doi:10.1182/blood.V97.8.23142001 97: 2314-2322   

 G. MannSaulius Butenas, Kevin M. Cawthern, Cornelis van't Veer, Maria E. DiLorenzo, Jennifer B. Lock and Kenneth 

induced blood coagulation−Antiplatelet agents in tissue factor

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

Antiplatelet agents in tissue factor–induced blood coagulationSaulius Butenas, Kevin M. Cawthern, Cornelis van’t Veer, Maria E. DiLorenzo, Jennifer B. Lock, and Kenneth G. Mann

Several platelet inhibitors were examinedin a tissue factor (TF)–initiated model ofwhole blood coagulation. In vitro coagula-tion of human blood from normal donorswas initiated by 25 pM TF while contactpathway coagulation was suppressed us-ing corn trypsin inhibitor. Products of thereaction were analyzed by immunoassay.Preactivation of platelets with the throm-bin receptor activation peptide did notinfluence significantly the clotting time orthrombin–antithrombin III complex (TAT)formation. Addition of prostaglandin E 1 (5mM) caused a significant delay in clotting(10.0 minutes) versus control (4.3 min-utes). The prolonged clotting time is cor-

related with delays in platelet activation,formation of TAT, and fibrinopeptide A(FPA) release. In blood from subjectsreceiving acetylsalicylic acid (ASA or as-pirin), none of the measured products ofcoagulation were significantly affected.Similarly, no significant effect was ob-served when 5 mM dipyridamole (Persan-tine) was added to the blood. Antagonistsof the platelet integrin receptor glycopro-tein (gp) IIb/IIIa had intermediate effectson the reaction. A 1- to 2-minute delay inclot time and FPA formation was ob-served with addition of the antibodies7E3 and Reopro (abciximab) (10 mg/mL),accompanied by a 40% to 70% reduction

in the maximal rate of TAT formation anddelay in platelet activation. The cyclic hepta-petide, Integrilin (eptifibatide), at 5 mM con-centration slightly prolonged clot timeand significantly attenuated the maxi-mum rate of TAT formation. The disrup-tion of the gpIIb/IIIa-ligand interaction notonly affects platelet aggregation, but alsodecreases the rate of TF-initiated throm-bin generation in whole blood, demonstrat-ing a potent antithrombotic effect superim-posedon theantiaggregationcharacteristics.(Blood. 2001;97:2314-2322)

© 2001 by The American Society of Hematology

Introduction

The importance of the platelet in normal hemostasis and in diseasestates, such as atherosclerosis and thrombosis, is well established.1-5 Aparticularly germane example is the platelet-rich thrombus, which formson fissured or ruptured atherosclerotic plaques.3-5 Although the slowformation of the underlying plaques is a progressive disease that servesto narrow the lumen of the vessel over an extended period of time, it isthe sudden and catastrophic superposition of the platelet-rich thromboticocclusion on these lesions that is central to nearly all ischemic coronaryevents. General acceptance that the ultimate occlusive event is throm-botic in nature has led to the development of strategies that attempt toameliorate, inhibit, or reverse this final phase of the coronarydisease process. Some of the newest strategies focus on inhibitionof platelet functions, which contribute to the formation of thethrombotic occlusion.

The generation of thrombin during the blood clotting processcan be divided into 2 semidiscrete intervals, the initiation andpropagation phases.6 During the initiation phase nanomolar amountsof thrombin and picomolar amounts of other coagulation enzymesare generated. Clot occurs at the inception of the propagationphase, which is characterized by rapid and near quantitativethrombin generation. By this point activation of platelets andcleavage of factor V and factor VIII are almost complete.7 Innormal blood, the thrombin activation process is independent ofplatelet or factor V activation, but is limited by the generation offactor Xa.6,7

Normal platelet function has been divided into 3 processes:adhesion, aggregation, and activation (including surface phospho-lipid rearrangements and secretory pathways8-11). These functionsbestow on the platelet its antihemorrhagic qualities and areregulated by a variety of mechanisms.12 The most potent plateletagonist isa-thrombin, the central enzyme of coagulation thatcontrols hemorrhage in vivo. Thrombin is generated from theinactive circulating precursor prothrombin via the tissue factor(TF) pathway of coagulation13-16; this serine protease activatesplatelets via specific cell surface receptors, leading to secretion,aggregation, changes in platelet morphology, and expression of aprocoagulant, phospholipid-equivalent, surface.9-11 Aggregatedthrombin-activated platelets form the basis of the thrombus innormal hemostasis and provide the surface on which the complex-dependent reactions of the TF pathway are localized. Furthermore,thrombin amplifies its own generation by activating circulatingplasma procofactors (factor V and factor VIII), critical componentsof the pathway’s proteolytic complex catalysts, which also serve tolocalize proteolytic activity to the surface of the activated plate-let.6,17 Thrombin converts fibrinogen by limited proteolysis tofibrin, which polymerizes throughout the growing thrombus render-ing added stability.18-20 The importance of TF-induced thrombingeneration on platelets in acute coronary syndromes is stronglysuggested by the observation that inflammatory cytokines canup-regulate TF expression in certain cells, which may be found in

From the Department of Biochemistry, College of Medicine, University ofVermont, Burlington.

Submitted April 20, 2000; accepted December 12, 2000.

Supported in part by RR00109, P01 HL 46703 (K.G.M.), PHS T32 HL07594-12(K.G.M.) from the National Institutes of Health, and by a TALENT stipend of theNetherlands Organization of Scientific Research (C.v.V.).

Portions of this work were presented at the 39th Annual Meeting of theAmerican Society of Hematology, December 5-9, 1997, San Diego, CA (Blood

90 [suppl 1]:29a, abstract 114).

Reprints: Kenneth G. Mann, Department of Biochemistry, College of Medicine,University of Vermont, Burlington, VT 05405-0068; e-mail: kmann@zoo.uvm.edu.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2001 by The American Society of Hematology

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atherogenic lesions, blood, or the vasculature.21-23 Therefore, theinterrelationships between platelet functions and thrombin genera-tion are central to an understanding of the thrombotic process incoronary syndromes.

