Fibrinolytic mechanisms and the development of thrombolytic therapy

Fibrinolytic Mechanisms and the Development of Thrombolytic ANTHONY P. FLETCHER,

Therapy*

M.D., NORMA ALKJAERSIG,

M.S. and SOL

SHERRY,

M.D.

St. Louis, Missouri

I

N recent years it has become apparent that the fibrinolytic process in man is in a much more dynamic state than previously recognized and, as a result, aberrations in its mechanisms may contribute significantly to the pathogenesis of disease. Such considerations, along with the interest in the development of thrombolytic agents, currently makes the study of fibrinolytic phenomena a most fertile area for clinical investigation. Investigative interest and the corresponding increase of publication has been of such explosive growth that any short review, particularly one covering a broad field of physiologic and pathologic phenomena, must necessarily be highly selective and reflect in great measure the authors’ views. t More detailed reviews [7-91 and symposium reports [X-72] are available to the interested reader. It will be our purpose to review current concepts concerning physiologic fibrinolytic and thrombolytic$ mechanisms, recent application of these concepts to the understanding and treatment of pathologic “fibrinolytic” states and finally, the presently controversial but nevertheless promising field of thrombolytic therapy. INTRODUCTORY

140,000 [73],$ which is distributed in both intravascular and extravascular spaces and is also found in various secretions. Conversion of plasminogen from its precursor state to the proteolytic enzyme plasmin (molecular weight 108,000) 0 is accompanied by the splitting off of peptides [78]. Plasmin is a proteolytic enzyme of the trypsin class and degrades many native protein substrates among which are fibrinogen, fibrin, factor v, factor VIII, some components of complement, corticotrophin, growth hormone and glucagon. Additional protein substrates, some of which are used for assay purposes include: casein, gelatin, /3 lactoglobulin, azocol hide powder and protamine complexes. PLASMINOGENACTIVATORS Plasminogen-plasmin conversion is an enzymatic reaction mediated by kinases or activators. Plasminogen activator is found in plasma (plasma activator), in urine (urokinase), in various tissues including the vessels (tissue activator) and in body secretions. Although the relationship or possible identity of these activators is presently unknown, there is physiologic evidence to suggest that urokinase may represent excreted plasma activator, and preliminary biochemical evidence suggests some similarity between tissue activator and urokinase. The simplest theory, which would account for these

CONSIDERATIONS

In viva fibrin lysis is controlled by an enzymatic process which involves the conversion of an enzyme precursor plasminogen into a proteolytic enzyme plasmin, a reaction mediated through activators or kinases. Plasminogen is a serum globulin with a molecular weight of

$ Although investigators are agreed that the S2o.w for plasminogen prepared by the acid extraction process [74-751 is approximately 4.3, disagreement as to the diffusion constant has caused one group of investigators to calculate a molecular weight of 83,800 [Xl. Plasminogen, prepared by DEAE column chromatography [77] which, unlike that prepared by acid extraction, is soluble at neutral pH also has an SzO,w of 4.3 at pH below 4 but physicochemical characterization of this apparently native protein is still incomplete.

t “Error of opinion may be tolerated where reason is left free to combat it.” THOMAS JEFFERSON, 1801. $ The term “thrombolysis” will be used to describe the reaction by which thrombi (or clots) are lysed. The will be used to designate bioadjective “thrombolytic” chemical moieties capable of inducing thrombolysis.

* From The Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri. This work was supported by Grant H 3745 from the National Heart Institute, U. S. Public Health Service, Bethesda, Maryland.

