Progress VOL XXXIV,

in

Cardiovascular

Diseases

NO 2

SEPTEMBER/OCTOBER

Thrombosis,

Fibrinoiysis, and Thrombolytic A Perspective

1991

Therapy:

Sol Sherry and Victor J. Marder

T

HROMBOSIS as a pathological entity is second to none in importance as an acute cause of serious disability and death. Furthermore, this importance will continue to increase as the average age of the population increases. Nevertheless, although a most serious public health problem, thrombosis does not receive the attention it deserves at the clinical Ievel. This is because the clinically oriented specialist views it as an independent finding within his/her domain of interest, rather than an entity that can complicate the vascular system at any site. Although there are attempts to develop a specialty of vascular medicine that could help view thrombosis as a holistic entity, it is likely that such vascular disease specialists will find it difficult to compete with the organ-oriented practitioners for the care of patients, except perhaps for peripheral vascular problems, or to significantly enhance basic research on thrombosis. The growth and development of thrombosis research in recent years has been remarkable and this has led to a balance between research and clinical practice of hemostasis and thrombosis by skilled investigators and physicians that exceeds any that existed before. Thrombosis is a very complex phenomenon; not only can thrombi differ in composition and be initiated by different mechanisms, but the process involves an extraordinary number of interrelationships between the vessel wall, platelets, the coagulation mechanism, various activators and inhibitors, the dynamics of blood flow, and the involvement of the plasminogen-plasmin enzyme system. There is no orderly process; rather, there are multiple actions going on simultaneously with modulating systems, in turn, influenced by various factors that determine Progress

in Cardiovascular

Diseases,

Vol XXXIV,

No 2 (September/October),

their contributions to the overall process. Thus, the speed, extent, and disposition of the thrombotic process is likely to differ from case to case and from organ to organ. If this complicated situation is to be understood, it must be separated into appropriate test models, each of which could be individually understood, then woven back into the fabric of the fully reconstituted, complex entity that is the hemostatic/ thrombotic system. One such part of the whole is the plasminogenplasmin enzyme system. Recent advances in thrombolytic therapy have stimulated basic research, which in turn has led to further improvements in therapeutics. The current status of physiological and clinical aspects in this field constitutes the focus for this symposium, the plasminogen-plasmin enzyme system now being recognized as a major modulator of the thrombotic process. Indeed, the thrombus is best viewed as being in a dynamic state between two opposing forces: continuing thrombus formation and thrombus resolution. It is the balance between these forces that determines the fate of the thrombus and the clinical outcome of the patient.

From the Department of Medicine, Temple University School of Medicine, Philadelphia, PA; and the Hematology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, Nk: Supported in purt by Grant No. HL-30616, National Heart, Lung and Blood Institute, National Instifu&es of Health, Bethesda, MD. Address reprint requests to Sol Sherry, MD, Distinguished Professor of ,Medicine, 4th Floor SFC, Temple Universiry School of Medicine, 3400 N Broad St, Philade?phia, PA 19140. Copyright o 1991 by W.B. Saunders Company 0033-0620/91/3402-0001$5.00/O 1991:

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The purpose of this symposium is to review the biology of the human plasminogen-plasmin enzyme system within the vascular tree and to summarize clinical results of its pharmacological activation in a variety of thrombotic disorders. Although usualIy referred to as the fibrinolytic enzyme system, its action under physiological circumstances is not restricted to the lysis of fibrin nor is it restricted to the vasculature. Plasminogen is ubiquitous in distribution, binding to many surfaces possessing specific binding sites. Local activation to plasmin can result in proteolysis of several important native substrates, for example, collagen and platelet membrane receptors, among others. In truth, the plasminogen-plasmin enzyme system represents a major endogenous proteolytic system used by the body for a number of regulatory functions, one of which is concerned with thrombus dissolution. THE BACKGROUND

PERIOD (1945-1959)

