Schaaf W, van Wijk RT, van der Meer J. Rapidenzym immunoassay of anti-streptokinase antibodies in human plasma. ClinicaChimica Acta 1993;218:121-9.

7. Sigwart U, Grbic M, Bachmann F. Measurement of antistreptokinase antibodies. JAm Coll Cardiol 1985;5:1500.

8. Lew AS, Laramee P, Ganz W. Reply to Sigwart et al. J Am Coll Cardiol1985;5:1500.

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Tissue-type Plasminogen Activator and Plasminogen ActivatorInhibitor in Patients With Acute Myocardial Infarction Treated WithStreptokinase

Johan Brügemann, Jan van der Meer (*), Erwin J.A.M. Göbel, Victor J.J. Bom (*) andKong I. Lie

Department of Cardiology and Division of Haemostasis, Thrombosis & Rheology (*),University Hospital Groningen, The Netherlands.

J Am Coll Cardiol 1992:19;179A (abstract)submitted

Abstract

We studied 46 patients with acute myocardial infarction (MI) who were treated withintravenous streptokinase (SK) and heparin≤4 hours after onset of chest pain. Coronaryangiography was performed about 24 (≤48) hours after therapy. Blood samples forassessment of fibrinolytic and coagulation parameters were collected on ice before therapyand on days 1 and 2 at 12.00 AM in order to eliminate circadian fluctuations in plasmalevels. Patency of the infarct related vessel was found in 36/46 (78%) patients. Baseline t-PA antigen and PAI activity levels were lower in patients with a non-patent compared tothose with a patent coronary artery: 6.0 vs 9.3 ng/ml, p=0.04, for t-PA antigen and 1.9 vs3.5 AU/ml, p=0.06, for PAI activity. On the first day after MI all patients showed anincreased level of t-PA antigen and PAI-activity. Thus, in patients with MI treated withSK, systemic endogenous t-PA levels are somewhat lower in patients with a non-patentvessel.

Introduction

The endogenous fibrinolytic system includes on the one hand the activators urokinase-type and tissue-type plasminogen activator (t-PA) and on the other the inhibitorsα2-antiplasmin and plasminogen activator inhibitor (PAI) (1). t-PA is localized in endothelialcells and is released into the blood in response to a variety of stimuli, such as thrombinformation, fibrin deposition, venous occlusion or ischemia. The level of t-PA activity inblood is regulated by fast-acting PAI liberated from plateletα-granules and/or fromendothelial cells. PAI is present in complex with t-PA or in a free active form. Total PAIcontent can be measured by the PAI antigen assay while PAI activity reflects functionalfree PAI (2,3). Plasma concentrations of both t-PA and PAI show a diurnal variation (4,5).Elevated PAI levels have been associated with non-successful therapy with recombinant t-PA (rt-PA) in acute myocardial infarction (MI) (6).

It is unknown if plasma levels of endogenous t-PA and PAI affect the outcome of

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treatment with streptokinase (SK) in patients with MI. These levels were determined in aprospective study in which coronary angiography was performed to determine success oftherapy.

Methods

Forty-six patients (age 28-78, mean 56 years) with electrocardiographically proven MIand symptoms less than 4 hours, were treated with 1.5 million Units (U) SK intravenously.SK was administered for 1 hour whereafter heparin was started in a dosage of 25,000U/24 hours for at least 1 day. Thereafter coronary angiography was performed todetermine patency of the infarct related vessel. Patients with TIMI-grades 0 or 1 wereconsidered to have occlusion of the infarct related vessel whilst those with grades 2 or 3to be patent (7).

Blood samples were collected on ice before therapy and at 12.00 AM on days 1 and 2in order to eliminate the circadian fluctuations of fibrinolytic parameters. Measurement oft-PA activity and t-PA antigen was performed on euglobulin fractions using the Coaset t-PA™ assay (KabiVitrum, Stockholm, Sweden) (8), and the Asserachrom t-PA™ enzyme-linked immunosorbent assay (ELISA) (Boehringer, Mannheim, Germany), respectively.PAI-1 antigen and PAI activity was performed by use of an ELISA purchased fromBiopool AB, Umea, Sweden, and by the chromogenic substrate method of the Berichrom-PAI™ assay (Behringwerke, Marburg, Germany) (9), respectively. Fibrinogen wasdetermined according to the method of Clauss (10). Plasminogen andα2-antiplasminassays were performed using a synthetic chromogenic substrate (11).

Statistical comparisons were calculated with the Student t-test.

Results

Repeated coronary angiography after 24 hours revealed patency of the infarct relatedartery in 36 of 46 patients. Following thrombolytic therapy, plasma levels of fibrinogen,plasminogen andα2-antiplasmin decreased in all patients irrespective of angiographicpatency at 24 hours (Table I). There was no significant difference in the extent of decreasein these coagulation parameters between patients showing patency or non-patency of theinfarct related vessel. Before therapy, t-PA antigen levels were significantly lower inpatients with a non-patent vessel (6.0 vs 9.3 ng/ml, p=0.04) and PAI activity levelsshowed this as a trend (1.9 vs 3.5 AU/ml, p=0.06). However, PAI levels did not exceedthe normal range. Within both groups a significant increase in t-PA antigen levels frombaseline to day 1 occurred (p<0.01). A similar increase of PAI antigen in both groups didnot reach statistical significance.

Discussion

During the first 2 to 3 days following treatment with SK because of MI, a temporarydecrease of fibrinogen, plasminogen andα2-antiplasmin in the plasma of patients has beenshown previously (12). Our results are in agreement with those findings. A significantincrease in the level of t-PA antigen and PAI activity, leading to a decrease of t-PA

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Table I Fibrinolytic parameters (mean±sd) in patients with acute myocardial infarctionbefore (0) and at day 1 (D1) and day 2 (D2) following treatment with streptokinase.

state patency (n=36) non-patency (n=10) refe-rencevalue

time 0 D1 D2 0 D1 D2

fibrinogeen (g/l) 3.5±1.0 1.2±0.7 3.1±1.2 3.6±0.9 1.4±1.6 4.3±1.9 1.7-3.5

plasminogeen (%) 104±16 51±15 70±17 103±13 47±15 68±13 70-130

α2-antiplasmin (%) 97±11 67±16 90±16 95±11 68±19 93±14 90-130

t-PA antigen (ng/ml) 9±7* 21±13+ 13±9 6±3* 19±7+ 10±6 3.8-4.5

t-PA activity (IU/ml) 1.5±2.1 0.9±0.4 1.0±0.5 0.9±0.4 1.1±0.4 1.0±0.3 1.0-1.8

PAI antigen (ng/ml) 17±17 24±18 13±8 10±11 18±11 9±4 27±16

PAI activity (AU/ml) 3.5±3.8 4.8±7.2 2.4±2.7 1.9±1.4 2.4±1.6 1.3±1.0 <5

[t-PA and PAI reference values were adapted from Angleton (ref. 5) and Sane/Loskutoff(ref. 17,18), respectively]

* : values between patency and non-patency, p=0.04+ : baseline versus day 1 values within the groups, p<0.01

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activity, has been observed following surgery. This has been called the postoperativefibrinolytic shutdown (13). The same investigators showed a rapid increase in PAI activityin patients with MI which illustrated the acute phase characteristics of PAI. In thePhysicians’ Health Study cohort, it was shown that study participants who developed MIhad a significantly higher mean t-PA antigen level (14). Hence, it was suggested that theendogenous t-PA antigen level increased as a consequence of coronary artery disease (15).We found pretreatment t-PA antigen levels in correspondence to the figures found inpatients with coronary artery disease (14,15). Interestingly, t-PA antigen levels in patientswith a non-patent vessel were lower compared to those patients with a patent vessel atcoronary angiography.