The multifaceted roles of the platelet in hemostasis providetargets for the development of strategies to arrest the occlusivephase of atherosclerotic disease. A number of inhibitors have beendescribed, which are directed at different aspects of plateletfunction. Prostaglandin E1 (PGE1) is a potent antagonist of plateletactivation and functions through the platelet prostaglandin receptorby up-regulating adenylate cyclase production of intracellularcyclic adenosine monophosphate (cAMP).12 The weaker plateletinhibitor aspirin (acetylsalicylic acid or ASA) has been found to bemildly effective at reducing the risk of coronary events or theirrecurrence.24,25 This effect is presumably due to aspirin’s role asirreversible inhibitor of the cyclooxygenase activity of prostaglan-din H synthase in the arachidonic acid pathway leading tothromboxane A2 (TXA2) synthesis for release on platelet activa-tion.26,27Inhibitors of the platelet integrin glycoprotein (gp) IIb/IIIahave been designed, which interfere with the ability of thesereceptors to bind fibrin(ogen), and thus to form the large platelet-fibrin aggregates, which serve as the foundation of the thrombus.28

Recently, the murine monoclonal antibody 7E3 (and the humanizedvariant, known as c7E3, Reopro, or abciximab) has provenclinically effective as an adjuvant in the management of high-riskpatients undergoing percutaneous transluminal coronary angio-plasty (PTCA).29-31 Similarly, a cyclic heptapeptide containing a“KGD” sequence has also been developed as a high-affinity mimicof the fibrinogen “RGD” sequence, which binds to the gpIIb/IIIareceptor (Integrilin, or eptifibatide). This peptidomimetic hasproven effective as an adjuvant in PTCA and in treatment ofunstable angina.32,33 Although the above agents or their analogsexhibit significant clinical benefits in treating acute coronarysyndromes, it is unclear whether their therapeutic properties arelocalized to the platelet aggregation function alone or extend tothrombin generation within the thrombotic occlusion. The goal ofthis study was to examine the relationships between therapeuticdoses of these reagents and TF-induced coagulation in wholeminimally altered human blood. This was accomplished using alaboratory model we have developed, which directly examinesvarious aspects of the coagulation reaction in freshly drawnhuman blood.7,34

Materials and methods

Materials

Recombinant human TF was provided as a gift by Drs Roger Lundblad andShu-Len Liu (Hyland Division, Baxter Healthcare, Duarte, CA). 1-Palmitoyl-2-oleoyl phosphatidylserine (PS) and 1-palmitoyl-2-oleoyl phosphatidylcho-line (PC) were purchased from either Sigma Chemical (St Louis, MO) orAvanti Polar Lipids (Birmingham, AL).D-Phenylalanyl-L-prolyl-L-argininechloromethyl ketone (FPRck)35 was obtained as a gift from HematologicTechnologies (Essex Junction, VT), synthesized in house, or purchasedfrom Calbiochem (San Diego, CA). Corn trypsin inhibitor (CTI) wasobtained either from Fluka (Ronkonkoma, NY) or prepared by a modifica-tion of the procedure of Hojima.34,36 PGE1 and dipyridamole (Persantine)were obtained from Sigma Chemical. PGE1 was diluted to 0.564 mM in95% ethanol and dipyridamole was solubilized at 2 mM in DMSO. Aspirin(1003 325-mg tablets) was obtained from a local grocery (HannafordFood and Drug Superstore, Rockland, ME). The antibody 7E3 was kindlyprovided by Dr Joseph A. Jakubowski (Thrombosis Research Division, EliLilly, Indianapolis, IN); stock concentration was 2 mg/mL in 20 mM

HEPES, 0.15 M NaCl, pH 7.4 (HBS). Reopro (abciximab) and Integrilin(eptifibatide) were gifts of Dr Uma Sinha, Cor Therapeutics (South SanFrancisco, CA). Stock concentrations of Reopro and Integrilin were 2mg/mL and 5 mM, respectively (solutions in HBS). The thrombin receptoractivation peptide (TRAP) was provided by Dr Paula Tracy (University ofVermont). Burro antihuman platelet factor 4 (PF4) immunoglobulin G(IgG) antibody was prepared as previously described.7 Horseradish peroxi-dase–labeled goat antihorse IgG antibody was purchased from SouthernBiotech (Birmingham, AL). The following analytes were estimated usingenzyme-linked immunosorbent assay (ELISA) kits obtained from themanufacturers according to the instructions provided: thrombin-antithrom-bin III (TAT) (Enzygnost TAT, Behring, Marburg, Germany); fibrinopeptideA (FPA) (Asserachrom FPA, Diagnostica Stago/American Bioproducts,Parsippany, NJ); platelet osteonectin (a gift from Dr Richard Jenny,Hematologic Technologies).

Human donors

All donors were recruited and advised according to a protocol approved bythe University of Vermont Human Studies Committee.7,34Healthy individu-als (aged 22-36) were selected so as to exclude donors with a personal orfamilial history of thrombosis/hemorrhage, or regular aspirin or drug use.All individuals exhibited values in the normal range for the prothrombintime (PT; 11.6-13.8 seconds), activated partial thromboplastin time (aPTT;27-36 seconds), and platelet counts (172 000-376 000/mL). For the ASAstudies, each donor was sampled before ASA treatment (control series—noASA for . 1 month before experiment) then again after 3 days of 325 mgASA twice a day (experimental series).

Preparation of TF/lipid reagent

Tissue factor (5 nM) was relipidated into small unilammelar vesicles37 of25% PS/75%PC (PCPS) (10mM total lipid) in HBS plus calcium (2 mM)for 30 minutes at 37°C. Concentrated sucrose (60% w/v) was subsequentlyadded to the relipidation mixture to 10% final to stabilize the vesicles forlong-term freezer storage (up to 12 months). Aliquots of the reagent (200mL) were lyophilized and stored at220°C, which could be rehydrated 60minutes before each experiment and used with reproducible results.

PT and aPTT assays

Assays were performed in plastic tubes (VWR Scientific, West Chester, PA,no. 60818-270) on 100-mL samples of fresh frozen, citrated human plasmausing the manual “tilt-tube” approach described by the manufacturer of thePT or aPTT reagent. When testing the effect of buffer or inhibitor, no morethan 10mL of the additive was mixed with 90mL plasma, to minimizedilution errors.

Coagulation in whole blood

The protocol used is a modification of Rand and colleagues,7 performed atthe Clinical Research Center, Fletcher Allen Health Care (Burlington, VT).For all platelet agents except the aspirin study (see below), studies ofclotting in freshly drawn, nonanticoagulated whole blood were performedsimultaneously in 2 series (16/series) of capped, polystyrene tubes modifiedto accept reagents as described.7 One series contained buffer only (controlseries), the other contained an appropriate amount of the antiplatelet agentunder investigation (experimental series). To suppress contact activation,CTI was added to all tubes (10mL, 5 mg/mL stock); in addition, the firsttube of each series of 16 tubes was designated the zero tube and waspreloaded with 1 mL 50 mM EDTA in HBS, pH 7.4, and 10mL 10 mMFPRck (diluted in 0.01 M HCl). As a control for possible TF in the bloodresulting from the draw or other sources, all but the last tube in each series(designated “2TF”) were preloaded with relipidated TF.