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Gel Phase Plasminogen

I

I1

I. Transport Mechanism Plasminogen

2. Plasminogen

Activation

Activation

Plasmin __ _ _ _ __ Antiplosmin ____----

Plasmin 3. Plasmin

Inhibition

Antiplasmin 4 Susceptible

Substrates

( Fibrinogen,

FactwsY

Usually FIG. 1. Schematic

representation

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by Inhibitors

1 Thrombolysis

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is that tissue activator is the primary source, that plasma activator is its product and that urokinase is its excretion form. However, evidence on this point is almost non-existent and highly purified urokinase displays a degree of biophysical heterogeneity that suggests the presence of several multimolecular form enzymes [ 191. Bacterial activators, of which the best known are streptokinase and staphylokinase, have also been isolated. Intensive developmental work has culminated in the preparation of highly purified streptokinase, which has undergone extensive clinical trial as a thrombolytic agent. Other modes of plasminogen activation include activation by trypsin [18] and autocatalytic activation in glycerol [ZO] or under other circumstances [27]. Some recent evidence [22,23] suggests that, in viva, Hageman factor (factor XII) may play some indirect role in plasminogen activation. Inhibitors. Plasma and other body fluids contain powerful inhibitors to plasmin which subserve important physiologic functions. These inhibitors [24-271 have not been adequately characterized but one is contained within the alphai and another within the alpha2 globulin plasma fractions. Norman’s evidence [26] would suggest that one inhibitor reacts immediately with plasmin, while the other reacts more slowly, in a definite proportion, as the result of a temperature sensitive reaction. The hypothesis that the inhibitory effect of plasma upon plasmin findings,

Protected

8YllI)

activation.

may be exerted by a dual mechanism would account for most of the known facts, but details are uncertain and the number and types of reactions leading to plasmin inhibition controversial (for review, [d]). Platelets also contain antiplasmin [28,29], and plasma may also destroy or inhibit plasminogen activator; whether this latter property is due to the presence of a specific inhibitor, enzymatic degradation or some other mechanism is hypothetical. Normally plasma antiplasmin averages approximately 5 casein inhibitory units per ml. and exceeds the plasminogen content of plasma (3.8 casein units per ml.) [30]. The Dual Phase Concept of Plasminogen Activation. Enzymatic reactions of the proteolytic type are essential to biological function. Nevertheless when such reactions occur in the extracellular compartments and involve enzymes, such as plasmin, which possess relatively nonspecific substrate requirements, they present peculiar hazards to the organism. Indeed untrammelled and inappropriate plasma proteolysis produces serious disease complications section on “Pathologic Plasma ( see later Proteolysis”). However in viva, because of certain biological properties of the plasminogen-plasmin this relatively non-specific enzyme system, normally produces a single highly specific action-that of fibrin lysis. The mechanism underlying this action [3,30-321 is shown schematically in Figure 1.

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Following its synthesis, plasminogen is distributed in the extravascular spaces and the plasma; its concentration usually is roughly proportionate to the fibrinogen concentration of the fluid in which it is found. When clotting occurs, either extra or intravascularly, the fibrinous deposit or thrombus will be found to contain, on analysis [37], substantial quantities of plasminogen which is in intimate spatial relationship with the fibrin fibrils. Although the physicochemical nature of the affinity between plasminogen on the one hand and fibrinogen and fibrin on the other has not been elucidated, the evidence for the existence of such a phenomenon is strong: not only do clots or thrombi formed from plasminogen-rich fluids always contain substantial concentrations of plasminogen, but clots rendered plasminogen-poor rapidly take up plasminogen on immersion in plasma [33]. Furthermore, fibrinogen is only separated with considerable difficulty from plasminogen except when specific solubilizing agents for plasminogen are used [34,3fl. Consequently, as shown in Figure 1, plasminogen in a plasma-clot system exists, in a physical sense, as a dual phase system; plasminogen in plasma constitutes the soluble phase and plasminogen in the clot the gel phase. As a result of this physical distribution, the biochemical consequences of plasminogen activation in the two phases are entirely different. Plasminogen activation in the soluble or plasma phase (provided that it is slow) produces no detectable effects on susceptible substrates contained in plasma, for plasmin is rapidly inhibited by plasma antiplasmin on its formation. (See center section of Fig. 1.) However, if rapid activation of soluble plasma plasminogen occurs, susceptible substrates, of which fibrinogen is the chief, will be destroyed and a severe coagulation defect and hemorrhagic diathesis may be produced (see later section). Plasminogen activation in the clot or gel phase (right hand section of Fig. 1) produces an entirely different result: fibrin is now the major substrate, and thrombolysis or clot dissolution proceeds, because of the intimate spatial relationship of clot plasminogen and fibrin fibrils, relatively independent of plasma inhibitors. In this manner the actions of the enzyme although themselves relatively nonplasmin, specific with respect to substrate requirements, may in vivo be restricted to a circumscribed locus and a particular substrate. These findings

et al.

