effect of streptokinase-activated...

STUDYON THE EFFECT OF STREPTOKINASE-ACTIVATEDPLASMIN(FIBRINOLYSIN) ON CLOTS IN VARIOUS STAGESOF

ORGANIZATION1.2

By NATHANBACK, JULIAN L. AMBRUS,C. LENORESIMPSON, ANDSIDNEY SHULMAN

(F1roni. The Roswell Park Memorial Institute and the Departmnents of Pharmnacology, Bacteri-ology and Iimmunology, and Pathology, University of Buffalo Medical School,

Buffalo, N. Y.)

(Submitted for publication July 25, 1956; accepted February 20, 1958)

In previous studies (1, 4), a method was de-veloped for the quantitative recording of the lysisof thrombi and emboli produced with I'31-labeledfibrinogen. Using this method, the in vivo fibri-nolytic activity of human and bovine plasmin wasdemonstrated (2). Other proteolytic enzymes(crude pancreatic protease, trypsin, carboxypep-tidase, papain and ficin) were ineffective in maxi-mal tolerated doses (1, 3, 4). Literature on thetherapeutic use of proteolytic enzymes has beenreviewed (1, 2). In all previous experiments,treatment was started one-half hour after forminglabeled clots in the experimental animals. Thequestion arose whether plasmin treatment wouldbe effective in dissolving organized clots andwhether there is a correlation between degree oforganization and plasmin-induced lysis. Thepreparations referred to as "plasmin" in this studyactually contain a variety of enzymatic activities;they are fibrinolytic, proteolytic and are able toactivate the fibrinolysin system. Presumably theyrepresent a mixture of plasmin, plasmin-activatorand free streptokinase. It is difficult to ascribetherapeutic activity to any one of these compo-nents. The purpose of these experiments is tostudy therapeutic indications and conditions forthe preparations currently undergoing clinicaltrial. Labeled clots were produced in several veinsof dogs at various time intervals. At the time oftreatment, these clots were of various ages. Dif-ferences in lysis were established by recording

1 Preliminary report presented before the Federationof American Societies of Experimental Biology, AtlanticCity, 1956 (Fed. Proc. 1956, 15, 396).

2 Supported by grants from The American Heart As-sociation, Parke Davis and Co., The Sharp and DohmeDivision of Merck and Co., and the United States PublicHealth Service.

disappearance of radioactivity and by histopatho-logic study.

MATERIALS AND METHODS

Materials and methods were similar to those de-scribed in previous studies (1). Under aseptic condi-tions, I3"-labeled purified bovine fibrinogen was intro-duced through side branches into isolated segments ofveins of dogs and clotted with the aid of thrombin.Semi-constricting aluminum wire ligatures were placedproximal to the clots and artery clamps isolating thesegment were released one-half hour after clot formation.Radioactivity over the area was recorded with the aidof a specially constructed lead shield, a scintillationcounter, an analytical radiation rate meter, and an Ester-line recorder.

Mongrel dogs, 10 to 16 Kg. in body weight, were used.At the time of the beginning of treatment, the clots variedin age from 30 minutes to 10 days. In most dogs twoclots of the same age were present; one was removedbefore institution of plasmin treatment. All other clotswere removed after the last treatment. Sections from atleast two areas of each clot were examined histologically.

Five daily intravenous doses of human plasmin, 30plasmin units (Loomis) per Kg.,3 were given. One mg.of the plasmin preparation used was found to contain 3fibrinolytic plasmin units (Loomis) or 0.098 proteolyticcasein unit (6). The specific activity was 0.28 caseinunit per mg. protein. This dose was previously found(2) to cause lysis of fresh clots but not to alter fibrino-gen and prothrombin levels. In most previous studies thedose was administered in four hour infusions; in thepresent experiments, rapid intravenous inj ections weregiven. In the previous studies, human plasmin was pre-pared from Cohn's Fraction III according to the methodof Kline (7). In the present study a simple streptokinase-activated acid extract of Fraction III was used, preparedby Dr. Werner Baumgarten, Sharp and Dohme Divisionof Merck and Co., West Point, Pa. The following in-formation was made available on the method of prepara-tion of this material. Fraction III, prepared by the

3A plasmin unit (Loomis) is defined as the quantityof enzyme needed to dissolve 0.6 ml. of a 0.3 per centpurified bovine fibrin clot at 450 C. in two minutes at pH7.2 (5).