For studies using platelet agents other than ASA, the platelet agentunder investigation was preloaded into the experimental series from a stockprepared at a concentration appropriate to give the desired final concentra-tion in the blood (typically 100- to 400-fold). Care was taken to avoidpremixing of the reagents prior to initiation. An equivalent volume ofsolvent/vehicle (HBS, pH 7.4, or 95% ethanol for the PGE1 or DMSO for

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dipyridamole studies) was preloaded into each tube of the correspondingcontrol series. Following venipuncture under the protocol approved by theHuman Studies Committee at the University of Vermont,7 the initial 2 mLblood was discarded, samples were drawn for blood analyses (completeblood count, PT, aPTT, blood chemistry, etc), and about 40 mL fresh wholehuman blood was drawn into a repeating pipette or fitted with a plasticbarrel syringe (Eppendorf Combi-Tip, Eppendorf, Westbury, NY). Another1 to 2 mL blood was discarded, and the reaction was initiated by rapiddistribution of the remainder (1 mL/tube) to each of the 16 preloaded tubesin each series (control and experimental). The final concentrations ofadditives in the various experiments are as follows: CTI (50mg/mL),relipidated TF (3.1 pM, 25 pM, and 200 pM), and, where appropriate, theplatelet agonist or antagonist (PGE1, TRAP, dipyridamole, and Integrilin 5mM; 7E3 and Reopro 10mg/mL).

For each of the ASA (aspirin) experiments, a similar procedure was usedas described above; however, only one series of 16 tubes was prepared asabove with no additions other than CTI and TF. Each of the control studieswas performed using normal donor whole blood (;20 mL) drawn prior toadministration of ASA; each of the experimental studies was performed ona separate day on blood from the same donor treated according to the ASAregimen described above (see “Human donors”).

For each series in all experiments, periodic quenching of each tube(except the zero tube) was carried out at predefined intervals after initiationusing EDTA and FPRck as described above (see discussion of zero tubes).Quenched samples were obtained for each series tracing the reactionprogress up to 20 to 30 minutes after initiation. The samples werecentrifuged to remove cellular components and clots, and an aliquot (200mL) of the resulting serum from each tube was filtered to remove cellularcontaminants for osteonectin assays (0.2mm Acrodisc, Gelman Sciences,Ann Arbor, MI). From the remaining serum, both reducing (1%b-mercap-toethanol) and nonreducing sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) samples were prepared (60mL serum, 190mL2% SDS-PAGE sample solution, heated exactly 5 minutes at 98°C), thenstored at220°C for further analysis (PF4). Western blot analysis of PF4was performed as previously described.7 The remaining serum and cellpellets/clots were aliquotted to screw cap tubes, frozen, then stored at270°C for immunoblot or immunoassay analysis.

Immunoassays

Commercial ELISAs for FPA, TAT, and plateleta-granule release (osteonec-tin) were performed according to manufacturers’protocols, with correctionsfor sample dilution by added quench solution (1.00 mL). Results wereanalyzed on a Vmax microtiter plate reader (Molecular Devices, MenloPark, CA) equipped with SOFTMax version 2.0 software and an IBMPersonal System 2 Model 30/286 PC. In each assay, a minimum of 5standard concentrations were run in duplicate, with duplicate sampledeterminations at each time point. The relationship between concentrationof standard and optical density was established by fitting the data to either a4-parameter or log-logit fit, as described by the manufacturer (MolecularDevices). For each of the analytes (TAT, FPA, and osteonectin), duplicatedata describing each time point for a given donor were averaged over allexperiments.

Results

Assay conditions developed by Rand and colleagues7 allow studiesof coagulation directly in whole human blood in vitro underconditions that attempt to model the physiologic coagulationreaction in vivo. A tripartite approach is central to this model, usingminimally modified whole blood initiated with limiting (pM)concentrations of relipidated TF (PCPS/TF ratio 2000:1) and CTIto suppress the contact pathway, which does not play a significantrole in the normal hemostatic reaction.13-16 Such conditions permitexamination of the reaction with clotting at times similar to thoseobserved with in vivo assays like the template bleeding time. The

conditions also produce a reaction that is sensitive to factor VIII,factor IX, and factor XI deficiencies known to be associated withhemophilias A, B, and C, and therefore provide a model ofhemostasis that is improved over traditional plasma assays such asthe PT and aPTT.38 Finally, use of whole human blood as thesubstrate for the reaction affords direct examination of hemostaticmechanisms on the platelet and cellular surfaces relevant to thereaction in vivo; this aspect is particularly important in allowingstudy of the effects of pharmaceutical agents (such as antiplateletdrugs) on the coagulation reaction in a safe ex vivo model.

At present, the whole blood model provides simultaneousmeasurements of clot formation, thrombin generation (via TAT),fibrin formation (via FPA), and platelet activation (via osteonectinrelease), among others.7 Of particular value have been the determi-nations of thrombin level over the course of the reaction, whichshow that thrombin generation proceeds in 3 distinct phases. Frominitiation of the reaction until the clot is observed, thrombin isproduced at very low rates (,1 nM/min). These low rates ofthrombin generation have been ascribed to the limiting concentra-tions of prothrombinase produced during this initiation phase6 ofthe reaction, limited by the small amounts of factor Xa beingproduced. However, at clot time significant platelet and cofactoractivation are observed, indicating that the low levels of thrombingenerated are sufficient to potentiate the system for an increase inthe rate of thrombin generation. This postclot time thrombingeneration, propagation phase, is largely driven by factor Xaderived from the intrinsic Xase during this phase. The rate ofthrombin generation during the propagation phase routinely ex-ceeds 50 to 100 nM/min. Clotting occurs coincident with theinception of the propagation phase.7,34 Finally, rates of thrombingeneration are observed to slow as a result of endogenousregulatory mechanisms in what is referred to as the terminationphase of the reaction. Analyses performed across these phases ofthe reaction provide a means of characterizing the effects ofexternal influences (such as antiplatelet agents) on the reactionas a whole.