Knowledge of the “instability” of blood clots has existed for over 150 years’ but it was Dastre, a half century later, who attributed this to fibrinolysis.* There was very slow progress in understanding this phenomenon, although this was taken advantage of in Russia by Yudin for providing incoagulable blood for transfusion purposes.3 Three observations were responsibIe for opening up this field of investigation and for initiating the explosion of knowledge that continues to this day. First, Tillett and Garner discovered that hemolytic streptococci secrete a substance that mediates the rapid lysis of human blood or plasma clots.4 Originally named streptococcal fibrinolysin, but subsequently renamed streptokinase, this substance was the first to have a demonstrated capacity to dissolve fibrin rapidly. Second, Milstone noted that streptococcal fibrinolysin did not dissolve a clot made with purified fibrinogen and thrombin but did dissolve a clot formed in the presence of human protein found in plasma “euglobulin.“5 The required plasma protein was named “plasma lysing factor.” Third, Christensen and Kaplan7 independently showed that the “plasma lysing factor” is the inactive precursor (plasminogen) of the proteolytic enzyme (plasmin) and that this enzyme. is activated in the presence of streptokinase. Christensen’s work was the more

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extensive and involved characterization of the various components, including the plasma inhibitor, antiplasmin.‘*’ The terminology for this system, proposed in 1945 by Christensen and MacLoedT has remained in effect ever since. The ability to rapidIy and strikingly induce fibrinolysis by activation of this native enzyme system spurred further experimentation that followed several directions. The physiological approach was initiated by Macfarlane and Biggs’ and subsequently pursued by Kwaan and McFadzean”; the study of tissue distribution and disposition of native tissue activators was initiated by Astrup” and subsequentIy by Todd”; pharmacologic studies were begun by von Kaulla13; and therapeutic application was pioneered by Tillett and Sherry and their associates.‘4-L6 Although phenomenology dominated general coagulation research for decades, much of the early work on the plasminogen-plasmin system used biochemical techniques, which were ideally suited for the study of enzymes. This approach fostered knowledge on the isolation, purification and characterization of components,‘7-L9 kinetics of the reactions? assay of components, including methods using synthetic substrates,*’ and the basis for which plasminogen activators rather than plasmin serve best for therapeutic purposes?* In 1959, a major review of this early work provided a conceptual basis for the action of this system under physiological conditions and for its manipulation for therapeutic purposes.23 THE PERIOD OF EXPANDING RESEARCH AND THERAPEUTIC DEVELOPMENT (1960-1979)

The introduction of a potentially powerful new treatment for thrombotic and embolic disorders, namely rapid dissolution of the offending vascular obstruction by administration of a plasminogen activator such as streptokinase, now made it possible for improved management of all forms of thrombo-occlusive disease. In turn, this therapeutic opportunity excited new interest in the basic aspects of the plasminogen-plasmin enzyme system and in its therapeutic evaluation and application. This also was a period of major technical development in the laboratory (chromatography for protein purification and characterization, peptide analyzers, ultracentrifugation, tissue culture, conventional