[30-32,361, besides greatly enhancing physiologic understanding of the in vivo actions of the plasminogen-plasmin system, provide the key to therapeutic thrombolysis. Application of In Vivo Plasminogen Activation. these concepts at the clinical level are illustrated by the data displayed in Figure 2. The left hand panel describes a study in a patient receiving streptokinase therapy; during the first three hours the effect of the activator infusion was predominantly on soluble phase plasminogen, and plasma plasminogen concentration fell to low levels while thrombolytic activity was not increased. Because soluble phase plasminogen was being activated rapidly, inhibition of plasmin by antiplasmin was only partially effective and the plasma fibrinogen fell 30 per cent. However when plasminogen had fallen to low levels, the fall of plasma fibrinogen was arrested yet a rapid increase of thrombolytic activity now occurred. The middle panel reveals a similar study in a patient receiving a urokinase infusion; in contrast to the finding urokinase induced prewith streptokinase, dominant gel phase plasminogen activation. Consequently thrombolytic activity was substantially increased with only a minor fall of plasma plasminogen. Clinical findings of this nature have confirmed the significance of an extensive in vitro investigation [37] concerning the relative fibrinogenolytic and fibrinolytic actions of various plasminogen activators in a plasma clot test system. This work, which made use of isotopically-labelled substrates, demonstrated that distinct and quantitatively important differences existed between various plasminogen activators relative to the ratio of their action on soluble and gel phase plasminogen. Moreover important evidence of increased gel phase plasminogen activation in the absence of soluble phase plasminogen activation has been derived from two studies in man, the one concerned with procedures enhancing thrombolytic activity [32] and the other with its occurrence in patients [38]. However in certain pathologic plasma proteolytic states, high levels of circulating activator will produce activation of plasminogen in both of any preferential action phases, regardless of the activator on gel phase plasminogen: the instructive investigations of Johnson and McCarty [39] and others [30,40,41] are pertinent in this regard. The right hand portion of Figure 2 displays AMERICAN

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FIG. 2. The effects of the intravenous administration of plasminogen plasminogen, fibrinogen and thrombolytic activity of patients (see text).

an important and often neglected characteristic of in vivo plasminogen-plasmin activation. It illustrates, in a patient given a streptokinase the converse phenomenon to that infusion, shown in the left hand panel of the figure. On the left, plasminogen activation was relatively rapid and susceptible substrates, chiefly fibrinogen, were degraded. On the right, plasminogen activation was induced at a rate slow enough to allow for almost complete inhibition of plasmin by plasma antiplasmin; as a result, substrate degradation was not detected over a six-hour period nor did significant thrombolytic activity develop. Yet it could be calculated that during the six-hour experimental period, over 5,000 casein units of plasmin had been formed in vivo, an amount approximately ten times as great as that employed by most advocates of plasmin therapy. These data, in concert with other investigasupport the view that, in vivo, tions [3>32], thrombolysis is a function of the plasminogen activator concentration locally present around which, by virtue of activating a thrombus, gel phase plasminogen, produces thrombolysis without necessarily disturbing soluble phase plasminogen; however activation of plasma plasminogen may also occur if the activator concentration is maintained at high levels. A contrary theory [42,4?] suggesting that preferential dissociation of the plasmin-antiplasmin complex may occur in the presence of fibrin and in this way produce thrombolysis in VOL.