Platelet activation and suppression prior to TF: TRAP and PGE 1

Under normal conditions, the TF-initiated reaction in whole blooddoes not appear to be platelet activation limited.39 In keeping withthis conclusion the current study found the reaction not to besignificantly accelerated by the platelet agonist, TRAP sequenceSFLLRN.40 The reaction initiated by 25 pM TF was studied innormal blood in the absence (■, Figure 1) and presence (F) ofTRAP (Table 1), which fully activated the platelets within 1 minuteof initiation as illustrated by osteonectin release (Figure 1B). In theabsence of the TRAP, clotting occurred at 3.1 minutes (arrow a) andwas accelerated slightly by addition of agonist (2.3 minutes, arrowb). However, this observed shortening of the clot time is most likelyattributable to increased platelet aggregation leading to an early“call” of the clot formation, because the clotting time is estimatedvisually and is the most subjective parameter among the evaluatedin this model of blood coagulation. This is supported by TATprofiles collected in the absence and presence of activation peptide,which show no significant alteration in thrombin generation(Figure 1A). Thus, TRAP had little effect on the thrombingeneration profile, despite significant prior platelet activation.Together with earlier results, demonstrating significant generationof the factor Va and platelet activation prior to clot time,7,34 thesedata reinforce the conclusion that in the TF-initiated reaction innormal blood (and presumably during physiologic coagulation aswell) platelet and factor V activation are not the limiting factors of

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TF-induced thrombin generation during the initiation phase. Rather,factor Xa appears to be the limiting component of the thrombin-generating prothrombinase complex.6

Like prostaglandin I2, PGE1 is a natural prostanoid, which

inhibits intracellular release of stored Ca11 and platelet activationby up-regulation of adenylate cyclase to form cAMP. The clinicalusefulness of this reagent is limited by its short half-life in thecirculation and an ability to stimulate peripheral vasodilation;however, some preliminary studies have been described withanalogs of this reagent for use in controlling pulmonary hyperten-sion.41,42 Figure 2describes results with PGE1 (5 mM) added invitro to whole blood initiated to clot by 25 pM TF. Inhibition of theTF pathway of blood coagulation is observed. The clotting times inthe control experiment and in the presence of PGE1 are indicated ineach panel of the figure (arrows a and b, respectively). In thepresence of PGE1, clot time is prolonged from 4.4 minutes to 9.3minutes. Thrombin generation, shown as TAT formation over thecourse of the experiment (Figure 2A), is severely affected by thisagent. In the absence of PGE1, thrombin generation is very slowduring the initiation phase (■, 0-4 minutes region ending at arrowa), but thereafter occurs in an explosive fashion with a maximumrate near 54 nM/min during the propagation phase of the reaction.This result is similar to those reported for our previous studies ofcoagulation in blood from normal donors.7 In the presence of PGE1

(F) the initiation phase is prolonged (clotting occurring at arrow b)and the maximum rate of TAT production during the propagationphase is significantly reduced (12 nM/min) (Table 1).

Release profiles of FPA for the control and in the presence ofPGE1 are compared in Figure 2B. The normal profile (■) exhibitslimited FPA formation until just before clot time (arrow a). Theburst of FPA slightly precedes the burst of thrombin during thepropagation phase (compare to Figure 2A), reaching 30% of themaximum FPA at clot time. FPA levels rapidly go to completionwithin a 3-minute period, well before peak thrombin levels aregenerated (,100 nM TAT). This profile is similar to thosedescribed elsewhere,33,34 and shows that FPA release is significantat limiting amounts of thrombin. Platelet inhibition by PGE1 (F)produces a delay in the initiation of FPA release, although at clottime FPA release is approximately 35% (arrow b) and proceeds at a

Figure 1. Whole blood coagulation and TRAP. Comparison of whole blood coagulationin the absence (f) and presence (F) of 5mM TRAP. Coagulation was initiated in wholeblood by 25 pM relipidated TF as described in “Materials and methods.” PanelArepresentsthrombin (TAT) generation, panel B platelet activation (osteonectin [ON] release).Arrow “a”indicates clotting times for the control experiment, arrow “b” for the TRAP experiment.

Table 1. Maximum rates of TAT formation, FPA generation, and osteonectin release during TF-initiated (25 pM) whole blood clotting

Agent Subject

Clotting time (min)

Maximum rate

TAT (nM/min) FPA (mM/min) Osteonectin (nM/min)

C E C E C E C E

TRAP 2 3.1 2.3 149 136 — — 8.2 8.0

5 mM 6 4.7 4.2 94 122 — — 3.9 4.0

Average 3.9 3.3 122 129 — — 6.0 6.0

PGE1 1 4.2 10.7 70 35 6.0 5.2 9.9 7.5

5 mM 2 4.4 9.3 54 12 3.4 3.2 11.8 9.0

Average 4.3 10.0 62 24 4.7 4.2 10.8 8.2

ASA 1 4.2 3.7 63 53 7.6 2.7 6.5 3.9

2 3 325 mg/d 2 3.1 4.1 51 46 4.6 2.7 5.5 2.7

3 5.1 3.6 105 102 3.2 7.5 4.7 4.7

Average 4.1 3.8 73 67 5.1 4.3 5.6 3.8

Dipyridamole 4 4.2 4.5 64 99 — — 9.7 16.8

5 mM 5 6.0 6.1 98 83 9.7 10.1 14.5 21.3

Average 5.1 5.3 81 91 9.7 10.1 12.1 19.0

7E3 1 5.0 6.5 67 42 11.4 4.6 6.5 7.1

10 mg/mL 2 3.1 3.7 51 31 4.6 5.2 7.8 10.5

Average 4.0 5.1 59 36 8.0 4.9 7.1 8.8

Reopro 1 4.0 5.9 73 47 8.3 4.2 13.0 8.1

10 mg/mL 2 4.5 6.7 90 27 5.4 4.1 4.1 4.0

Average 4.2 6.3 81 37 6.8 4.1 8.6 6.0

Integrin 1 4.2 4.8 76 18 8.2 4.5 5.9 9.3

5 mM 2 3.7 4.3 43 26 10.5 4.0 16.8 16.5

Average 3.9 4.5 59 22 9.3 4.2 11.3 12.9

C indicates control; E, experiment.

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rate that is similar to that for the control (3.4mM/min in controlexperiment and 3.2mM/min in the presence of PGE1). Therefore,PGE1 inhibits the onset of explosive fibrin formation, presumablyresulting from the reduced levels of thrombin generated during theinitiation phase of the reaction in the presence of this inhibitor.