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PERSPECTIVE

and scanning electron microscopy, gel electrophoresis, immunologic identification) and in the clinic (angiography, venography, intravascular catheterization, etc), which made it possible to expand the information pool qualitatively and quantitatively. During this period there was further clarification of the details of the fundamental mechanisms of thrombolysis,24 striking advances in the biochemistry of plasminogen and its activation,Z-34 description of an intrinsic system for plasminogen activation that complemented the more obvious extrinsic system,3s3’ elucidation of the role of streptokinase in mediating the activation of plasminogen,29334 purification and characterization of tissue plasminogen activator,3*-40 isolation and characterization of native inhibitors to plasmin,41-44 and elucidation of fibrinogen structure, its fragmentation by plasmin and the effect of these products on blood coagulation4’-” The detailed biochemical and molecular data of this period provided support and further insights for the broad conceptual outlines that were formulated in a major review published in 1959.23 At that time, there was recognition of the local orientation of the plasminogenplasmin enzyme system within the vascular bed. According to this understanding, fibrin deposition, anoxia, or vasoactive substances stimulated endothelial cell release of activator into the involved area. Because the concentration of endothelial cells per unit of vascular volume in small vessels was much greater than in large vessels, fibrinolytic activity in the capillary bed was more intense than in the larger vessels. This explained why thrombus formation was more common in arteries and veins, because the same thrombogenic stimulus would easily outbalance the fibrinolytic response. In contrast, fibrin formed within the capillary bed due to local vascular injury or filtered out by the microcirculation after formation in the blood is subjected to high concentrations of plasminogen activator, resulting in prompt lysis of the fibrin and reperfusion of the capillaries. It was apparent that the total plasmin inhibitory activity of plasma far exceeds the plasmin concentration that could be produced by such local activation of plasminogen. Thus the presumed role for the antiplasmin(s) was to prevent the escape of plasmin into the systemic circulation. A similar

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role probably exists for the inhibitors of endogenously released plasminogen activators (PAIs). This mechanism helps to circumscribe the action of the plasminogen-plasmin system to sites of vascular injury or vascular involvement, even while potent fibrinolytic activity is induced locally. As for the ability of plasma to dissolve fibrin (fibrinolysis) or a thrombus (thrombolysis), this is due to the activator concentration of plasma not to its plasmin contem2’ This special selectivity of the plasminogen-plasmin system toward fibrin dissolution without inducing fibrinogen proteolysis by plasmin is due to the binding of plasminogen to fibrin. The amount of plasmaplasminogen bound to fibrin during clot formation is relatively small (only about 4% of the available plasma plasminogen), but when converted to plasmin directly on the fibrin surface, there are no other substrates available to compete for its action and no effective neutralization of its action by plasmin inhibitors. Thus the notable feature of this enzyme system in vivo is not a selective action of plasmin but rather a selective binding of plasminogen to fibrin. Under physiological conditions this provides a mechanism by which plasmin restricts its effects to one substrate (fibrin) and in a relatively inhibitor-free environment. However, activity of this enzyme system in vivo is determined by the local concentration of plasminogen activator released by endothelial cells in response to local stimulatory events. Today we understand more clearly the molecular basis’* for the selective action of this nonspecific proteolytic enzyme system, a process that is finely tuned for enhancement on a surface, such as fibrin, but ineffective with regards to activation of plasminogen in plasma. The clinical advances kept pace with the biochemistry, physiology, and pharmacology, with notable contributions including development of the first nonantigenic thrombolytic agent (urokinase) for clinical medicine,53”5 introduction of antifibrinolytic agents for the treatment of various bleeding disorders,5b-64discovery of prourokinase,“.” extensive controlled trials with streptokinase and urokinase for acute myocardial infarction, pulmonary embolism and deep vein thrombosis,6”73 and formal approval of plasminogen activators for general clinical use.74 These important studies laid the founda-

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tion for the current phase of greatly expanded clinical progress, primarily the result of involvement by the cardiology community. THE COMING OF AGE FOR THROMBOSIS RESEARCH AND THE EXPLOSION OF CLINICAL TRIALS (1980-1990)