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activators

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the absence of activator, appears to lack a sound biochemical basis [31,37]; it is inadequate to explain many well established experimental and clinical phenomena and further evidence to support its validity is lacking. Site and Mechanism of Plasminogen Activator Release. Although plasminogen activator may be extracted [&I from all tissues, except the liver and placenta, and some tissues are notably richer in this substance than others [45,&l, evidence is lacking to suggest a specific site or sites for activator synthesis. However, there is clinical evidence [47-491 to indicate that the liver may be a site for activator destruction. This is supported by observations on cirrhotic patients who responded differently from normal subjects to a stimulus (nicotinic acid or electroshock) designed to elicit thrombolytic activity. Not only did the cirrhotic patients show a much greater response to the stimulus, but the response was also of much longer duration [50,57]. An important problem concerns the stimulus for, and the nature of, plasminogen activator release. Essentially, there is uncertainty as to whether the constant but low level of plasminogen activator, present in normal plasma [38], is sufficient to induce lysis of microthrombi, or whether the deposition of a microthrombus causes a local release of activator. Intellectually, the theory of local activator release as a response to fibrin deposition or thrombosis is an attractive one, for it would provide a ready explanation for experiments

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such as those in which infusion of thrombin or thromboplastin failed to induce obvious intravascular coagulation [52,53], and those in which pulmonary emboli, produced in rabbits, were lysed without detectable evidence of systemic plasminogen activation [54-561. Kwaan, Lo and McFadzean [57-60] have been particularly zealous in championing the view that thrombus formation itself stimulated local activator release through a mechanism triggered by the lysis of platelets undergoing viscous metamorphosis. However, some of their experimental findings have not been confirmed, and the general significance of the work is still uncertain since the degree of activator release studied was always moderate and sometimes slight. Moreover Astrup and co-workers [67] have demonstrated that, in arteries, plasminogen activator is located primarily in the adventitia rather than in the media and intima; concentration in the latter sites would be expected if local mobilization of activator was a reality. Relationship of Thrombotic Phenomtxa and Failure of Plasminogen System Activity. From a broad etiologic viewpoint, clinical thrombosis may be regarded as arising from disease of the vessel wall, hypercoagulability of blood (either primary or secondary, e.g., due to stasis, etc.), or by inhibition or failure of plasminogen-plasmin system activity. Since factors enhancing the coagulability of blood are amply reviewed in other papers in this symposium, we shall restrict our comment to possible failure or inhibition of plasminogen system activity as a cause of thrombotic phenomena; this aspect of the subject has been the focus of considerable speculation. Unfortunately, to date, because of the technical difficulties inherent in the problem of assaying plasma thrombolytic activity, only semiquantitative methods for measurement of this parameter have been employed, and data on its clinical variation are meager and unsatisfactory. However there is agreement that, in general, plasma thrombolytic activity appears to be somewhat reduced in patients suffering from either present or past thrombotic episodes [62-66]. The problem is one of considerable difficulty for, although quantitative methods using radiochemical assays [38] are available, they are complex and may not possess sufficient sensitivity for measuring the reduced levels inferred to exist in some types of disease.

et al.

However an indirect approach, utilizing urokinase as an indirect assay of urinary measure of presumed plasma activator levels, has yielded provocative results. In patients suffering from disease in which thrombotic episodes are common, e.g., carcinomatosis and heart failure, urokinase excretion rates were significantly decreased as compared to control subjects (P < 0.001); however, in patients recovering from myocardial infarction urokinase excretion rates were significantly (P < 0.001) increased [67]. The findings with respect to carcinomatosis have been confirmed [68], but further evidence as to the validity of the approach is required. An extremely interesting case history of a patient who suffered from multiple clinical thrombotic episodes and who showed an apparent great increase of a plasma plasminogen activator inhibitor has been published by Nilsson et al. [69]. Confirmatory work is eagerly awaited. However this phenomenon would seem to be rare, as our laboratory, using methods essentially similar to those of Nilsson, has so far failed to find a similar patient among many who were admitted to the hospital with a history of multiple thrombotic episodes. The possible relationship of altered plasminogen system activity to the onset of thrombosis has many physiologic and pathologic implications, particularly with respect to the problem of atherosclerosis; it is regrettable that current knowledge is so incomplete. Perhaps the main difficulty in this respect stems from the present uncertainty as to whether fibrinogen turnover involves a phase of continuous intravascular fibrin deposition with subsequent lysis (the so-called hemostatic balance theory [Z]), and whether, if such is the case, fibrin lysis occurs through the mediation of circulating plasminogen activator or through locally released activator. If the latter mechanism is of predominant importance, present assay methods may be inadequate to detect its activity. Because of these uncertainties, the equally important field of blood lipids and their influence on plasminogen system activity has been greatly impeded and knowledge is confined to in vitro experiments [70-74 and some highly artificial in vivo situations [73,74]. Pathologic Plasma Proteolytic States. A clinical syndrome characterized by a coagulation defect, hypofibrinogenemia (sometimes “afibrinogenemia”), often an associated hemorrhagic diathesis AMERICAN