The effect of PGE1 on platelet activation is evident in theplatelet osteonectin and PF4 release profiles (Figure 2C and D,respectively). The normal profile of osteonectin release (■) shows

that platelet activation begins just before the clot formation and isabout 50% complete at clot time (arrow a), consistent withobservation of Rand and coworkers.7 However under the influenceof PGE1 (F), little or no plateleta-granule release is detectableuntil after clot time (arrow b), and the profile is delayed overall by 6minutes (versus control). Similarly, PF4 release in the presence ofPGE1 (Figure 2D; lower lane) is delayed by approximately 6minutes when compared with control experiment (upper lane). Asimilar effect of 5mM PGE1 on the clotting time, TAT and FPAformation, and osteonectin release was observed for the blood ofanother healthy volunteer (Table 1).

In summary, the results suggest that PGE1 at this concentrationinhibits the TF-initiated reaction in whole blood in vitro bysuppressing formation of the membrane sites required for complexenzyme assembly. Thus, thrombin generation during both theinitiation and propagation phases becomes platelet activationdependent and is reduced by PGE1. The results obtained usingPGE1 in this model system resemble those obtained in whole bloodfrom thrombocytopenic patients with platelet counts less than11 000/mL.39

Aspirin (ASA) and dipyridamole (Persantine)

No inhibitory effects on the TF pathway of blood coagulation werediscerned in 2 of 3 individuals ingesting aspirin (Table 1). In fact,clotting time in whole blood initiated by 25 pM TF appeared onaverage to be slightly shorter in the presence of the drug than incontrol experiment (3.8 and 4.1 minutes, respectively); however,the degree of variability in these experiments precludes conclu-sions concerning any aspirin accelerating effect. For individual “1”aspirin administration decreased clotting time by 0.5 minute,whereas for individual “3” this decrease reached 1.5 minutes(Figure 3). Similarly, thrombin generation (Figure 3A) and fibrinconversion (Figure 3B) profiles for these 2 individuals were shiftedto the left in the presence of the drug (F) in comparison with thecorresponding cases in the absence of ASA (■), whereas osteonec-tin release was not significantly influenced by aspirin administra-tion (Figure 3C). For individual “2” the clotting time wasprolonged by 1 minute after aspirin administration (3.1 and 4.1minutes, respectively). The maximum rates of TAT generation, FPAformation, and osteonectin release appeared on average to beslightly slower than normal (Table 1); however, the variability inthese assays does not allow any conclusion concerning decelerationof these processes by ASA.

These studies failed to find a significant role for aspirin at thedose used in attenuating TF-initiated coagulation and underscorethe complexity of the physiologic aspirin effect. The data suggestthat aspirin’s therapeutic effect does not arise primarily from directmodulation of platelet procoagulant function in the TF pathway tothrombin generation because aspirin does not appear to have anymeasurable effect on platelet activation in this system. This may bedue to 2 factors. First, platelet activation in this model system isthrombin mediated as underscored by the observation that additionof nanomolar quantities of hirudin completely inhibit plateletactivation. Second, thrombin, a potent platelet agonist, overridesthe weak inhibitory effects of aspirin via the TXA2 pathway.

Dipyridamole (Persantine) is a pyrimidopyrimidine derivativethat inhibits platelet aggregation, but the mechanism of action ofthis compound has been controversial.43 It has been established thatplasma concentrations of this derivative are 2 to 6mM after oraladministration of pharmaceutical doses. An in vitro addition of 5mM dipyridamole to whole blood only marginally affected theclotting time, increasing it by 0.1 to 0.3 minute versus control

Figure 2. Whole blood coagulation and PGE 1. Comparison of whole bloodcoagulation in the absence (f) and presence (F) of 5 mM PGE1. Coagulation wasinitiated in whole blood by 25 pM relipidated TF as described in “Materials andmethods.” Panel A represents thrombin (TAT) generation, panel B fibrin (FPA)formation, and panels C and D platelet activation (osteonectin [ON] and PF4 release,respectively). Arrow “a” indicates clotting times for the control experiment, arrow “b”for the PGE1 experiment. Upper lane in panel D represents control experiment andlower lane represents PGE1 experiment.

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(Table 1). Neither thrombin generation, FPA formation, nor os-teonectin release was significantly altered by the presence of thisplatelet antagonist.

Inhibitors of gpIIb/IIIa

Inhibitors of the gpIIb/IIIa complex on the platelet surface aredesigned to block the interactions between fibrinogen and platelets,inhibiting the formation of platelet aggregates.28 We examined theeffect of the murine antibody 7E3, the chimeric human/murineantibody Reopro (abciximab) and cyclic heptapetide Integrilin(eptifibatide) on blood coagulation induced by 200 pM, 25 pM, and3.1 pM TF.

In studies of whole blood with TF concentration of 200 pM, theclotting time was 1.25 minutes (Figure 4; arrows a-c). At thisinitiator concentration no significant effects of gpIIb/IIIa antago-nists on the blood coagulation process were observed. Clotting

times and maximum rates of TAT formation were similar in thecontrol experiment (■) and in the presence of 10mg/mL Reopro(F) or 5 mM Integrilin (Œ).

At a TF concentration of 25 pM both forms of antibody (7E3,Figure 5; and Reopro, Table 1) at 10mg/mL modestly delayed clotformation (Figure 5, arrow b). On average, 7E3 prolonged theclotting time from 4 to 5.1 minutes and Reopro from 4.2 to 6.3minutes (Table 1). This reflects a modest reduction in thrombingeneration versus normal in the presence of the inhibitors duringthe initiation phase of the reaction. With Integrilin (Figure 6), clotformation was delayed slightly (by 0.6 minute; arrow b) versuscontrol (arrow a). Consistent with this observation, the initiationphase of thrombin generation (3.9 minutes) was slightly prolongedby this inhibitor (to 4.5 minutes).

Thrombin generation during the propagation phase wassignificantly suppressed by all 3 gpIIb/IIIa inhibitors. With 7E3(Figure 5A,F), the maximum thrombin generation rate follow-ing clot formation was approximately 60% of that observed inthe control experiment (31 nM/min versus 51 nM/min, for theexperiment presented in Figure 5A). Reopro decreased themaximum rate of TAT formation on average by 54% (Table 1).Similarly, in the presence of Integrilin this rate was decreasedfrom 59 nM/min to 22 nM/min, a more than 60% reduction(Table 1; Figure 6A).