Elevated PAI activity levels have been reported in patients showing a non-patent infarctrelated coronary artery 3 days after treatment with rt-PA or urokinase (6,16). The TAMI-investigators, who studied thrice the number of patients compared to Barbash et al., foundonly a weak correlation between a low PAI activity level and patency of the infarct relatedvessel when determined 90 minutes after rt-PA therapy (17). The limited level ofagreement between these findings was suggested to relate to the different points in time atwhich angiography was performed. Average physiologic PAI antigen and PAI activitylevels amount approximately 27 ng/ml and <5 IU/ml, respectively (17,18). In contrast,pharmacologic rt-PA levels range from 1 to 3 µg/ml (order of multiplication >1000).Therefore it is likely that PAI activity levels are overwhelmed during rt-PA treatment.However, because of the short half-life of the systemic rt-PA concentration followingtherapy, it remains possible that PAI plays a role after termination of the infusion of thisdrug. A local high PAI concentration, due to release of this protein by thrombin-mediatedactivated platelets at the culprit lesion, could be responsible for blunting the initialthrombolytic success. Supporting evidence for this theory can be derived from recentexperiments in a rabbit model of thrombosis which showed that monoclonal antibodies toPAI enhanced rt-PA mediated thrombolysis (19).

Our study showed that in patients with MI treated with SK, systemic levels of t-PAantigen are lower in case of non-patency of the infarct related vessel. This may explain thehigher coronary patency rate after a combination of rt-PA and SK in patients with MIcompared to treatment with a single thrombolytic agent (20,21). Unfortunately, however,the hypothesis of superiority of a combination of thrombolytic agents versus thrombolyticmonotherapy was not confirmed in the recent GUSTO-trial (22). Systemic PAI levels donot appear of significant importance for the outcome of thrombolytic therapy with SK.Whether or not systemic levels of t-PA affect the patency rate of infarct related vesselsfollowing SK therapy in patients with MI needs further study.

References

1. Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis.Blood 1991;78:3114-24.

2. Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987;69:381-7.

3. Erickson LA, Ginsberg MH, Loskuthoff DJ. Detection and partial characterization of

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an inhibitor of plasminogen activator in human platelets. J Clin Invest 1984;74:1465-72.

4. Andreotti F, Davies GJ, Hackett DR, Khan MI, De Bart ACW, Aber VR, Maseri A,Kluft C. Major circadian fluctuations in fibrinolytic factors and possible relevance totime of onset of myocardial infarction, sudden cardiac death and stroke. Am JCardiol 1988:62;635-7.

5. Angleton P, Chandler WL, Schmer G. Diurnal variation of tissue plasminogenactivator and its rapid inhibitor (PAI-1). Circulation 1989;79:101-6.

6. Barbash GI, Hod H, Roth A, Miller HI, Rath S, Zahav YH, Modan M, Zivelin A,Laniado S, Seligsohn U. Correlation of baseline plasminogen activator inhibitoractivity with patency of the infarct artery after thrombolytic therapy in acutemyocardial infarction. Am J Cardiol 1989;64:1231-5.

7. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial: phase Ifindings. N Engl J Med 1985;312:932-6.

8. Nilsson K, Rosen S, Friberger P. A new kit for the determination of of tissuelasminogen activator and its inhibitor in blood. Fibrinolysis 1987;1:163-8.

9. Stief TW, Lenz P, Becker U, Heimburger N. Determination of plasminogen activatorinhibitor (PAI) capacity of human plasma in presence of oxidants: a novel principle.Thromb Res 1988;50:559-73.

10. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung desFibrinogens. Acta Haematol 1957;17:237-46.

11. Friberger P, Knös M, Gustavsson S, Aurell L, Claeson G. Methods for determinationof plasmin, antiplasmin and plasminogen by means of substrate S2251. Haemostasis1978;7:138-45.

12. Monassier J-P, Hanssen M. Hematological effects of anisoylated plasminogenstreptokinase activator complex and streptokinase in patients with acute myocardialinfarction, interim report of the IRS II study. Drugs 1987;33 (suppl.3):247-52.

13. Kluft C, Verheijen JH, Jie AFH, Rijken DC, Preston FE, Sue-Ling HM, Jespersen J,Aasen AO. The postoperative fibrinolytic shutdown: a rapidly reverting acute phasepattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma.Scand J Clin Lab Invest 1985;45:605-10.

14. Ridker PM, Vaughan DE, Stampfer MJ, Manson JE, Hennekens CH. Endogenoustissue-type plasminogen activator and risk of myocardial infarction. Lancet1993;341:1165-8.

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15. Jansson JH, Nilsson TK, Olofsson BO. Tissue plasminogen activator and other riskfactors as predictors of cardiovascular events in patients with severe angina pectoris.Eur Heart J 1991;12:157-61.

16. Sakamoto T, Yasue H, Ogawa H, Misumi I, Masuda T. Association of patency ofthe infarct-related coronary artery with plasma levels of plasminogen activatorinhibitor activity in acute myocardial infarction. Am J Cardiol 1992;70:271-6.

17. Sane D, Stump D, Topol E, Sigmon K, Kereiakes D, George B, Mantell S, Macy E,Collen D, Califf RM. Correlation between baseline plasminogen activator inhibitorlevels and clinical outcome during therapy with tissue-type plasminogen activator foracute myocardial infarction. Thromb Haemost 1991;65:275-9.

18. Loskutoff DJ, Sawdey M, Mimuro J. Type 1 plasminogen activator inhibitor. ProgHemost Thromb 1989;9:87-115.

19. Levi M, Biemond BJ, van Zonneveld A-J, ten Cate JW, Pannekoek H. Inhibition ofplasminogen activator inhibitor-1 activity results in promotion of endogenousthrombolysis and inhibition of thrombus extension in models of experimentalthrombosis. Circulation 1992;85:305-12.

20. Califf RM, Topol EJ, Stack RS, Ellis SG, et al. for the TAMI Study Group.Evaluation of combination thrombolytic therapy and timing of cardiac catheterizationin acute myocardial infarction. Results of thrombolysis and angioplasty inmyocardial infarction-Phase 5 randomized trial. Circulation 1991;83:1543-56.

21. Grines CL, Nissen SE, Booth DC, Gurley JC, Chelliah N, Wolf R, Blankenship J,Branco MC, Gurley JC, Bennett KA, DeMaria AN, and the Kentucky acutemyocardial infarction trial (KAMIT) group. A prospective, randomized trialcomparing combination half-dose tissue-type plasminogen activator and streptokinasewith full-dose tissue-type plasminogen activator. Circulation 1991;84:540-9.

22. The GUSTO Investigators. An international randomized trial comparing fourthrombolytic strategies for acute myocardial infarction. N Engl J Med 1993;329:673-82.

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Lipoprotein(a) Levels in Myocardial Infarction Treated withAnistreplase: No Prediction of Efficacy but Inverse Correlation withPlasminogen Activation in Non-Patency

Johan Brügemann, Jan van der Meer, Hans L. Hillege, Adrian J. van Boven, Jasper J. vanDoormaal (*), Pieter A. de Graeff (*), and Kong I. Lie

Departments of Cardiology and Internal Medicine (*), University Hospital Groningen, TheNetherlands

Thromb Res 1992;65:S98 (abstract)Eur Heart J 1992;13:27 (abstract)Int J Cardiol 1994, in press

Abstract

The aim of this study was to investigate whether failure of thrombolytic treatment mightbe due to inhibition of fibrinolysis by high lipoprotein(a) levels. Fifty-eight patients withacute myocardial infarction were treated intravenously within 4 hours after onset ofsymptoms with anistreplase (30 U) and heparin (30.000 IU/24 h). Blood samples formeasurement of coagulation parameters were taken before and 1.5 hours after treatment.Coronary angiography was performed after 48 hours. Levels of lipoprotein(a) weremeasured 6 months after discharge from hospital. The patency rate was 74% (43/58).Median lipoprotein(a) levels were not different between the patients with a patent andthose with a non-patent vessel (10 and 8 mg/dl, respectively), however, in patients with anon-patent infarct related vessel, a significant inverse correlation was found between thelipoprotein(a) level and the decrease of plasminogen in the first 1.5 hours after treatment.It is concluded that high lipoprotein(a) levels, although not directly associated with a pooroutcome of anistreplase therapy, might contribute to insufficient fibrinolysis in patientswith a non-patent infarct related vessel.