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Fibrinolytic Mechanisms and Thrombolytic and manifestations of whole blood or plasma “fibrinolysis” (spontaneous lysis of whole blood or plasma clots) has long been recognized. The condition, usually of acute onset, may develop following surgery, particularly thoracic surgery; may be associated with obstetric complications, particularly abruptio placenta; may develop rarely during the course of neoplastic disease but more commonly if the prostate is involved; and may complicate the course of hepatic cirrhosis or certain other medical conditions, most frequently in their terminal stages [for references see 4,471. More recently an iatrogenic form, arising secondarily to thrombolytic therapy [30,39-47, 75,751, has assumed considerable importance. Previously such terms as “pathologic fibrinolytic state” or “fibrinolysis as a disease state,” have been used to describe this condition because the diagnosis was usually suggested by the observation of rapid whole blood or plasma clot lysis in specimens obtained from the patient. However since this phenomenon involves the action of proteolytic rather than strictly fibrinolytic enzymes, and rapid whole blood or plasma clot lysis is not invariably present, descriptive terms involving the word proteolytic rather than fibrinolytic are preferable. Descriptions such as “pathologic proteolytic state” or “excessive plasma proteolytic activity” not only define the condition more accurately but focus attention on its wider ramifications. The pathogenesis of the severe coagulation defect and sometimes fatal hemorrhagic diathesis that may develop in association with pathologic plasma proteolytic states has hitherto been obscure. Although many specific coagulation factors, notably factors v, VIII and IX, prothrombin and plasma thromboplastin, have been reported as susceptible to plasmin degradation, the evidence has been contradictory and agreement as to susceptible substrates limited to factors v and VIII. Fibrinogen is another plasmin substrate, and many patients with this syndrome have been reported to show afibrinogenemia. Consequently the coagulation defect has been attributed usually to specific depletion of coagulation factors complicated by hypo- or afibrinogenemia. However recent investigation [47,75,76] of the iatrogenically-induced coagulation disorders that may complicate thrombolytic therapy with plasminogen activators has thrown fresh light on this anomaly and suggested means for its more effective treatment. Thrombolytic therapy, VOL.

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with streptokinase, causes rapid activation of plasminogen so that within three to six hours of starting the infusion, plasma plasminogen is reduced to zero or trace levels [31]. During this phase of rapid plasminogen activation, plasma fibrinogen* may fall some 30 per cent, and onestage prothrombin and thrombin clotting times are increased to a variable degree. There is no significant alteration in platelet count or capillary fragility, but factor v concentration is reduced to approximately 50 per cent of normal [39,47]. Occasionally, if plasminogen activation occurs very rapidly, these findings may be grossly exaggerated, and a clinically evident hemorrhagic diathesis develops with a predilection for traumatized or diseased tissue [39]. An important parameter of this coagulation defect and, in conjunction with other data, the key to its elucidation has been the observation that a close correlation always existed between increase of the one-stage prothrombin time and the thrombin clotting time: a relationship showing a high degree of statistical significance in a large series of patients [39]. This suggested, together with the findings of a relatively normal recalcification time [47], that the main coagulation anomaly occurred at the stage of fibrinogen-fibrin conversion by thrombin. Since fibrinogen was the major plasma protein degraded during plasma proteolytic states [39], and its proteolysis products increase the thrombin clotting time of plasma or fibrinogen solution, to which they are added [78,79], it seemed probable that the formation of fibrinogen proteolysis products, rather than any other cause, might underlie the development of the coagulation defect. This hypothesis was confirmed by demonstrating that (1) the laboratory features of the coagulation anomaly could be reproduced in vitro by the addition of fibrinogen proteolysis products (prepared by plasmin digestion) to plasma; (2) the plasma of patients, exhibiting the defect contained fibrinogen proteolysis products; and (3) calculation of fibrinogen proteolysis rates in patients treated with strepto* Conventional plasma fibrinogen methods yield erroneously low values in patients receiving thrombolytic agents or suffering from pathologic plasma proteolytic states. Modification of standard fibrinogen methods, which yield accurate results in these circumstances, have been described [30,77], and their use is essential in patient management for otherwise standard methodology will suggest that many patients are suffering from q’afibrinogenemia” when such is not the case.