Of greatest importance in the surge of information regarding the therapeutic potential of thrombolytic agents was the rediscovery of coronary thrombosis as the responsible cause of the sustained ischemic event underlying acute myocardial infarction” and the direct angiographic evidence that either local or systemic infusion of a plasminogen activator induces early reperfusion.76.77With the evident advantage of rapid delivery of agent afforded by intravenous administration, data quickly followed that clearly demonstrated functional and clinical benefit in the form of limitation of infarct size78-80and reduced mortality.79~81-85Because acute myocardial infarction is second to none as a major public health problem in the aging population, thrombolytic therapy for this disorder has gained worldwide recognition and has led to the development of second generation thrombolytic agents.86-90Each plasminogen activator has in turn spawned a unique area of research interest in relation to its pharmacology, clinical use, and, where appropriate, human biology. Again, the extraordinary progress during this period was made possible by new scientific techniques, the most striking of which was in the discipline of molecular biology involving recombinant DNA technology and immunology. New studies allowed for definition of PAI’s,~~-~~more accurate and specific measurements of circulating activators and inhibitors and their complexes95-101and of antifibrinolytic activities in response to physiological and abnormal stimulastructural analyses of the proteins,31’87.‘08-“2 ~ion,1me’07 relationships of plasminogen and plasminogen activator on vascular surfaces and understanding of the effects of plasmin formation at these sites,“3~‘z’ identification of hereditary molecular deficiency or dysfunctional states and their clinical effects,1u-129 improvements in drug design for second and third generation thrombolytic agents,Bp90and the preparation of large quantities of highly purified native activators for therapeutic purposes.87.13a’” Also, new drug regimens have been tested for the

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treatment of pulmonary embolism135-137and peripheral arterial thrombo-occlusive events,138-142 and clinical investigations have been initiated in other coronary syndromes, such as unstable angina and non-Q wave infarctions. Insights from clinical trials have reaffirmed that aged thrombi are more resistant to lysis than fresh thrombi or emboli, as is now evident in the treatment of acute myocardial infarction,120~‘43and that bleeding is more the result of hemostatic plug dissolution,‘” less related to the extent of hypofibrinogenemia.w,7’,145S16 Other data have altered previously held concepts. For example, we now recognize that striking fibrinogenolysis may be an advantage in thrombolytic therapy rather than a disadvantage because it does not cause more severe or more frequent bleeding complications but is associated with a lower likelihood of rethrombosis.‘Z03’4”1a Furthermore, mortality reduction in myocardial infarction may not depend solely on infarct size reduction but may also depend on late reperfusion (after the infarct is completed)‘47~‘49~151and that 90minute patency rates have provided misleading information, because they do not correlate with observations on myocardial salvage or mortality reduction.‘47,‘52-‘55 There has been recognition of the importance of early rethrombosis as a significant problem in achieving lasting vascular patency and full clinical benefit. Early rethrombosis is not prevented by heparin,156 but early and continued heparin infusion and oral aspirin may both improve survival rates after acute myocardial infarction.83 None of the plasminogen activators administered via the intravenous route has documented clinical superiority in safety or efficacy over other agents for thrombotic disorders, especially for acute myocardial infarction, and each has its respective advantages and disadvantageS~0.120,152.157,158 One exception to this rule may exist, as urokinase has been claimed to be the for local perfusion of periphagent of choice’59X160 eral arterial thrombo-occlusions. Whichever agent is applied for acute myocardial infarction, the best therapy is applied early after symptom onset, because treatment initiated after the opportunity to salvage heart muscle achieves less impressive results in mortality reduction. To fulfill the potential that thrombolytic ther-

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apy offers will require mechanisms for instituting therapy as quickly as possible. THE NEXT

DECADE

(1991-2000)