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EFFECT OF PLASMIN ON CLOTS OF VARIOUS AGES

alcohol fractionation method of Cohn, was extracted withacid, adjusted to pH 7.8, and to each equivalent of 40 mg.of Fraction III, 13,000 Christensen units of streptokinasewas added (U. S. Patent 2,666,729). Wefound that whenlyophilized samples of the above material were dilutedwith increasing volumes of physiological saline, fibrino-lytic activity first increased, then decreased. Such bell-shaped dilution versus activity curves could be duplicatedby adding excessive amounts of streptokinase to plasmino-gen prepared by the Kline method. This was interpretedas indicating that the preparation used in this study con-tained a considerable amount of free streptokinase. Itis known that most batches of streptokinase are pooractivators of dog plasminogen. On the other hand Sherry,Titchener, Gottesman, Wasserman, and Troll have shown(8) that streptokinase-activated human plasmin prepara-tions may activate dog plasminogen. It is presently amatter of controversy whether this effect of streptokinase-activated human plasmin is due to the presence of "acti-vator" (9), or to the activating ability of a hypotheticalplasminogen-streptokinase complex (10, 11), or a plasmin-streptokinase complex (4, 11).

Blood samples were taken before, and at various timeintervals after, plasmin injections for hematological andbiochemical analyses as in previous studies (14). Pro-thrombin time is reported as clotting index: the ratio ofthe prothrombin time prior to treatment to the prothrom-bin time during and after plasmin administration. Inmost experiments as in previous studies (1-4) prothrom-bin time was also determined after adding bovine fibrino-gen, so as to eliminate the effect of fibrinogenopenia onprothrombin time determinations.

RESULTS

Clot lysisFigure 1 shows an experiment in which a dog

with one-half hour, one, two and three day oldclots was treated with daily injections of 30 plas-min units (Loomis) per Kg. of human plasmin.The first point on the graph indicates radioactivityover the thrombus at the time of clot formation;the second represents radioactivity immediately

FIG. 1. EFFECTOF PLASMIN ON ONE-HALFHOUR, ONE, TWOANDTHREEDAYOLDCLOTS, CLOTTING INDEX, FIBRINOGEN LEVEL ANDWHITE BLOODCELL COUNT

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N. BACK, J. L. AMBRUS, C. L. SIMPSON, AND S. SHULMAN

FIG. 2. EFFECT OF PLASMIN ON ONE-HALF HOURAND10 DAYOLD CLOTS, CLOT-TING INDEX, FIBRINOGEN LEVEL ANDWHITE BLOODCELL COUNT

before initiation of treatment. It can be seen that1131-labeled clots up to the age of two days com-pletely disappeared during the five day treatmentperiod. Only slight lysis was evidenced in the

TABLE I

Effect of clot age on lysis

Mean of %lysisand standard error

Age of No. of Treatment of mean at end ofclots clots of dogs treatment period

i hr. 23 saline 23.54 ± 3.17(control)

10 days 4* 30 plasmin 23.72 3.755 days 4 units (Loomis) 22.43 ± 2.633 days 10 per Kg. body 20.49 3.552 days 6 weight 80.17 4.261 day 6 80.53 4.792hr. 6t 97.42 1.10

* Eight additional clots gave similar results but are notincluded since somewhat different methods were used.

t Three additional clots gave similar results after treat-ment with more purified plasmin preparations.

three day clot. It should be remembered, how-ever, that statistical analysis of lysis of all three dayold clots versus control clots revealed no significantdifference. Figure 2 illustrates an experiment inwhich a dog with two 10 day old clots and one-halfhour old clot was treated. Radioactivity over thehalf-hour old clot disappeared as in the previousexperiment, but the 10 day old clots showed no

significant lysis compared to clots in untreateddogs (2). Results with five day old clots wereidentical with those of 10 day old clots. Fresh1131-labeled fibrinogen was prepared before eachseries of experiments. Thus, clots of various agesproduced in the same dog had various startinglevels of radioactivity. No relationship was foundbetween base radioactivity levels and degree oflysis.