Fibrinolytic Patholoqico

Mechanisms I

and Thrombolytic

Etiologicol Agent

Plasma

Therapy--Fletcher

Biochemical

Cooqulotion

Consequence

Defect

et al.

Clinic01 Presentation

Proteolytic Stote

Pure

Form

Activator

2. Some Clinic01

_

Fibrinogen

Plosminogen

I. Iotrogenic

--+

Defective

_

Hemorrhagic Diothesis

Fibrin

Proteolysis

Polymerization

t+

Cases

Complex

Form

I, Obstetric

Cases

2.Surgicol

Cases

Moteriol

Activator

FIG. 3. Pathogenesis

Tendency

lntrovosculor

Thromboplostic +

tt

Coagulation

-W

Proteolysis-

of the various types of pathologic

kinase were in agreement with those predicted from the coagulation data [47]. Purification studies on the fibrinogen digests were undertaken and a large molecular weight fibrinogen fragment (5.27 S~O,~), stable against was isolated: this fraction alone plasmin, reproduced the coagulation anomaly when added to plasma [751. In vivo the proteolysis product was found to have a 50 per cent plasma clearance time of 9.5 hours [N]. The proteolysis product had no inhibitory actions on thrombin, but inhibited the polymerization and gelling of fibrin monomer, and its concentration in the final clot was found to parallel the severity of the coagulation defect [751. Electron microscopy confirmed the hypothesis of disordered clot of polymermorphology [76], and determination ization rates, using fibrin monomer, permitted further quantitation of the polymerization anomaly [80,81]. Consequently the biochemical lesion in the iatrogenically-induced coagulation defect is that of fibrinogen proteolysis; the proteolysis products circulate in the plasma and directly inhibit coagulation, even though plasma fibrinogen is usually in the low normal range, and deficit of specific coagulation factors is confined to relatively unimportant falls in factors v and VIII. This coagulation defect, of unique pathogenesis, has been termed defective fibrin polymerization [82,75]. These observations have clinical significance, not only with respect to the pathogenesis of the

Further

Hemorrhagic

to

Introvos-

Polymerization

proteolytic

+

’

Diothesis

Leoding

to

Thrombotic Complication

states seen in patients.

hyperplasminemic coagulation defect, but also with regard to its treatment. The demonstration that the 50 per cent plasma clearance rate of the abnormal fibrinogen fragment is approximately 9.5 hours indicates that the coagulation defect will spontaneously resolve provided that the hyperplasminemic state is controlled. Preliminary clinical trial with epsilon aminocaproic acid, an inhibitor of plasminogen activation which may be administered intra[83,841, venously, has yielded promising results [85,86] : as predicted, control of pathologic plasma proteolysis is followed by a disappearance of the coagulation anomaly within a period of twelve to twenty-four hours. However not all hypofibrinogenemic states are due to release of plasminogen activator alone, for, as was admirably demonstrated by Soulier and colleagues [87], hypofibrinogenemia following surgical intervention is commonly the result of both plasminogen activator and thromboplastic material release from traumatized tissue. Similarly the work of Schneider [S&89] and others [90] indicates that the hypofibrinogenemia complicating obstetric emergencies, such as abruptio placenta, may be partly attributable to release of thromboplastic material from the placental site (for a fuller discussion and further references see [4,47,97]). The consequences of this dual etiologic mechanism are shown in Figure 3 and indicate that too vigorous or too early treatment, of what may be a largely secondary pathologic plasma AMERICAN