Impressive progress has been made in the last 45 years to realize the potential of plasminogen activators as valid therapeutic agents for all manner of thrombotic disease.12’ Yet, issues remain to be settled, therapy is far from ideal, and even accepted and proven indications are not fully applied to the patient population. There still is vigorous debate as to whether one plasminogen activator is preferable to another, despite the lack of clinical data to justify such a choice. Thus, a major challenge for the clinician wilI be to choose between three, four, or more excellent plasminogen activators and a multitude of regimens using activators with various anticoagulants, as all therapeutic options are not likely to have been compared with any single study. The action of plasminogen activators on the thrombus, blood, and hemostatic plug is shown in Fig 1, which schematically compares results obtained with the more “fibrinogen-sparing” (“fibrin-selective”) agents, such as rt-PA and prourokinase (scu-PA) with those that induce a marked degradation of plasma fibrinogen, such as streptokinase, urokinase, and anisoylated plasminogen-streptokinase activator complex (APSAC).“” For patients treated 3 or more hours after symptom onset, the more fibrinogensparing (fibrin-selective) agents induce reperfusion more rapidly than the agents that induce a potent lytic state. However, along with the lesser degree of lytic state associated with rt-PA therapy, there is also a greater tendency for rethrombosis and reocclusion and the cumulative patency rates of all of the agents at 3 hours14’ or at 24 hours’“’ after treatment induction are the same. Thus, the greatest need for improving the efficacy of streptokinase, urokinase, and APSAC is to achieve more rapid thrombolysis and reperfusion, whereas that for rt-PA is to achieve more lasting patency, free of the greater tendency for reocclusion. Although single-chain urokinase (scu-PA) similar to single-chain rt-PA has a fast action in vivo for lysing thrombi, it differs from rt-PA when administered intravenously in large amounts, for example 80 mg is equivalent to

93

12.4 million urokinase units. With such large doses, a portion of the activator is rapidly converted to urokinase in the circulation. The result is a systemic lytic state that is much more extensive than observed with rt-PA and a low reocclusion rate.162.‘63Whether amelioration of efficacy will result from better adjunct anticoagulant treatment, from a new regimen of plasminogen activator, from combined use of two plasminogen activators,‘64.‘” or from a new plasminogen activator altogether, awaits further study. New plasminogen activators include unique proteins that combine a catalytic site with an antifibrin antibody,“’ mutant deletions of rt-PA that have a prolonged half-life in blood,‘67 and a recombinant bat salivary gland plasminogen activator with an exceptionally high fibrinogen-sparing tendency.‘68 However, in vitro biochemical advantage and even superiority in animals or in human vascular reperfusion studies do not necessarily ensure functional or clinical superiority of such innovative agents over existing activators. An especially recalcitrant negative aspect of treatment is the increased risk of hemorrhage over and above that which attends heparin treatment.74.‘69”70 None of the agents thus far used in human disease lacks this bleeding propensity. Although an agent with exceptionally high fibrinogen-sparing capacity has not yet been available for treatment and theoretically could be safer, it seems unlikely that hemostatic plugs will resist their action and protect against bleeding from sites of vascular injury.‘58 The most dreaded complication is intracranial hemorrhage, an event that has been noted in patients with a prior history of a cerebrovascular accident, especially of hemorrhagic nature. Perhaps the last frontier for potential use of thrombolytic treatment is in patients who have fresh thrombotic occlusions of the carotid cerebral, basilar, or vertebral arteries. Long a subject of clinical research interest,“‘.“’ a renewed effort in patients with this potential indication concentrates on very early treatment and on ruling out a nascent hemorrhagic component with computerized tomographic scanning.‘73,‘74 If the early encouraging data hold,“’ then perhaps a subset of patients with thrombotic cerebrovascular accidents can be identified and saved from disabling or life-threatening disease.

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A

B

C ‘,

.:.. :.

;.:

,::.:.. . . . .