Introduction

Lipoprotein(a) is a genetically determined plasma lipoprotein variant firstly described byBerg in 1963 (1). The lipid composition is similar to that of low-density lipoprotein. Theprotein moiety consists of apolipoprotein B-100 linked covalently to apolipoprotein(a),which is a glycoprotein with a striking homology to plasminogen (2). The distribution oflipoprotein(a) plasma concentrations in the Caucasian population is independent of age anddiet and highly skewed for both men and women, with mean levels of 17.0 and 15.5mg/dl, respectively. Two-thirds of white subjects have lipoprotein(a) levels below 16mg/dl (3,4). The lipoprotein(a) level is a stable biological characteristic which is notinfluenced by life style or drug use (5). Lipoprotein(a) is an independent risk factor for

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coronary artery disease, largely unrelated to established endocrine-metabolic andanthropometric risk factors (6-8).

It has been shown in vitro that lipoprotein(a) competes with plasminogen for the samebinding sites on endothelial cells and fibrin (9-11). In addition, several authors haveclaimed interference of the streptokinase and tissue-type plasminogen activator mediatedactivation of plasminogen by lipoprotein(a) (12-15). Until now, no studies have reportedon the influence of lipoprotein(a) levels on the clinical efficacy of the streptokinasecontaining drug anistreplase.

The aim of the study was to assess, firstly, the relation between steady statelipoprotein(a) levels and patency of the infarct related vessel after thrombolytic treatment,and secondly, the influence of lipoprotein(a) levels on coagulation parameters, in particularplasminogen activation.

Materials and Methods

Fifty-eight consecutive caucasian patients (47 men, 11 women) with proven acutemyocardial infarction (mean age 57 years, range 34-71), who were treated with anintravenous bolus-injection of 30 U anistreplase (SmithKline Beecham), within 4 hoursafter the onset of chest pain, were studied. Adequate anticoagulation with intravenousheparin was started 4-6 hours after thrombolytic treatment and continued for 48 hours.Subsequently coronary angiography was performed to assess patency of the infarct relatedartery. Patency was documented according to the score used in the Thrombolysis inMyocardial Infarction (TIMI) trial (16). Patients with grade 0 or 1 were considered tohave occlusion of the infarct related vessel whilst those with grades 2 or 3 to be patent.

Venous blood samples were collected before treatment, and after 1.5 hours, and placedon ice in 1/10 volume 3.05% trisodium citrate for measurement of fibrinogen, plasminogenand α2-antiplasmin. Assays were performed immediately or plasma was stored at -80oCfor later analysis. Fibrinogen was determined according to the method of Clauss (17),plasminogen andα2-antiplasmin were determined according to the method of Friberger(18).

Approximately 6 months after myocardial infarction, blood samples for lipoprotein(a)assay were collected in tubes containing citrate. Specimens were stored at 2-8oC untilassessment. Levels of apo(a) were measured by a two-site apolipoprotein(a) immunoradiometric assay kit purchased from Pharmacia diagnostics AB, S-75182 Uppsala,Sweden. The lipoprotein(a) detection limit is 0.02 Unit apolipoprotein(a)/dl whereas 1 Unitof apolipoprotein(a) is equal to 0.7 mg lipoprotein(a). A concentration of up to 5 g/l ofplasminogen gives no measurable crossreactivity in the assay. Intra-assay variabilityamounts 6.3%.

Statistical analysis: Values of the normally distributed coagulation parametes areexpressed as mean (SD). Lipoprotein(a) levels, which distribution is skewed, are expressedas median (range). Levels of coagulation parameters within the two groups were comparedwith a paired t-test, for comparisons between the groups an unpaired t-test was used.Lipoprotein(a) values between the patients with a patent and a non-patent coronary arterywere compared with the Mann-Whitney U-test. For comparison of the normally distributedhemostatic parameters with the skewed distribution of lipoprotein(a), the Spearman rank

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correlation (R) coefficient was calculated. Because the logarithm of lipoprotein(a) withvalue zero does not exist, zero was replaced for 0.14 because that was the detection limitof the assay. P-values of less than 0.05 were considered significant.

Results

Fifty-eight patients were retrospectively classified into 2 groups. Forty-three patientsshowed a patent whilst 15 patients showed a non-patent infarct related artery (Table I).The patients were comparable for initial values of fibrinogen (normal range 1.7-3.5 g/l),plasminogen (normal range 70-130 %) andα2-antiplasmin (normal range 90-130 %). Inboth the patent and the non-patent groups there was a statistically significant decrease ofthese parameters between the baseline value and the value at 1.5 hours after anistreplasetherapy. At 1.5 hours, there were statistical significant differences between the 2 groupsfor each of these 3 parameters. Mean values of fibrinogen in the patent and the non-patentgroups were 0.0 versus 0.9 g/l, respectively; plasminogen 11 versus 31 %, respectively;and α2-antiplasmin 3 versus 19 %, respectively. Fifty-five of the 58 patients wereavailable for lipoprotein(a) assessment. The remaining 3 patients, who did show a patentvessel at angiography, were not sampled due to logistical reasons. The distribution oflipoprotein(a) levels among the subject population was highly skewed. Median and meanlipoprotein(a) levels were 14 and 27 mg/dl (range 0-184), respectively. Levels oflipoprotein(a) below the limit of detection, which is approximately 0.014 mg/dl, werefound in 14 subjects, 10 with a patent and 4 with a non-patent coronary artery,respectively. Unmeasurable lipoprotein(a) levels were assigned 0.14 before logtransformation was performed. Extremely high levels of lipoprotein(a) were found in 2patients with a patent coronary artery (116 and 184 mg/dl) and in 1 patient with a non-patent coronary artery (112 mg/dl). Lipoprotein(a) levels were not statistically differentbetween patients with successful and unsuccessful thrombolysis. For patients with a patentvessel the median level of lipoprotein(a) was 10 mg/dl (range 0-184) and for patients witha non-patent vessel, 8 mg/dl (range 0-112). The decrease (delta) of fibrinogen, plasmin-ogen andα2-antiplasmin levels from before to 1.5 hours after thrombolytic therapy wasnot significantly rank correlated with the lipoprotein(a) levels in patients with a patentvessel. In these patients, the coefficients of rank correlation (R) of lipoprotein(a) withdelta-fibrinogen, delta-plasminogen and delta-α2-antiplasmin were -0.09, 0.03 and 0.05,respectively. However, in patients with a non-patent vessel, the coefficients of rankcorrelation (R) were -0.38 (delta-fibrinogen), -0.57 (delta-plasminogen, p<0.05) and -0.52(delta-α2-antiplasmin, p<0.10). The log transformed lipoprotein(a) level and the associateddelta-plasminogen percentage in patients with a patent and non-patent vessel are depictedin Figure I.

Discussion

The relation of cardiovascular disease with elevated plasma lipoprotein(a) levels isconsidered to be due to antifibrinolytic effects of the plasminogen-like apolipoprotein(a) inlipoprotein(a) (9). Plasma lipoprotein(a) levels are steady during life, but in patients withmyocardial infarction, its level is raised, reflecting the acute phase characteristics of the

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Table I Coagulation parameters before and at 1.5 hours after thrombolytic therapy, andlipoprotein(a) levels at 6 months after myocardial infarction, in patients stratified topatency of the infarct related artery 48 hours after treatment.

patent (n=43) non-patent (n=15) p-value#

fibrinogen (g/l)beforeafter

3.1 (1.1)0.0 (0.2)*

3.0 (0.4)0.9 (1.2)*

NS<0.01

plasminogen (%)beforeafter

97 (19)11 (14)*

100 (13)31 (25)*

NS<0.01

α2-antiplasmin (%)beforeafter

93 (15)3 (5)*

92 (10)19 (21)*

NS<0.01

lipoprotein(a) (mg/dl) 10 (0-184) 8 (0-112) NS

* p<0.01 vs baseline (before therapy); # p-values for patent and non-patent comparisons.NS denotes not significant. Fibrinogen, plasminogen andα2-antiplasmin are expressed asmean (SD); lipoprotein(a) is expressed as median (range).