Both inhibitory antibodies produced delays in FPA release(Figure 5B; 7E3;F) of approximately 1 to 2 minutes relative tocontrol (■), reflective of the prolonged initiation phase and delay inclot formation. This delay in the FPA profiles is consistent with theinfluence of these inhibitors on the TF pathway reaction during itsinitiation phase. Although the FPA release is delayed versuscontrol, the ultimate levels and rates of FPA formation are similarto those observed in the absence of the inhibitors. FPA release in thepresence of 5mM Integrilin (Figure 6B) is also consistent with thedepressed thrombin generation although clotting data suggest thatthis inhibitor has a lesser effect on the initiation phase than theantibodies at this TF concentration. Integrilin did not significantlyinhibit the rate, duration, or maximum level of FPA release (F)compared with the control (■).

Platelet osteonectin (Figure 5C for 7E3) and PF4 release(Figure 5D) are delayed versus normal (■) in the presence ofinhibitory antibodies (F). With Integrilin, osteonectin release(Figure 6C) is slightly delayed (F) versus normal (■). Themaximum rates of osteonectin release are not affected by thepresence of these inhibitors (Table 1).

Figure 3. Whole blood coagulation and ASA. Comparison of whole bloodcoagulation before (f) and after (F) administration of ASA, 2 3 325 mg/d for 3consecutive days. Coagulation was initiated in whole blood by 25 pM relipidated TFas described in “Materials and methods.” Panel A represents thrombin (TAT)generation, panel B fibrin (FPA) formation, and panel C platelet activation (osteonec-tin [ON] release). Arrow “a” indicates clotting times before ASA administration, arrow“b” after.

Figure 4. Thrombin (TAT) generation in whole blood initiated by 200 pM TF.Coagulation was initiated by relipidated TF as described in “Materials and methods”in the absence of any adjuvant (f), in the presence of 5 mM Integrilin (Œ) and in thepresence of 10 mg/mL Reopro (F). Arrow “a” indicates clotting time for controlexperiment, arrow “b” for Integrilin experiment, and arrow “c” for Reopro experiment.

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In summary, the antibody inhibitors of the gpIIb/IIIa com-plex influence thrombin generation, fibrin formation, and plate-let activation in both the initiation and propagation phases ofthe reaction in whole blood induced by 25 pM TF. The largesteffect appears to be on the propagation phase of the reaction.Integrilin also has a pronounced effect on the propagation phase,with a more limited effect on the initiation phase of thrombingeneration.

Experiments were also performed at a lower (3.1 pM) TF

concentration. Under these conditions both agents (Integrilin andReopro) prolonged the clotting time from 8.2 to 14.3 minutes(Integrilin) and almost 20 minutes (Reopro) and depressed themaximum levels of TAT formed (data not shown).

Discussion

Studies from this laboratory and others7,34,44-46using physiologi-cally relevant systems to model the TF pathway have demonstratedthat thrombin generation is not uniform over time but occurs indistinct phases. During an initiation phase, low rates of thrombingeneration (,1 nM/min) are observed as a result of low rates offactor Xa produced primarily by the factor VIIa-TF com-plex.6,7,34,44-46Subsequently, enhanced levels of factor Xa are made

Figure 5. Whole blood coagulation and 7E3. Comparison of whole bloodcoagulation in the absence (f) and presence (F) of 7E3 (10 mg/mL). Coagulation wasinitiated in whole blood by 25 pM relipidated TF as described in “Materials andmethods.” Panel A represents thrombin (TAT) generation, panel B fibrin (FPA)formation, and panels C and D platelet activation (osteonectin [ON] and PF4 release,respectively). Arrow “a” indicates clotting times for the control experiment, arrow “b”for the 7E3 experiment. Upper lane in panel D represents control experiment andlower lane represents 7E3 experiment.

Figure 6. Whole blood coagulation and Integrilin. Comparison of whole bloodcoagulation in the absence (f) and presence (F) of 5 mM Integrilin. Coagulation wasinitiated in whole blood by 25 pM relipidated TF as described in “Materials andmethods.” Panel A represents thrombin (TAT) generation, panel B fibrin (FPA)formation, and panel C platelet activation (osteonectin [ON] release). Arrow “a”indicates clotting times for the control experiment, arrow “b” for the Integrilinexperiment.

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available by participation of the factor IXa–factor VIIIa complex,38

and a burst of thrombin generation (. 50 nM/min) occurs during apropagation phase of the reaction. Clot formation, the usualend-point of in vitro assays, occurs approximately at the transitionbetween the initiation and propagation phases and coincides withthe onset of the thrombin burst.7,34 By this point in the reaction inwhole blood, activation of platelets and cleavage of factor V andfactor VIII are significant.7

In normal blood, the limiting component in the ultimateactivation of prothrombin to thrombin is not platelet or factor Vactivation, but factor Xa generation.6,7Thus, as anticipated, preacti-vation of platelets with TRAP has little effect on thrombingeneration.

In contrast, the potent platelet inhibitor PGE1 significantlyprolongs the initiation phase of thrombin generation, which isreflected in a doubling of the clotting time and delays in the FPArelease and thrombin generation. PGE1 also delays platelet degranu-lation (osteonectin release), with accompanying reduction in thepresentation of platelet surface receptors for procoagulant enzymecomplex formation thus impairing coagulation complex assem-bly.10,11This impaired complex assembly in the presence of PGE1 isreflected by a decreased thrombin generation rate during thepropagation phase to less than half that observed normally.Ultimately, platelet activation and fibrin formation, though de-layed, proceed to completion despite the reduced prothrombinactivation rates. Thus, although thrombin levels are sufficient tofully activate platelets and cleave fibrinogen at maximal ratesduring the propagation phase, suppression of platelet activation bythe potent antagonist PGE1 imparts a substantial platelet activationdependency to the process of thrombin generation.

Aspirin (ASA) at clinically relevant doses did not alter clottingtime, thrombin generation, fibrin formation, or platelet activation inblood induced to clot by addition of TF. This result is similar to thatnoted by Kjalke and associates who reported no effect of aspirinadministration on the thrombin generation in a reconstituted systemin the presence of platelets when the reaction was initiated by TF.47

Thus, at the doses used, aspirin does not overcome the thrombinactivation of the platelets, the agonist principally responsible forplatelet activation in the TF-initiated reaction. Similarly, theaddition of dipyridamole (Persantine) to whole blood in vitro atpharmacologic concentration43 has no effect on the processesoccurring during TF-induced blood coagulation in this model.