Fig 1. Relative effects of plasminogen activators in a vessel occluded with thrombus and in which a site of prior trauma is sealed with a hemostatic plug. Two groups of plasminogen activator are compared. those with the attribute of greater “fibrinogen-sparing” (“fibrin selectivity“) (t-PA and scu-PA) and those with more potent effects on the blood coagulation and flbrinolytic proteins (SK, UK, APSAC). The latter group causes a greater decrease in plasma fibrinogen, as indicated schematically by the lower density of dots, but both types of plasminogen activator induce bleeding at the same rate and to the same degree by virtue of hemostatic plug dissolution. Assuming the occluded vessel is a coronary artery in a patient with acute myocardial infarction, reperfusion follows at approximately equal rates and speed for “fresh” thrombi of 2 hours duration, but more rapid lysis is achieved with the more fibrinogen-sparing (fibrin-selective) agents when thrombi are older than 3 hours. Thus, at phase A (approximately 90 minutes after initiation of treatment), t-PA and scu-PA will have achieved a higher rate of vascular reperfusion than SK, UK, and APSAC, whereas with t-PA the blood fibrinogen concentration will not have decreased as much. However, this does not occur with scu-PA, because it is partially converted to UK in the circulation, resulting in a potent lytic state. By 3 to 24 hours after treatment initiation (phases Band C), thrombus exposed to the nonfibrinogen-sparing agents also dissolves, producing a stable condition of a striking lytic state and persistent vascular patency. With t-PA, there is a higher tendency for vascular reocclusion (shown in phase C), perhaps related to the lesser degree of fibrinogen degradation and blood hypocoagulability. However, scu-PA therapy is associated with blood hypocoagulability and a low incidence of rethrombosis. The final patency status (70% to 80%) resulting from slower, progressive and stable reperfusion induced by SK, UK, and APSAC is equivalent to that which follows more rapid reperfusion counterbalanced by increased rethrombosis associated with &PA or scu-PA.

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The greatest potential for expanding clinical benefit to many more patients does not require an innovative plasminogen activator, a new biochemical insight, or a large-scale clinical trial. At present, less than 15% of patients with acute myocardial infarction receive thrombolytic treatment with any agent,‘76 but an expanded program of thrombolytic treatment may be possible, for example, using early administration by ambulance personnel.“’ In addition, if

increased use in the at-risk population could be achieved within 2 or 3 hours after symptom onset, then significantly more patients could be spared short (1 month) or long-term (1 year) lethal outcomes from acute myocardial infarction. Such an accomplishment would represent the full fruition of research on the plasminogenplasmin enzyme system, initiated nearly 60 years ago with the identification of streptococcal fibrinolysin.4

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17. Kline DL: Purification and crystallization of plasminogen (profibrinolysin). J Biol Chem 204:949-955,1953 18. Alkjaersig N, Fletcher AP, Sherry S: Activation of human plasminogen. 1. Spontaneous activation in glycerol. J Biol Chem 233:81-85,1958 19. Shulman S, Alkjaersig N, Sherry S: Physicochemical studies on human plasminogen (profibrinolysin) and plasmin (fibrinolysin). J Biol Chem 233:91-97,1958 20. Alkjaersig N, Fletcher AP, Sherry S: Activation of human plasminogen. 2. A kinetic study of activation with trypsin, urokinase and streptokinase. J Biol Chem 233:8690,1958 21. Troll W, Sherry S, Wachman J: Action of plasmin on synthetic substrates. J Biol Chem 208:85-93,1954 22. Alkjaersig N, Fletcher AP, Sherry S: The mechanism of clot dissolution by plasmin. J Clin Invest 38:1086-1095, 1959 23. Sherry S, Fletcher AP, Alkjaersig N: Fibrinolysis and fibrinolytic activity in man. Physiol Rev 39:343-382, 1959 24. Wiman B, Collen D: Molecular mechanisms of physiological fibrinolysis. Nature 272:549-550,1978 25. Summaria L, Hsieh B, Robbins KC: The specific mechanism of activation of human plasminogen to plasmin. J Biol Chem 242:4279-4283,1967 26. DeRenzo EC, Siiteria PK, Hutchings BL, et al: Preparation and properties of highly purified streptokinase. J Biol Chem 242:533-542, 1967 27. Deutsch DG, Mertz ET: Plasminogen: Purification from human plasma by affinity chromatography. Science 170:1095-1096,197O 28. Rickli EE, Cuendet PA: Isolation of plasmin-free human plasminogen with N-terminal glutamic acid. Biochim Biophys Acta 250:447-451, 1971 29. Castellino FJ, Soditz JM, Brockway WJ, et al: Streptokinase. Methods Enzymol45:244-257, 1976 30. Robbins KC: The human plasma fibrinolytic system: Regulation and control. Mol Cell Biochem 20:149-157, 1978 31. Sottrup-Jensen L, Claeys H, Zajdel M, et al: The primary structure of human plasminogen: Isolation of two lysine-binding fragments and one “mini”-plasminogen (MW 38,000) by elastase-catalyzed specific limited proteolysis, in Davidson JF, Rowan RM, Samama MM, et al (eds): Progress in Chemical Fibrinolysis and Thrombolysis, vol 3. New York, NY, Raven Press, 1978, p 191 32. Dayhoff MO: Atlas of Protein Sequence and Structure, vol 5. Silver Spring, MD, National Biomedical Research Foundation, 1978, p 91 (suppl3)