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compound (19). In case of thrombolytic treatment, plasma lipoprotein(a) levels temporarilydecrease about 50% (20). Following myocardial infarction, lipoprotein(a) levels return tobaseline after at least 3 months (21). In order to avoid these confounding effects onlipoprotein(a) levels, we measured the steady state level at 6 months after acutemyocardial infarction.

Anistreplase is a long-acting streptokinase derivate which use has been associated with avery low early reocclusion rate (22). Its efficacy appeared not to be influenced byconjunctive intravenous heparin therapy (23). Therefore, coronary angiography 48 hoursafter administration of anistreplase was considered appropriate.

Interference of lipoprotein(a) with the thrombolytic activity of streptokinase and tissue-type plasminogen activator has been demonstrated in vitro by several authors (12-15). Inclinical practice, however, no relation between the lipoprotein(a) level and outcome ofthrombolytic therapy has been shown (24-26). Our results are in agreement with thesefindings as the median lipoprotein(a) level in patients with a patent and a non-patentinfarct related vessel were about the same. That lipoprotein(a) levels are in some wayaffecting endogenous fibrinolysis in the clinical situation was recently shown in survivorsof myocardial infarction who did not receive thrombolytic therapy (27). In plasma samplesobtained 23 months after the event, significantly elevated lipoprotein(a) levels were foundin patients with a non-patent infarct related coronary artery compared to those whoshowed patency. It was suggested that, probably due to an elevated level of lipoprotein(a),intrinsic fibrinolysis was impaired resulting in non-patency of the infarct related vessel.Our finding of an inverse correlation between lipoprotein(a) levels and the decrease ofplasminogen levels in the first 1.5 hours following thrombolytic therapy in patients with anon-patent coronary vessel, supports this hypothesis of impaired fibrinolysis. Thus,lipoprotein(a) may interact with fibrinolysis following thrombolytic treatment, but only ina complex manner which does not necessarily lead to failure of therapy. Other factorswhich impair or inhibit coronary thrombolysis are probably morphology related and/or dueto plasma constituents such as anti-streptokinase antibodies and plasminogen activatorinhibitor (28). The relative contribution of lipoprotein(a) compared to these otherparameters needs to be determined.

Acknowledgment

We are grateful to Dr. GJM Boerma, Dept. of Clinical Chemistry, Academic HospitalDijkzigt Rotterdam-The Netherlands, for determining the lipoprotein(a) levels.

References

1. Berg K. A new serum type system in man: the Lp system. Acta Pathol MicrobiolScan 1963;59(suppl):369-382.

2. Eaton DL, Fless GM, Kohr WJ, McLean JW, Xu Q-T, Miller CG, Lawn RM, ScanuAM. Partial amino acid sequence of apolipoprotein(a) shows that it is homologous toplasminogen. Proc Natl Acad Sci USA 1987;84:3224-3228.

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3. Albers JA, Cabana VG, Warnick GR, Hazzard WR. Lp(a) lipoprotein: Relationshipto sinking pre-ß lipoprotein, hyperlipoproteinemia, and apolipoprotein B. Metabolism1975;24:1047-1054.

4. Guyton JR, Dahlen GH, Patsch W, Kautz JA, Gotto AM. Relationship of plasmalipoprotein lipoprotein(a) levels to race and to apolipoprotein B. Arteriosclerosis1985;5:265-272.

5. Scanu AM, Lawn RM, Berg K. Lipoprotein(a) and atherosclerosis. Ann Intern Med1991;115:209-218.

6. Kostner GM, Avogaro P, Cazzalato G, Marth E, Bittolo-Bon G, Quinci GB.Lipoprotein Lp(a) and the risk for myocardial infarction. Atherosclerosis 1981;38:51-61.

7. Dahlen GH, Guyton JR, Attar M, Farmer JA, Kautz JA, Gotto AM. Association oflevels of lipoprotein Lp(a), plasma lipids and other lipoproteins with coronary arterydisease documented by angiography. Circulation 1986;74:758-765.

8. Sundell IB, Nilsson TK, Hallmans G, Hellsten G, Dahlén GH. Interrelationshipsbetween plasma levels of plasminogen activator inhibitor, tissue plasminogenactivator, lipoprotein(a), and established cardiovascular risk factors in a NorthSwedish population. Atherosclerosis 1989;80:9-16.

9. Scott J. Thrombogenesis linked to atherogenesis at last? Nature 1989;341:22-23.

10. Harpel PC, Gordon BR, Parker TS. Plasmin catalyzes binding of lipoprotein(a) toimmobilized fibrinogen and fibrin. Proc Natl Acad Sci USA 1989;86;3847-3851.

11. Gonzalez-Gronow M, Edelberg JM, Pizzo SV. Further characterization of the cellularplasminogen binding site: evidence that plasminogen 2 and lipoprotein(a) competefor the same site. Biochemistry 1989;28:2374-2377.

12. Loscalzo J, Weinfeld M, Fless GM, Scanu AM. Lipoprotein(a), fibrin binding, andplasminogen activation. Arteriosclerosis 1990;10:240-245.

13. Karàdi I, Kostner GM, Gries A, Nimpf J, Romics L, Malle E. Lipoprotein(a) andplasminogen are immunochemically related. Biochim Biophys Acta 1988;960:91-97.

14. Edelberg JM, Gonzalez-Gronow M, Pizzo SV. Lipoprotein a inhibits streptokinase-mediated activation of human plasminogen. Biochemistry 1989;28:2370-2374.

15. Rouy D, Grailhe P, Nigon F, Chapman J, Anglés-Cano E. Lipoprotein(a) impairsgeneration of plasmin by fibrin-bound tissue-type plasminogen activator, in vitrostudies in a plasma milieu. Arteriosclerosis and Thrombosis 1991;11:629-638.

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16. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial: phase Ifindings. N Engl J Med 1985;312:932-936.

17. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung desFibrinogens. Acta Haematol 1957;17:237-246.

18. Friberger P, Knös M, Gustavsson S, Aurell L, Claeson G. Methods for determinationof plasmin, antiplasmin and plasminogen by means of substrate S2251. Haemostasis1978;7:138-145.

19. Maeda S, Abe A, Seishima M, Makino K, Noma A, Kawade M. Transient changesof serum lipoprotein(a) as an acute phase protein. Atherosclerosis 1989;78:145-150.

20. Hegele RA, Freeman MR, Langer A, Connelly PW, Armstrong PW. Acute reductionof lipoprotein(a) by tissue-type plasminogen activator. Circulation 1992;85:2034-2038.

21. Stubbs P, O’Connor B, Noshirwani K, Seed M. Changes in lipoprotein(a) [Lp(a)]concentration in the peri- and post myocardial infarction period. Eur Heart J1991;12:139 (abstr).

22. Relik-van Wely L, Visser RF, van der Pol JMJ, et al. Angiographically assessedcoronary arterial patency and reocclusion in patients with acute myocardial infarctiontreated with anistreplase: Results of the anistreplase reocclusion multicenter study(ARMS). Am J Cardiol 1991;68:296-300.

23. O’Connor CM, Meese R, Carney R, et al. for the DUCCS Group. A randomized trialof intravenous heparin in conjunction with anistreplase in acute myocardialinfarction: The Duke university clinical cardiology study (DUCCS) 1. J Am CollCardiol 1994;23:11-18.