The inhibitors of the platelet gpIIb/IIIa receptor exhibit some-what different behaviors in influencing the TF-initiated reaction. Inreactions initiated by 25 pM TF, antibodies 7E3 (the murine form)and Reopro abciximab or c7E3 (the chimeric murine/human form)each provide a modest prolongation of the initiation phase evi-denced by slight delays in clotting time and delays in both FPA andplatelet osteonectin release profiles. However, both 7E3 andReopro significantly reduce thrombin generation during the propa-gation phase of the reaction. Despite reduced thrombin generationthe ultimate maximum rates of FPA formation and osteonectinrelease were observed. Both the fibrin and platelet reactionsreached completion. These observations of reduced thrombingeneration in the presence of anti-gpIIb/IIIa antibodies are consis-tent with those described in a defibrinated plasma model.48

The cyclic heptapeptide Integrilin (eptifibatide) slightly delayedthrombin generation at 25 pM TF during the initiation phase,resulting in a slightly prolonged clotting time. However, atpharmacologic concentration the influence of Integrilin was lessthan that observed for the 7E3 and Reopro antibodies. Integrilin,similarly to antibody inhibitors, reduced the rate of thrombingeneration during the propagation phase of the reaction.

At higher TF concentrations (200 pM), neither Reopro norIntegrelin at pharmacologic concentrations affected clot formation,clotting times, or the thrombin generation profiles. At the lowerinitiator concentration (3.1 pM TF) the efficiencies of bothgpIIb/IIIa inhibitors is enhanced. Under these conditions, bothIntegrilin and Reopro significantly prolonged the clotting time.These significant prolongations of clotting time may explain thebleeding observed with these agents.49-51

Overall, the gpIIb/IIIa inhibitors appear to significantly influ-ence either the quantitative or qualitative expressions of theprocoagulant complexes leading to thrombin.

Acknowledgments

We thank Sara G. Paradis and Joshua D. Dee for the technicalassistance. We thank Dr Shu Len Liu and Dr Roger Lundblad forproviding us with TF; Dr Joseph A. Jakubowski for providing 7E3;Dr Uma Sinha for providing Reopro and Integrilin; Dr Paula Tracyfor providing TRAP; and Dr Richard Jenny for providing osteonec-tin ELISA kits and FPRck.

References

1. Weiss HJ. Antiplatelet therapy: properties ofplatelets. N Eng J Med. 1978;298:1344-1347.

2. Ware AJ, Coller BS. Platelet morphology, bio-chemistry and function. In: Beutler E, LichtmanMA, Coller BS, Kipps TJ, eds. Williams Hematol-ogy. 5th ed. New York, NY: McGraw-Hill;1995:1161-1176.

3. Fuster V, Badimon L, Badimon JJ, Chesebro JH.The pathogenesis of coronary artery disease andthe acute coronary syndromes (1). N Engl J Med.1992;326:242-250.

4. Fuster V, Badimon L, Badimon JJ, Chesebro JH.The pathogenesis of coronary artery disease andthe acute coronary syndromes (2). N Engl J Med.1992;326:310-318.

5. Badimon L, Badimon JJ, Fuster V. Pathogenesisof thrombosis. In: Verstraete M, Fuster V, TopolEJ, eds. Cardiovascular Thrombosis: Thrombo-cardiology and Thromboneurology. 2nd ed. Phila-delphia, PA: Lippincott-Raven; 1998:23-44.

6. Butenas S, van ’t Veer C, Mann KG. Evaluation ofthe initiation phase of blood coagulation using

ultrasensitive assays for serine proteases. J BiolChem. 1997;272:21527-21533.

7. Rand MD, Lock JB, van ’t Veer C, Gaffney DP,Mann KG. Blood clotting in minimally alteredwhole blood. Blood. 1996;88:3432-3445.

8. Hartwig JH. Platelet morphology. In: Loscalzo J,Schaefer AI, eds. Thrombosis and Hemorrhage.2nd ed. Baltimore, MD: Williams & Wilkins; 1998:207-228.

9. Bevers EM, Comfinius P, Van Rijn JLML, HemkerHC, Zwaal RFA. Generation of prothrombin con-verting activity and the exposure of phosphatidyl-serine at the outer surface of platelets. Eur J Bio-chem. 1982;122:429-436.

10. Schroit AJ, Zwaal RFA. Transbilayer movement ofphospholipids in red cell and platelet membranes.Biochim Biophys Acta. 1991;1071:313-329.

11. Zwaal RFA, Schroit AJ. Pathophysiologic implica-tions of membrane phospholipid asymmetry inblood cells. Blood. 1997;89:1121-1132.

12. Kroll NM, Sullivan R. Mechanisms of platelet acti-vation. In: Loscalzo J, Schafer AI, eds. Thrombo-

sis and Hemorrhage. 2nd ed. Baltimore, MD: Wil-liams & Wilkins; 1998:261-291.

13. Davie EW, Fujikawa K, Kisiel W. The coagulationcascade: initiation, maintenance and regulation.Biochemistry. 1991;30:10363-10370.

14. Rapaport SI, Rao LVM. The tissue factor path-way: how it has become a ‘prima ballerina.’Thromb Haemost. 1995;74:7-17.

15. Mann KG, Krishnaswamy S, Lawson JH. Sur-face-dependent hemostasis. Semin Hematol.1992;29:213-226.

16. Jenny NS, Mann KG. Coagulation cascade: anoverview. In: Loscalzo J, Schafer AI, eds. Throm-bosis and Hemorrhage. 2nd ed. Baltimore, MD:Williams & Wilkins; 1998:3-27.

17. Pieters J, Lindhout T, Hemker HC. In situ-gener-ated thrombin is the only enzyme that effectivelyactivates factor VIII and factor V in thromboplas-tin-activated plasma. Blood. 1989;74:1021-1024.

18. Blomback B. Fibrinogen and fibrin—proteins withcomplex roles in hemostasis and thrombosis.Thromb Res. 1996;83:1-75.

ANTIPLATELET AGENTS IN BLOOD COAGULATION 2321BLOOD, 15 APRIL 2001 z VOLUME 97, NUMBER 8

For personal use only. by guest on June 9, 2013. bloodjournal.hematologylibrary.orgFrom

19. Doolittle RF. Fibrinogen and fibrin. Annu Rev Bio-chem. 1984;53:195-229.

20. Sixma JJ, van den Berg A. The haemostatic plugin haemophilia A: a morphological study of hae-mostatic plug formation in bleeding time skinwounds of patients with severe haemophilia A.Br J Haematol. 1985;58:741-753.

21. Bevilacqua MP, Pober JS, Majeau GR. Interleukin1 (IL-1) induces biosynthesis and cell surface ex-pression of procoagulant activity in human vascu-lar cells. J Exp Med. 1984;160:618-623.