Table I summarizes the results of all clots in-vestigated in this study. It appears that under theconditions of these experiments and at the dose

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k * . A X

FIG. 3. THREEDAY OLD CLOT WITH FNDOTHELIAL COVERINGOVERTHE ENDS (HEMATOXYLINEosIN 420 X)

level used, clots three days of age or older can not

effectively be dissolved with human plasmin.

Histopathologic findings

The histological findings were not uniformthroughout the length of a clot. Despite this itwas possible to grade the clots histologically andit was found that the histological picture correlatedclosely with their known age. The most importantfactor in grading was the degree of endothelialand fibroblastic activity and subsequent vasculari-zation and fibrosis. The ends of the clot andspaces where the clot did not adhere firmly to thevessel wall were largely covered with end'otheliumby the third day (Figure 3) and about this timea few fibroblasts were occasionally present in thesubstance of the clot (Figure 4). Vascular spaces

were formed soon after this (Figure 5). By thetenth day all clots were fully vascularized and many

were only partially attached to the vessel wall withblood flowing around them (Figure 6). A sec-

ond factor in grading was the change in the clotitself from the early mixture of fibrin and red cellsto a more homogeneous mass from which the red

cells gradually disappeared. Van Giesen stainrevealed no collagen formation before the tenthday.

It was not possible to distinguish the clots whichhad received plasmin therapy after three or moredays of existence from the control clots of similarage. Where treatment was given earlier, the clotappeared to be less advanced than expected at thetime of removal. In some vessels where completedisappearance of the original isotopically-labeledfibrin structure was indicated by measurement ofradioactivity, histologic examination revealed thepresence of relatively fresh nonlabeled clots. Itseemed probable that lysis of the original clot hadbeen followed by formation of a fresh thrombus.Control clots of the same age showed little decreaseof radioactivity levels. A varying degree of in-flammatory reaction was always present in thevessel wall as a result of the manipulations neces-sary in the daily measurement of radioactivity.This may have influenced the reformation of theclot after the initial lysis. Occasionally, organiza-tion appeared less advanced than expected in thepresence of severe necrosis of the vessel wall.

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FIG. 4. FOURDAY OLDTHROMBUSWITH FIBROBLASTS IN

SUBSTANCEOF CLOT (HE-MATOXYLIN FosiN 420 X)

Biochemnical findings

In previous experiments (1, 2), human plas-min, prepared according to the method of Klineand activated with streptokinase in a dose of 30plasmin units (Loomis) per Kg. (4) had no ef-fect on the fibrinogen and prothrombin level ifgiven as a four hour intravenous infusion. Inthis study, acid extracts of Cohn's Fraction IIIactivated with streptokinase were given by rapidintravenous injection. This resulted in a fall offibrinogen levels as well as clotting index afterthe first injection. These changes did not usu-

ally occur during subsequent injections, but oc-

casionally reappeared at the fifth daily injection.

Heniatologic findings

Leukopenia was observed after each injection.No decrease of this change occurred during pro-

longed daily treatment. Leukopenia does not ap-

pear to be due to the enzymatic effects of plasminsince plasminogen was shown to cause the samechanges (2), and since Guest, Murphy, Bodnar,Ware, and Seegers (12) produced similar effectswith inactivated bovine plasmin. Red blood cellcounts, hematocrit and hemoglobin fluctuatedwithin normal daily limits.

Blood pressure

In many dogs, carotid blood pressure was re-corded during and after administration of plasmin.Rapid infusions of 30 plasmin units (Loomis) perKg. produced a small decrease of blood pressure(7 to 10 mm. Hg) which returned to normalwithin 10 minutes. In previous studies (2), agradual decrease in the hypotensive activity wasnoticed during daily treatment with plasmin.

DISCUSSION

It appears from this and previous studies (2)that during daily human plasmin treatment, resist-ance gradually develops toward its fibrinolytic,hypotensive. fibrinogenopenic and prothrombino-penic, but not its leukopenic effects.