24. Armstrong VW, Neubauer C, Schütz E, Tebbe U. Lack of association between raisedserum Lp(a) concentration and unsuccessful thrombolysis after acute myocardialinfarction. Lancet 1990;ii:1077.

25. Hodenberg E von, Kreuzer J, Hautmann M, Nordt T, Kübler W, Bode C. Effects oflipoprotein(a) on success rate of thrombolytic therapy in acute myocardial infarction.Am J Cardiol 1991;67:1349-1353.

26. Qiu S, Théroux P, Genest J, Solymoss BC, Robitaille D, Marcil M. Lipoprotein(a)blood levels in unstable angina pectoris, acute myocardial infarction, and afterthrombolytic therapy. Am J Cardiol 1991;67:1175-1179.

27. Moliterno DJ, Lange RA, Meidell RS, et al. Relation of plasma lipoprotein(a) toinfarct artery patency in survivors of myocardial infarction. Circulation 1993;88:935-

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940.

28. Brügemann J, van der Meer J, de Graeff PA, Lie KI. Anti-streptokinase antibodiesinhibit fibrinolytic effects of anistreplase in acute myocardial infarction. Am JCardiol 1993;72:462-464.

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Reocclusion Three Months after Successful Thrombolytic Treatment ofAcute Myocardial Infarction with Anisoylated PlasminogenStreptokinase Activator Complex (APSAC)

Bert H. Takens, Johan Brügemann, Jan van der Meer (*), Peter den Heijer, Kong I. Lie

Departments of Cardiology and Haematology (*), University of Groningen, Groningen,The Netherlands

Eur Heart J 1988;9:10 (abstract).Am J Cardiol 1990;65:1422-4.

Abstract

Thirty consecutive patients with acute myocardial infarction (AMI) were treated withanisoylated plasminogen streptokinase activator complex (APSAC) within 4 hours afteronset of symptoms. After 1½ and 48 hours, patency of the infarct related vessel and thequantitative degree of residual diameter stenosis were studied by selective coronaryangiography. Ventriculograms were made to determine the global left ventricular ejectionfraction. Patients showing patency at 48 hours were reevaluated angiographically after 3months. At 1.5 and 48 hours after APSAC administration patent vessels weredemonstrated in 65 and 69% of patients respectively. Mean residual stenosis decreasedsignificantly from 56±11% at 1.5 hours to 46±13% at 48 hours (p<0.01). Patients notresponding to thrombolytic therapy showed significant deterioration of the left ventricularfunction during the first 48 hours after AMI. Side effects were minor and mainlyassociated with invasive procedures. Despite adequate oral anticoagulation,angiographically documented reocclusion at 3 months amounted 28%. Reocclusion,however, was neither associated with clinically documented reinfarction, nor with adecrease in the left ventricular ejection fraction. Our study shows that APSAC is aneffective thrombolytic agent in AMI but that late reocclusion may occur. Oralanticoagulants appear to be less effective in the prevention of reocclusion in the treatmentregimen after thrombolysis.

Introduction

Coronary thrombosis is the commonly accepted cause of acute myocardial infarction(AMI) (1). Intravenous thrombolytic treatment with streptokinase is well established aseffective therapy for improving survival (2,3) and preserving left ventricular function (4,5).During the last decade, new intravenous thrombolytic drugs such as recombinant tissue-type plasminogen activator and anisoylated plasminogen streptokinase activator complex(APSAC) have emerged. Each of these drugs is effective (6,7). Only APSAC can be givenas a slow bolus injection. Because of its long plasma half-life, APSAC exhibits prolonged

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action, a property that has been related to a low incidence of early reocclusion (8).However, little is known about reocclusion in the subsequent period. We therefore studied3-months reocclusion rate in patients with AMI treated with APSAC.

Methods

Patients: Patients who were 70 years or younger were eligible if they had the onset ofsymptoms suggestive of AMI within the previous 4 hours. In addition,electrocardiographic ST-segment elevation of at least 0.1 mV in≥1 of the standard leadsor at least 0.2 mV in≥2 of the precordial leads in a 12-lead registration was required.Symptoms had to be unresponsive to sublingual glyceryl trinitrate. Patients were excludedif contraindications for thrombolytic treatment were present.

Study Protocol: Treatment was started with nitroglycerin and lignocaine infusionfollowed by a bolusinjection of 100 mg prednisolone. Subsequently, 30 U of APSAC(Eminase™-Beecham) was given intravenously in 4-5 minutes. Intravenous therapy withheparin was supplied 4-6 hours after APSAC in a dosage of 30.000 IU in 24 hours andwas continued until an adequate level of anticoagulation had been achieved withacenocoumarol which was started after 48-72 hours. Oral anticoagulation was continuedfor at least 3 months. Antiplatelet therapy was not part of the treatment regimen. Heartcatheterisation by means of the Judkins technique was performed 1.5 (range 1 to 3) and 48hours (range 36 to 60) after the administration of APSAC in all patients. Patency andpercentage of residual diameter stenosis of the infarct related vessel were assessed bycoronary angiography. Patency was documented according to the score used in theThrombolysis In Myocardial Infarction Study (6). Patients with grade 0 or 1 perfusionwere classified as occluded, those with grades 2 and 3 as patent. Quantification ofcoronary residual stenosis was realized using the computer-assisted cardiovascularangiography analysis system (9,10). Global left ventricular ejection fraction was assessedby quantitative analysis of the left ventricular angiograms in the right anterior obliqueprojection. In patients with a patent vessel at 48 hours, heart catheterisation was repeatedat 3 months. Twelve lead electrocardiograms were made and blood samples were collectedfor creatine kinase level determination before treatment and serially after theadministration of APSAC.

Statistical analysis: Results for continuous variables are presented as mean ± standarddeviation. The Student’s unpaired t-test was used to assess differences between patientswith patent and non-patent vessels. Comparisons within each group were made with thepaired Student t-test. A p-value <0.05 was considered significant.

Results

Thirty consecutive patients (23 men, 7 women) ranging in age from 33 to 70 years(mean 55) with AMI (15 anterior, 15 inferior) participated in the study. The mean overalldelay between onset of symptoms and thrombolytic treatment was 2.6±0.9 hours. Onepatient died in cardiogenic shock within 1.5 hours after treatment with APSAC. Threepatients were not subjected to angiography at 1.5 hours for logistic reasons. Seventeeninfarct related coronary vessels in 26 patients at 1.5 hours were patent (65%). Forty-eight

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hours after the administration of APSAC, patency was found in 20 of 29 patients (69%),including the aforementioned 3 patients (Table I). No reocclusion occurred between 1.5and 48 hours. There was no significant relation between outcome of thrombolytic therapyand duration of symptoms. One patient, with a patent right coronary artery, underwentemergency bypass surgery soon after the coronary angiography at 48 hours because ofsymptoms of anterior myocardial ischemia. Another patient with a patent left anteriordescending artery at 48 hours died suddenly at home within 3 months. Two patients with anon-patent infarct related vessel died between the second day and the end of the 3 monthsfollow up period. They both had cardiac failure.

Angiography after 3 months was repeated in 18 patients who had a patent vessel after48 hours. The patient who underwent bypass surgery was excluded. Patency wasdemonstrated in 13 (72%). Reocclusion was found in 5 patients (1 right coronary artery, 4left anterior descending coronary artery). In none of these 5 patients did a documentedreinfarction occur.

The mean percentage diameter stenosis in patent infarct related arteries decreasedsignificantly between 1.5 and 48 hours from 56±11 to 46±13% (p<0.01). No furthersignificant improvement was found after 3 months when 40±14% residual coronarydiameter stenosis was demonstrated in the 13 patent vessels.