22. Bevilacqua MP, Pober JS, Majeau GR, Fiers W,Cotran RS, Gimbrone MA Jr. Recombinant tumornecrosis factor induces procoagulant activity incultured human endothelium: characterizationand comparison with the actions of interleukin 1.Proc Natl Acad Sci U S A. 1986;83:4533-4537.

23. Bevilacqua MP, Gimbrone MA Jr. Inducible endo-thelial functions in inflammation and coagulation.Semin Thromb Hemost. 1987;13:425-433.

24. Patrono C. Aspirin as an antiplatelet drug. N EnglJ Med. 1994;330:1287-1294.

25. Vane JR. Inhibition of prostaglandins as a mecha-nism of action for aspirin-like drugs. Nat New Biol.1971;231:232-235.

26. Thomgreen M, Shafi S, Bom GVR. ThromboxaneA2 in skin-bleeding-time blood and in clotted ve-nous blood before and after administration ofacetylsalicylic acid. Lancet. 1983;1:1075-1078.

27. Patrono C, Ciabattoni G, Pinca E, et al. Low doseaspirin and inhibition of thromboxane B2 produc-tion in healthy subjects. Thromb Res. 1980;17:317-327.

28. Coller BS, Peerschke EL, Scudder LE, SullivanCA. A murine monoclonal antibody that com-pletely blocks the binding of fibrinogen to plate-lets produces a thrombasthenic-like state in nor-mal platelets and binds to glycoproteins IIb and/orIIIa. J Clin Invest. 1983;72:325-328.

29. The EPIC Investigators. Use of a monoclonal an-tibody directed against the platelet glycoproteinIIb/IIIa receptor in high-risk coronary angioplasty:the EPIC investigation. N Engl J Med. 1994;330:956-961.

30. The EPILOG Investigators. Platelet glycoprotein

IIb/lIIa receptor blockade and low-dose heparinduring percutaneous coronary revascularization.N Engl J Med. 1997;336:1689-1699.

31. The CAPTURE Investigators. Randomized pla-cebo-controlled trial of abciximab before and dur-ing coronary intervention on refractory unstableangina: the CAPTURE study. Lancet. 1997;349:1429-1435.

32. Schulman SP, Goldschmidt-Clennont PJ, TopolEJ, et al. Effects of Integrilin, a platelet glycopro-tein IIb/IIIa receptor antagonist, in unstable an-gina: a multicenter trial. Circulation. 1996;94:2083-2089.

33. The IMPACT-II Investigators. Randomized pla-cebo-controlled trial of effect of eptifibatide oncomplications of percutaneous coronary interven-tion: IMPACT-II. Lancet. 1997;349:1422-1428.

34. Cawthern KM, van ’t Veer C, Lock JB, DiLorenzoME, Branda RF, Mann KG. Blood coagulation inhemophilia A and hemophilia C. Blood. 1998;91:4581-4592.

35. Kettner C, Shaw E. D-Phe-Pro-ArgCH2Cl-A selec-tive affinity label for thrombin. Thromb Res. 1979;14:969-973.

36. Hojima Y, Pierce JV, Pisano J. Hageman factorfragment inhibitor in corn seeds: purification andcharacterization. Thromb Res. 1980;20:149-162.

37. Barenholz Y, Gibbes D, Litman BJ, Goll J,Thompson TE, Carlson FD. A simple method forthe preparation of homogeneous phospholipidvesicles. Biochemistry. 1977;16:2806-2810.

38. Osterud B, Rapaport SI. Activation of factor IX bythe reaction product of tissue factor and factorVII: additional pathway for initiating blood coagu-lation. Proc Natl Acad Sci U S A. 1977;74:5260-5264.

39. Cawthern KM, Pennucci JJ, Mann KG, BrandaRF. The effect of thrombocytopenia on the tissuefactor pathway [abstract]. Blood. 1998;92 (suppl1):36a.

40. Vu TK, Hung DT, Wheaton VI, Coughlin SR. Mo-lecular cloning of a functional thrombin receptorreveals a novel proteolytic mechanism of receptoractivation. Cell. 1991;64:1057-1068.

41. FitzGerald GA, Patrono C. Antiplatelet drugs. In:

Verstraete M, Fuster V, Topol EJ, eds. Cardiovas-cular Thrombosis: Thrombocardiology andThromboneurology. 2nd ed. Philadelphia, PA: Lip-pincott-Raven; 1998:121-139.

42. Okano Y, Yoshioka T, Shimouchi A, Satoh T, Ku-neida T. Orally active prostacyclin analogue inprimary pulmonary hypertension. Lancet. 1997;349:1365.

43. FitzGerald GA. Dipyridamole. N Engl J Med.1987;316:1247-1257.

44. Lawson JH, Kalafatis M, Stram S, Mann KG. Amodel for the tissue factor pathway to thrombin; I:an empirical study. J Biol Chem. 1994;269:23357-23366.

45. van ’t Veer C, Mann KG. Regulation of tissue fac-tor initiated thrombin generation by the stoichio-metric inhibitors tissue factor pathway inhibitor,antithrombin-III, and heparin cofactor-II. J BiolChem. 1997;272:4367-4377.

46. Jones KC, Mann KG. A model for the tissue factorpathway to thrombin, II: a mathematical simula-tion. J Biol Chem. 1994;269:23367-23373.

47. Kjalke M, Oliver JA, Monroe DM, et al. The effectof active site-inhibited factor VIIa on tissue factor-initiated coagulation using platelets before andafter aspirin administration. Thromb Haemost.1997;78:1202-1208.

48. Reverter JC, Beguin S, Kessels H, Kumar R,Hemker HC, Coller BS. Inhibition of platelet medi-ated, tissue factor-induced thrombin generationby the mouse/human chimeric 7E3 antibody: po-tential implications for the effect of c7E3 Fabtreatment on acute thrombosis and “clinical reste-nosis.” J Clin Invest. 1996;98:863-874.

49. Winkley JM, Adams HP Jr. Potential role of abcix-imab in ischemic cerebrovascular disease. Am JCardiol. 2000;85:47C–51C.

50. Ali A, Patil S, Grady KJ, Schreiber TL. Diffuse al-veolar hemorrhage following administration oftirofiban or abciximab: a nemesis of platelet gly-coprotein Iib/IIIa inhibitors. Catheter CardiovascInterv. 2000;49:181-184.

51. Vahdat B, Canavy I, Fourcade L, et al. Fatal cere-bral hemorrhage and severe thrombocytopeniaduring abciximab treatment. Catheter CardiovascInterv. 2000;49:177-180.

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