Resistance to lysis is apparent from the obser-vation that most of the fibrinolytic effect is ob-tained within 24 hours after the first injection ofplasmin, the remaining portions of the clot exhibit-ing less and less lysis during the subsequent daysof treatment (2). It was found that the ability ofplasmin at a dose level of 30 plasmin units(Loomis) per Kg. to dissolve a clot is closely cor-related with the degree of organization of the clot;the older the clot, the less lysis. It may be sig-nificant that even by the third day, most of thefree edges of the clot were covered with a layer ofendothelial cells (see Figure 3). No extensivehistochemical study was made of the changes inthe fibrin mass but collagen was not demonstratedbefore the tenth day. Williams (13), in a detailedstudy of the organization of artificially producedarterial clots, noted rapid endothelialization, oc-

curring in the first 48 hours. He demonstrated a

change in the staining reaction accompanying con-

densation of the fibrin after four days. Collagenappeared in the second week. It seems probablethat the presence of endothelial cells may act as

a physical barrier between the plasmin and the

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FIG. 5. FIVE DAY OLD CLOT, SHOWINGVASCULARIZATION (HEMATOXYLIN EosIN 420 X)

FIG. 6. TEN DAY OLD CONTROLCLOT WITH EXTENSIVE VASCULARIZATION AND PARTIALDETACHMENTFROMTHE VESSEL WALL (HEMATOXYLIN EOSIN 200 X)

Note fresh blood flowing through vessel between clot and wall.

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fibrin clot. In addition, increased density of thefibrin may also impair the effect of plasmin.

The above factors may explain resistance to ly-sis, but are certainly inadequate to account for theresistance toward biochemical and hypotensivechanges. A study of changes in antiplasmin levelsand antiplasmin antibody formation is presentlyunder way. Results to date indicate that althoughchronic treatment with plasmin will induce fluctu-ations in these factors, they are insufficient to ex-plain the degree of resistance encountered.

From the therapeutic point of view, it appearsthat acute thrombo-embolic disorders present bet-ter indications for plasmin therapy than chronicconditions. Payling-Wright (14) has suggestedthe possibility that cortisone given simultaneouslywith anticoagulants may delay organization ofclots and increase the rate of recanalization ofthrombosed vessels in rabbits. On the other hand,there are indications that cortisone may increaseantiplasmin production or activity thus decreasingnormal fibrinolytic activity of the blood (15).

After successful lysis of acute clots, reformationwas observed in spite of continued treatment withplasmin. The experimental procedure called foropening the wound daily and inserting a lead shieldand scintillation counter in the area. This obvi-ously resulted in some degree of trauma and repre-sented a possible source of infection despite dailyadministration of antibiotics. In addition, inflam-matory reactions might have contributed to throm-bus formation. These new clots were nonradio-active and were detected only upon autopsy. Thefact that they did not lyse during daily treatmentwith plasmin (although they were fresh, unorgan-ized clots) may be an expression of the generalresistance developed toward plasmin as discussedabove. Under most clinical conditions, stimuli forclot reformation are undoubtedly less than in theexperimental arrangement reported here. Thepossibility of clot reformation after successfulfibrinolytic therapy should be considered, particu-larly if a) the conditions originally responsible forclot formation still persist, and b) .the temporarypresence of the clot caused additional injury to thevessel wall.

CONCLUSIONS

Venous clots were produced with I131-labeledfibrinogen in dogs at various time intervals. Five

daily treatments with 30 plasmin units (Loomis)per Kg. of human plasmin completely lysed clotsless than three days old, but did not affect thoseolder than three days. Histologic examinationrevealed that three days after formation, the clotis already largely separated from the blood streamby a layer of endothelial cells which may preventplasmin from reaching the clot. Increased clotdensity may also be a factor in the clot becomingresistant to treatment.

In some instances, although the original radio-active clot completely disappeared, reformation ofnew, nonradioactive thrombi took place. Traumamight have been a major cause of this phenome-non. Reformation of the clot occurred in spite ofdaily administrations of plasmin. This may berelated to the developing resistance of the animalto this agent as shown by decrease in changes ofthe fibrinogen levels, clotting index and bloodpressure response (2, 4, 16).