Left ventricular ejection fraction differed significantly between patients with a patentand a non-patent vessel at 1.5 hours (67±14 and 49±22%; p<0.05) and 48 hours (59±16and 41±20%; p<0.02) after treatment with APSAC. Patients with an occluded vessel alsoshowed a significant decrease between 1.5 and 48 hours after treatment (Table II). Thesepatients with an occluded vessel were not reassessed at 3 months. No significant decreaseof the left ventricular ejection fraction was found in 5 patients demonstrating reocclusionat 3 months; values at 48 hours and 3 months were 53±14 and 57±14%, respectively.

Neither creatine kinase levels, nor creatine kinase-MB levels were significantly differentin patients with a patent or an occluded vessel. However, the time to peak level of eachwas significantly shorter in patients with a patent vessel.

Mild bleeding, mainly at puncture sites, occurred in 9 patients. Two patients received ablood transfusion for moderate bleeding. A purpuric rash developed in 1 patient at the 5thday after treatment. A skin biopsy showed this to be due to a non specific vasculitis.

Discussion

In our study 3 patients did not undergo angiography at 1.5 hours. However, thesepatients showed rapid relief of chest pain, quick improvement of electrocardiographicrepolarization changes and an early peak of the creatine kinase-MB. These findings havebeen shown to be strongly predictive for patency of an infarct related vessel (11).Therefore, it seems likely that in case of angiography of all patients, the patency results at1.5 and 48 hours after APSAC would have been similar. Forty-eight hours after treatmentwith APSAC 69% of the patients in our study had a patent infarct related vessel. Thisfigure corresponds to the 60 to 70% of thrombolytic success reported in a review byMarder and Sherry (8). These authors estimated the incidence of reocclusion aftertreatment with APSAC to be 10%. However, this low percentage was derived from a

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Table I Patency rates in course of 3 months

coronary artery time of angiography

No. 1.5 hours 48 hours 3 months

LAD 14 8/11 11/14 6/10

RCA 11 6/11 6/11 4/5

CX 4 3/4 3/4 3/3

patency (%) 17/26* (65) 20/29 (69) 13/18# (72)

LAD: Left anterior descending, RCA: Right coronary artery, CX: Left circumflex

*: no angiography was obtained from 3 living patients because of logistic reasons, 1patient died <1.5 hours

#: only patients with a patent infarct related vessel at 48 hours were examined 3 monthsafter treatment, 1 patient underwent coronary artery bypass surgery and 1 patient died

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Table I Global left ventricular ejection fraction (%)

time

result of coronary angiography

patent occluded p-value#

1.5 hours 67±14 49±22 <0.05

48 hours 59±16 41±20* <0.02

3 months 62±15

# = between group comparisons, * = p <0.01 vs baseline

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limited number of patients in whom angiography was repeated 24 hours after treatment(12,13). We found no reocclusion between 1.5 and 48 hours. A similar result was obtainedin a study of 99 patients with AMI also treated with APSAC. In these patientsangiography was performed at 1.5 and 24 hours after treatment (14). The low rate of earlyreocclusion after treatment with APSAC is probably due to a long lasting extensivefibrinogenolysis. In such a hypocoagulable state a more complete lysis of the residualthrombus can be elicited, as was also demonstrated in our study. Thorough lysis was oneof the factors postulated to be preventive for reocclusion (15).

Only 1 study reported reocclusion data 4 weeks after therapy with APSAC (16). Theseinvestigators found reocclusion in 5 of 37 (14%) of the infarct related vessels. However,data of some of the patients were lacking, which may partly explain the low percentage. Inour study angiographic reocclusion at 3 months was found in 5 of 18 patients (28%). Thisrelatively high rate, despite adequate oral anticoagulation, is disappointing. Recently it wasshown in animal experiments that platelets have a vital role in reocclusion afterthrombolysis (17). Thus, instead of oral anticoagulants, platelet inhibitors might have beena more appropriate approach to prevent reocclusion. Since the Second International Studyof Infarct Survival, the issue of aspirin in the treatment of AMI is also acknowledged (3).

It should be emphasized that none of the patients with reocclusion at 3 months haddocumented reinfarction or decrease of the left ventricular ejection fraction. Obviously,reocclusion after initial successful therapy with APSAC appears not to be as deleterious ascomplete failure of treatment. This could be explained by the restriction of the cardiacarea at risk in the acute phase through reperfusion and subsequent adaptation of theventricle by collateral blood supply.

The significant decrease in coronary residual diameter stenosis in the first 48 hours aftersuccessful treatment corresponds with the findings in studies with streptokinase (18) orrecombinant tissue-type plasminogen activator (19). Their thrombolytic effect persists for alonger period than might be expected from their plasma half-life.

Thrombolytic therapy of AMI with APSAC has been shown to preserve the leftventricular ejection fraction compared to conventional heparin therapy (20). In our studythis only occurred in patients responding to APSAC, whereas a significant decrease of theleft ventricular function during the first 48 hours was seen in patients resistant tothrombolytic treatment. Thus, it appears highly desirable to achieve reperfusion of anoccluded coronary vessel in the acute phase of AMI in order to improve myocardialfunction.

Bleeding complications were infrequent and considered not serious in most patients.One case of vasculitis, which resolved quickly and spontaneously, was seen. Vasculitisresults from APSAC’s antigenic properties and has been described earlier in a limitednumber of patients (21).

In this study APSAC proved to be effective and safe. Early reocclusion was absent butreocclusion after 3 months was found in 28% of the patients. Failure to achieve patency,however, was more deleterious than failure to maintain patency. To avoid reocclusion,therapy with aspirin might be more appropriate instead of oral anticoagulants. This shouldbe confirmed in a controlled trial in patients with AMI after thrombolytic therapy.

Acknowledgment

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We are grateful to Beecham Research Laboratories Amstelveen, The Netherlands forsupporting this study.

References

1. DeWood MA, Spores J, Notske R, Mouser LT, Burroughs R, Golden MS, Lang HT.Prevalence of total coronary occlusion during the early hours of transmuralmyocardial infarction. N Engl J Med 1980;303:897-902.

2. Gruppo Italiano per lo Studio della Streptochinasi nell’Infarto Miocardico (GISSI).Long-term effects of intravenous thrombolysis in acute myocardial infarction: finalreport of the GISSI Study. Lancet 1987;2:871-874.

3. ISIS-2 Collaborative Group. Randomized trial of intravenous streptokinase, oralaspirin, both, or neither among 17.187 cases of suspected acute myocardialinfarction: ISIS-2. Lancet 1988; 2:349-360.

4. The ISAM Study group. A prospective trial of intravenous streptokinase in acutemyocardial infarction (ISAM): mortality, morbidity, and infarct size at 21 days. NEngl J Med 1986;314: 1465-1471.

5. White HD, Norris RM, Brown MA, Takayama M, Maslowski A, Bass NM,Ormiston JA, Whitlock T. Effect of intravenous streptokinase on left ventricularfunction and early survival after acute myocardial infarction. N Engl J Med1987;317:850-855.

6. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial: phase Ifindings. N Engl J Med 1985;312:932-936.

7. AIMS Trial Study Group. Effect of intravenous APSAC on mortality after acutemyocardial infarction: preliminary report of a placebo controlled clinical trial. Lancet1988;1:545-549.

8. Marder VJ, Sherry S. Thrombollytic therapy: current status (first of two parts). NEngl J Med 1988;318:1512-1520.

9. Reiber JHC, Serruys PW, Kooijman CJ, Wijns W, Slager CJ, Gerbrands JJ,Schuurbiers JCH, den Boer A, Hugenholtz PG. Assessment of short-, medium-, andlong-term variations in arterial dimensions from computer-assisted quantitation ofcoronary cineangiograms. Circulation 1985;71:280-288.

10. Zijlstra F, van Ommeren J, Reiber JHC, Serruys PW. Does the quantitativeassessment of coronary artery dimensions predict the physiologic significance of acoronary stenosis? Circulation 1987;75:1154-1161.