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33. Castellino FJ, Violand BN: The fibrinolytic systembasic considerations. Prog Cardiovasc Dis 21:241-254, 1979 34. Castellino FJ: A unique enzyme-protein substrate modifier reaction. Plasministreptokinase interaction. Trends Biocbem Sci 41-51979 35. Kluft C, Trumpi-Kalshoven MM, Jie AFH, et al: Factor XII-dependent fibrinolysis. A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII and kallikrein dependent plasminogen proactivator. Thromb Haemost 41:756-773,1979 36. Colman RW: Activation of plasminogen by human plasma kallikrein. Biochim Biophys Acta 35:273-279,1969 37. Mandle R Jr, Kaplan AP: Hageman factor substrates. Human plasma prekallikrein: Mechanism of activation by Hageman factor and participation in Hageman factordependent fibrinolysis. J Biol Chem 252:6097-6104,1977 38. Binder BR, Spragg J, Austen KF: Purification and characterization of human vascular plasminogen activator derived from blood vessel perfusates. J Biol Chem 254:19982003,1979 39. Cole ER, Bachmann F: Purification and properties of a plasminogen activator from pig heart. J Biol Chem 25213729-3737, 1977 40. Rijken DC, Wijngaards G, Zaal DeJong M, et al: Purification and partial characterization of plasminogen activator from human uterine tissue. Biochim Biophys Acta 580:140-153,1979 41. Mullertz S, Clemmensen I: The primary inhibitor of plasmin in human plasma. Biochem J 159:545-553, 1976 42. Wiman B, Cohen D: Purification and characterization of human antiplasmin, the fast-acting plasmin inhibitor in plasma. Eur J Biochem 78:19-26,1977 43. Wiman B, Collen D: On the mechanism of the reaction between human a,-antiplasmin and plasmin. J Biol Chem 254:9291-9297,1979 44. Moroi M, Aoki N: Isolation and characterization of a,-plasmin inhibitor from human plasma: A novel proteinase inhibitor which inhibits activator-induced clot lysis. J Biol Chem 251:5956-5965,1976 45. Fletcher AP, Alkjaersig N, Sherry S: Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (“fibrinolytic”) states. I. The significance of fibrinogen proteolysis and circulating fibrinogen breakdown products. II. The significance, mechanism and consequences of defective fibrin polymerization. III. Demonstration of abnormal clot structure by electron microscopy. J Clin Invest 41:896-948,1962 46. Latallo ZS, Fletcher AP, Alkjaersig N, et al: Inhibition of fibrin polymerization by fibrinogen proteolysis products. Am J Physiol202:681-686,1962 47. Triantaphyllopoulus DC: Anticoagulant effect of incubated fibrinogen on blood coagulation. Can J Biochem Physiol36:249-259,1958 48. Nussenzweig V, Seligmann M, Grabar P: Les produits de degradation du fibrinogene human par la plasmine. II. Etude immunologique: Mise en evidence d’anticorps anti-fibrinogene natif posstdant de specifites differentes. Ann Inst Pasteur 100:490-508,1961 49. Marder VJ, Shulman NR, Carroll WR: High molecular weight derivatives of human fibrinogen produced by plasmin. I. Physicochemical and immunological characteriza-

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