On the basis of these findings, it seems that bet-ter therapeutic results may be expected from in-travenous treatment with plasmin in acute ratherthan chronic thromboembolic disorders. In thelatter, results would appear to depend on the de-gree of organization of the clot.

ACKNOWLEDGMENT

This study was suggested by Dr. George E. Moore,Director, Roswell Park Memorial Institute, for whose ad-vice the authors wish to express their gratitude. Theassistance of J. W. Byron and Donna Halloran is grate-fully acknowledged.

REFERENCES

1. Ambrus, J. L., Back, N., Mihalyi, E., and Ambrus,C. M. Quantitative method for the in vivo testingof fibrinolytic agents: Effect of intravenous trypsinon radioactive thrombi and emboli. Circulat.Res. 1956, 4, 430.

2. Back, N., Ambrus, J. L., Goldstein, S., and Harrisson,J. W. E. In vivo fibrinolytic activity and pharma-cology of various plasmin (fibrinolysin) prepara-tions. Circulat. Res. 1956, 4, 440.

3. Ambrus, J. L., Back, N., Goldstein, S., Ambrus, C. M.,and Harrisson, J. W. E. In vivo fibrinolytic ef-fect of various proteolytic enzymes; quantitativetests employing iodine"3'-labeled clots. In press.

4. Ambrus, J. L., Ambrus, C. M., Back, N., Sokal, J. E.,and Collins, G. L. Clinical and experimental stud-ies on fibrinolytic enzymes in Ann. N. Y. Acad.Sci. 1957, 68, 97.

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5. Loomis, E. C., George, C., Jr., and Ryder, A. Fi-brinolysin: Nomenclature, unit, assay, preparationand properties. Arch. Biochem. 1947, 12, 1.

6. Remmert, L. F., and Cohen, P. P. Partial purificationand properties of a proteolytic enzyme of humanserum. J. biol. Chem. 1949, 181, 431.

7. Kline, D. L. Studies on the purification and activa-tion of plasminogen (fibrinolysin). Yale J. Biol.Med. 1954, 26, 365.

8. Sherry, S., Titchener, A., Gottesman, L., Wasserman,P., and Troll, W. The enzymatic dissolution ofexperimental arterial thrombi in the dog by trypsin,chymotrypsin and plasminogen activators. J. clin.Invest. 1954, 33, 1303.

9. Mullertz, S., and Lassen, N. An activator system inblood indispensable for formation of plasmin bystreptokinase. Proc. Soc. exp. Biol. (N. Y.) 1953,82, 264.

10. Sherry, S. The fibrinolytic activity of streptokinaseactivated human plasmin. J. clin. Invest. 1954, 33,1054.

11. Kline, D. L., and Fishman, J. B. Plasmin-the hu-

moral protease in Ann. N. Y. Acad. Sci. 1957, 68,25.

12. Guest, M. M., Murphy, R. C., Bodnar, S. R., Ware,A. G., and Seegers, W. H. Physiological effectsof a plasma protein: Blood pressure, leucocyteconcentration, smooth and cardiac muscle activity.Amer. J. Physiol. 1947, 150, 471.

13. Williams, G. Experimental arterial thrombosis. J.Path. Bact. 1955, 69, 199.

14. Payling-Wright, H. Factors influencing the re-canalisation of experimentally thrombosed bloodvessels in Proc. First International Conference onThrombosis and Embolism. Basel, Benno Schwabe& Co., 1954, p. 565.

15. Stefanini, M., and Gendel, B. R. Influence of fibri-nolysin on survival "in vivo" of various coagulationfactors and favorable effect of cortisone in "fibri-nolytic purpura." Clin. Res. Proc. 1953, 1, 5.

16. Back, N., and Ambrus, J. L. Mechanism of acquiredresistance to plasmin (fibrinolysin). Changes inanti-plasmin levels during intravenous plasmintherapy. J. Pharmacol. exp. Therap. 1957, 119,130.

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