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11. Hackworthy RA, Sorensen SG, Fitzpatrick PG, Barry WH, Menlove RL, RothbardRL, Anderson JL. Effect of reperfusion on electrocardiographic and enzymaticinfarct size: Results of a randomized multicenter study of intravenous anisoylatedstreptokinase activator complex (APSAC) versus intracoronary streptokinase in acutemyocardial infarction. Am Heart J 1988;116:903-914.

12. Marder VJ, Rothbard RL, Fitzpatrick PG, Francis CW. Rapid lysis of coronary arterythrombi with anisoylated plasminogen streptokinase activator complex: treatment bybolus intravenous injection. Ann Intern Med 1986;104:304-310.

13. Monk JP, Heel RC. Anisoylated plasminogen streptokinase activator complex(APSAC): a review of its mechanism of action, clinical pharmacology andtherapeutic use in acute myocardial infarction. Drugs 1987;34:25-49.

14. Relik-van Wely L, van der Pol JMJ, Visser RF, Aarts FJEM, Drost H, Vet AJTM,Klomps HC, Ekelen WAAJ, van den Berg F. A preliminary report on theangiographic assessed patency and reocclusion in patients treated with APSAC foracute myocardial infarction. A Dutch Multicentre Study (abstr). Eur Heart J1988;9:8.

15. Fuster V, Badimon L, Cohen M, Ambrose JA, Badimon JJ, Chesebro J. Insights intothe pathogenesis of acute ischemic syndromes. Circulation 1988;77:1213-1220.

16. Kasper W, Meinertz T, Wollschläger H, Bonzel T, Wolff P, Drexler H, Hofmann T,Zeiher A, Just H. Coronary thrombolysis during acute myocardial infarction byintravenous BRL 26921, a new anisoylated plasminogen-streptokinase activatorcomplex. Am J Cardiol 1986;58:418-421.

17. Yasuda T, Gold HK, Fallon JT, Leinbach RC, Guerrero JL, Scudder LE, Kanke M,Shealy D, Ross MJ, Collen D, Coller BS. Monoclonal antibody against the plateletglycoprotein (GP) IIb/IIIa receptor prevents coronary artery reocclusion followingreperfusion with recombinant tissue-type plasminogen activator in dogs. J Clin Invest1988;81:1284-1291.

18. Hackett D, Davies G, Maseri A. Pre-existing coronary stenosis in patients with firstmyocardial infarction are not necessarily severe. Eur Heart J 1988;9:1317-1323.

19. Schmidt WG, Uebis R, v Essen R, Effert S, Erbel R, Meyer J, Rutsch W, Schartl M,Schmutzler H, on behalf of the European Co-operative Study Group for RecombinantTissue-Type Plasminogen Activator. Residual coronary stenosis after thrombolysiswith rt-PA or streptokinase; acute results and 3 weeks follow-up. Eur Heart J1987;8:1182-1188.

20. Bassand JP, Machecourt J, Cassagnes J, Anguenot T, Lusson R, Borel E, PeycelonP, Wolf E, Ducellier D, for the APSIM Study Investigators. Multicenter trial of

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appendix 8

intravenous anisoylated plasminogen streptokinase activator complex (APSAC) inacute myocardial infarction: effects on infarct size and left ventricular function. JAm Coll Cardiol 1989;13:988-997.

21. Bucknall C, Darley C, Flax J, Vincent R, Chamberlain D. Vasculitis complicatingtreatment with intravenous anisoylated plasminogen streptokinase activator complexin acute myocardial infarction. Br Heart J 1988;59:9-11.

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Samenvatting

Nederlandse samenvatting

Na een plotselinge afsluiting van een kransslagader door een bloedstolsel krijgt hetstroomgebied van dat vat geen bloed meer aangevoerd. Bij blijvende belemmering zalvervolgens een deel van de hartspier afsterven hetgeen een hartinfarct genoemd wordt.Afhankelijk van de plaats van afsluiting zal er veel of weinig schade optreden. Somsontstaat acuut pompfalen al of niet met de fatale ritmestoornis kamerfibrilleren.

Twee maatregelen hebben geleid tot een afname aan de sterfte aan het hartinfarct: hetopzetten van hartbewakingsafdelingen en het invoeren van de electrische defibrillatie. Eenverdere vermindering van niet alleen de sterfte maar ook van de ziekteverschijnselen, werdverkregen door toepassing van bloedstolsel-oplossende (thrombolytische) therapie. Dezebehandeling werd in 1933 voor de eerste keer beschreven en klinische experimentenwerden al in 1940 in de Verenigde Staten verricht. Het heeft nog tot 1986 geduurd, hetjaar waarin de Italiaanse GISSI-1 studie gepubliceerd werd, tot deze therapie algemeengeaccepteerd werd. Kort daarna werd de Britse ISIS-2 studie gepubliceerd die leidde tot dehuidige standaard-therapie bij het dreigend hartinfarct, n.l. het thrombolyticumstreptokinase, aangevuld met aspirine (en heparine). Deze behandeling is echter niet altijdsuccesvol. Falen van thrombolytische therapie kan berusten op het niet opengaan òf hetsnel weer dicht gaan van het geopende bloedvat. Het toedienen van thrombolytica kan ookgepaard gaan met bloedingsbijwerkingen die een gunstig effect kunnen overschaduwen.

In dit proefschrift worden factoren beschreven die de effectiviteit van dethrombolytische therapie beïnvloeden. In de eerste plaats is dit de tijd die verloopt tussenhet krijgen van klachten en de aanvang van de behandeling. Hoe langer (een deel van) dehartspier verstoken is van zuurstof, hoe meer cellen afsterven. Thuisbehandeling leek hetmeest geschikt om dit tijdverlies zo veel mogelijk te verkleinen. Dit was de reden om inGroningen hier een onderzoek naar te doen. Door logistieke problemen bij het vaststellenvan de diagnose hartinfarct trad er echter een tijdverlies van gemiddeld 24 minuten op,terwijl de transporttijd naar het ziekenhuis slechts gemiddeld 9 minuten bedroeg. Derhalvekwamen wij tot de conclusie dat in een stad als Groningen, een patiënt die verdacht wordtvan een dreigend hartinfarct, beter onverwijld naar het ziekenhuis getransporteerd kanworden (appendix 1). Na aankomst in het ziekenhuis dient tijdverlies door protocollairebehandeling zoveel mogelijk voorkomen te worden.

Al in de 50er jaren werd gesuggereerd dat bij een thrombolytische behandeling afbraakvan fibrinogeen in het bloed vooraf gaat aan afbraak van fibrine in het stolsel. Mennoemde dit een systemisch lytische status. Wij toonden inappendix 2 aan dat het nietontstaan van zo’n systemisch lytische status gekoppeld is aan een niet succesvollethrombolytische therapie. Bij afbeelding van de kransslagader tijdens hartcatheterisatiewordt dan een niet-doorgankelijk vat gezien. Het meten van het fibrinogeen in het bloedna infusie van anistreplase of streptokinase is derhalve zinvol om aanvullende therapie tegeven voordat onomkeerbaar weefselverlies opgetreden is.

Al eerder werd aangenomen dat antistoffen gericht tegen het lichaamsvreemde enzymstreptokinase, de werkzaamheid hiervan teniet zouden kunnen doen. Anti-streptokinaseantistoffen (aSKa) worden gevonden bij gezonde mensen na doormaken van (keel)infectiesmet streptococcen maar ook bij patiënten die al eerder behandeld zijn met streptokinase of

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anistreplase. Bepaling van aSKa was al langer mogelijk maar nam veel tijd in beslag.Hierdoor kon de uitslag van de test niet gebruikt worden om de therapie bij te sturen. Eendergelijke eenvoudige en snelle test werd op het stollingslaboratorium van het AZGontwikkeld (appendix 3). De klinische bruikbaarheid van deze test moet overigens nogwel verder in de praktijk bevestigd worden. De hypothese dat een hoge aSKa spiegel inhet bloedplasma vooraf aan een behandeling met streptokinase het optreden van eensystemisch lytische status en een lokaal thrombolytisch effect verhindert, werd bevestigdin appendix 4. Bij patiënten met aanwijzingen voor een hartinfarct die eerder metstreptokinase of anistreplase behandeld zijn, moet daarom recombinant weefselplasminogeen activator (rt-PA) overwogen worden. Bij patiënten met een dreigendhartinfarct zou de nieuwe test standaard uitgevoerd kunnen worden om hoge spiegels vanaSKa in het bloed snel te kunnen vaststellen. De uitslag van de test zou dan richtingkunnen geven aan de verdere behandeling (appendix 5).

Plasminogeen activator inhibitor (PAI) is een eiwit dat een rol speelt in de bloedstollingvan de mens. Een overmaat van deze remmer zou een behandeling met rt-PA kunnenverstoren. Wij onderzochten of PAI ook betekenis had als er streptokinase gegeven werd.Dit was niet het geval (appendix 6). De gemeten t-PA spiegels gaven wel aanleiding totspeculatie t.a.v. de door verschillende onderzoeksgroepen gemelde mogelijk groterewerkzaamheid van een combinatie van streptokinase plus rt-PA bij het hartinfarct.

Bloedplasma bevat naast eiwitten (proteinen) ook vetten (lipiden). Met eiwit omhuldevetpartikels in het bloed worden daarom lipoproteinen genoemd. Een betrekkelijk kortbekend lipoproteine is het lipoproteine (a) [Lp(a)]. Dit Lp(a) vertoont grote gelijkenis metplasminogeen wat de voorloper is van het stolseloplossende plasmine. Nadat anderen inhet laboratorium aantoonden dat hoge Lp(a) spiegels een nadelig effect hadden opplasminogeen activatie, suggereert ons onderzoek dat bij patienten die na een poging totthrombolyse een dicht kransvat vertonen dit mogelijk mede te wijten zou kunnen zijn aaneen verminderde plasminogeen activatie door Lp(a) (appendix 7).

Tenslotte werd met herhaald kransslagaderonderzoek bekeken of door de behandelinggeopende vaten ook open bleven in verloop van de tijd. Daartoe werd eenhartcatheterisatie verricht 1.5 uur, 48 uur en 3 maanden na een thrombolytische therapie.Na toepassing van anistreplase bleken kransslagaders die 1.5 uur na behandeling openwaren dit ook te zijn 48 uur na behandeling. Kortom er was geen sprake van vroegereocclusie. Echter, ondanks adequate antistollingstherapie bleek na 3 maanden dat circa30% van de aanvankelijk open vaten, zonder duidelijke symptomen die passen bij eenhartinfarct, weer dicht (appendix 8). Met andere woorden er was een aanzienlijke "stille"reocclusie. Mogelijk is er dan inmiddels sprake van een zijdelingse (collaterale) circulatiedie symptomen voorkomt.

Samengevat kan gezegd worden dat de appendices 1 t/m 5 direct van praktischebetekenis kunnen zijn bij de thrombolytische behandeling van patiënten met een dreigendhartinfarct. Appendices 6 t/m 8 lijken vooralsnog meer van theoretisch belang maarkunnen richting geven aan verder onderzoek. De relatieve bijdrage van anti-streptokinaseantistoffen, plasminogeen activator inhibitor en lipoproteine(a) ten opzichte van elkaar zoubeter gekwantificeerd moeten worden. Nieuwe stollings- en/of bloedplaatjesremmendestoffen zoals beschreven in hoofdstuk IV zullen waarschijnlijk op korte termijn deeffectiviteit van een thrombolytische behandeling nog verder vergroten.

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Dankwoord

Dankwoord

Een proefschrift schrijf je niet alleen. Een aantal mensen wil ik noemen die eensubstantieële bijdrage aan dit werk geleverd hebben.

Professor Lie vroeg mij in 1988 te participeren in het Groningse thrombolyseonderzoek. Tevens liet hij mij toe tot de cardiologie opleiding. Graag wil ik hem als eerstebedanken voor het geschonken vertrouwen.

De kiem voor het beschreven onderzoek is gelegd door Bert Takens. Samen met hemwerd de reocclusie-studie en de Groninger Ambulance Studie uitgevoerd. Deze"prehospital-thrombolyse" studie was gekenmerkt door een uitstekende samenwerking vande Groninger huisartsen, ambulancediensten en ziekenhuizen. Nadat Bert als cardioloognaar het Martini-ziekenhuis vertrokken was, waren er gegevens en ideeën achtergeblevenzoals bv. de voorspellende waarde van het plasma fibrinogeen voor een niet succesvolletherapie. Deze relatie werd beschreven en gepubliceerd samen met Jan van der Meer.Onze combinatie, samen met Pieter de Graeff als eindredacteur bleek productief. Vrijwelalle artikelen werden in eerste t/m zoveelste versie door Jan op adequate wijze vancommentaar voorzien. Het doet mij genoegen dat hij ook paranimf bij de promotie wildezijn. Dr. Pieter de Graeff, hij noemde zichzelf wel eens mijn nemesis (Gr. myth.: Godinvan de wraak en van de gerechtigheid) kreeg vaak de al wat opgeschoonde versies van deartikelen onder ogen. Hij redigeerde deze en later ook de inleidende hoofdstukken inwederom enkele rondes hetgeen de leesbaarheid flink vergroot heeft. Een ware referent.Dr. Leemhuis, mijn opleider in Leeuwarden, toonde begrip voor mijn schrijverij tijdensmijn "interne" tijd. Erwin Göbel, prima collega assistent, verzamelde in die tijd op hetAZG een deel van de bloedmonsters voor het PAI-artikel, soms ook in de nacht (..).Mogelijk kan ik nog eens wat terugdoen. Hans Hillege, arts-biostatisticus, berekend algeruime tijd voor cardiologie promovendi op adequate wijze de p-waarden. Dat gaat goeden geruisloos en mag wel eens vermeld worden. Dr. Yvonne Hoogeveen, biologe,corrigeerde telkens op plezierige wijze binnen enkele dagen het Engels (en en-passant destrekking) van de artikelen, de brieven en de inleiding. Dr. Victor Bom, biochemicusverbonden aan het stollingslaboratorium, ontwikkelde en verbeterde samen met Wim vander Schaaf de anti-streptokinase antistoffen bepaling. Sandra Geerards, stollingsanaliste,deed de t-PA en PAI bepalingen. Ad van Boven, cardioloog, leverde de data voor hetlipoproteine(a) artikel. Jacob Pleiter, in welk dankwoord ontbreekt hij, maakte gratis deLp(a) figuur. De Sandoz Research Stichting maakte het mij mogelijk om in 1992 de PAIgegevens in Dallas te presenteren. De grafische kwaliteit van dit boekje komt op rekeningvan mijn broer Sjef. Jullie allemaal, en de elders genoemde medeauteurs van de artikelen,wil ik bedanken voor de onontbeerlijke bijdrage aan dit proefschrift.

De promotor professor Halie wil ik bedanken voor z’n opbouwende kritiek op hetmanuscript. Promotor professor Verheugt is in Nederland een autoriteit op het gebied vanthrombolyse en antithrombotica gebruik in de cardiologie. Dat hij (op afstand) promotorwilde zijn beschouw ik als een eer. De promotiecommissie, de professoren ten Cate,Reitsma en van der Werf wil ik bedanken voor het beoordelen van het proefschrift.

Tenslotte Isabelle, levensreisgenoot, ieder voor zich maar toch ook samen doen wij deopleiding cardiologie. Ook jij bent natuurlijk paranimf maar eigenlijk sta je me alle anderedagen ook terzijde en daar ben ik zeer gelukkig mee.

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