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Fibrinolytic Mechanisms and Thrombolytic proteolytic state, may result in a thrombotic complication. * Consequently the treatment of patients in whom intravascular coagulation is a possibility (particularly obstetric emergencies and those on cardiac bypass), should be as conservative as the clinical circumstances permit. Conservative management is often discouraged, because standard rapid laboratory assays for plasma fibrinogen yield erroneously and correspondingly alarmingly low values in this condition.? In our experience the only rapid fibrinogen method of sufficient accuracy to guide treatment is an immunologic one (Fr test, Hyland Laboratories, California) and with its aid, conservative management is greatly facilitated. S@ec$c Local Actions of Plasminogen Activator. Following the detection of plasminogen activators in various secretions and particularly in the urine [96], it was suggested that their presence might subserve a useful function in preventing obstruction of duct passages or the urinary tract by fibrinous deposits. Conversely, were urokinase to lyse fibrinous deposits in the urinary tract, it would be expected that fibrinous clots formed after trauma (and serving a hemostatic function) would also undergo lysis, thus leading to protracted hematuria. This hypothesis, of considerable clinical importance, has only recently been investigated in man [93,94]. The problem was approached through the use of epsilon aminocaproic acid, a potent inhibitor of plasminogen activation [83,84], which is excreted and strongly concentrated in the urine [97-991. Consequently the administration of epsilon aminocaproic acid, in sufficient dosage to inhibit the urokinase activity of urine, could be expected to reduce the degree and duration of hematuria following injury. This hypothesis was tested in patients undergoing relatively severe and uniform trauma to the urinary tract occasioned by prostatectomy. * It is well recognized that knowledge of therapeutic complications is often incomplete because of the natural and understandable reluctance of physicians to report single cases of therapeutic misadventure. The literature contains only a single case report [92] of a thrombotic complication following the fibrinogen treatment of a pathologic plasma proteolytic state, yet by very limited inquiry, the authors learned of six further cases. Similarly, while we have emphasized the potential thrombotic hazards of epsilon aminocaproic acid therapy in inappropriate circumstances [93,94], there is presently only one case report in the literature supporting this prediction [95], yet we know of five similar cases. t See footnote on page 743. VOL.

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Randomized administration of epsilon aminocaproic acid to patients operated on by the transurethral and suprapubic routes showed a striking and statistically significant reduction (fourfold for transurethral series and twofold for the suprapubic cases) of postoperative hematuria in the epsilon aminocaproic acid treated subjects compared to control subjects [93,94]: these results recently have been confirmed by others [ 704. Moreover epsilon aminocaproic acid therapy in postoperative prostatectomy patients, whose course has been complicated by severe hematuria of long duration, has been attended by success [94] and this therapy has proved to be a useful adjunct in the management of this otherwise intractable condition. Consequently, while urokinase fulfills an important function in preventing obstruction to the urinary tract by fibrinous deposits, this action may, under some circumstances, lead to impairment of hemostatic function. Possibly similar considerations obtain in the case of other organs, which secrete fluids containing plasminogen activator. THERAPEUTIC

THROMBOLYSIS

The high incidence of thromboembolic disease in man, especially in the elderly, with its heavy concomitant mortality and crippling morbidity, has stimulated work towards the therapeutic goal of in vivo thrombus or embolus dissolution by enzymatic means. That such an approach is feasible has been clearly established by animal experiment, and a high degree of success in the lysis of experimentally-induced arterial and venous “thrombi” has been reported with a variety of enzymatic agents [7,707-1031. However numerous side effects, particularly severe coagulation defects, have been prominent. Furthermore, the transfer of animal experimental results to the realm of human therapeutics has required the reinvestigation of many phenomena and the development of sophisticated biochemical concepts unnecessary at the animal experimental stage. In fact, successful animal experimental work has proved to be so relatively easy, and work in man so greatly different, that inapplicable and erroneous concepts, derived from animal experiment, have, at times, impeded clinical work. Progress in the therapeutic application of thrombolytic therapy has been extremely uneven. While considerable clarification of basic

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problems, underlying the therapeutic approach, has been achieved, results at the applied therapeutic level have been far less impressive and sometimes disappointing. Because many of the basic problems are now sufficiently understood to encourage considerable optimism in the future development of this type of therapy, the last section of this manuscript deals with some of the practical difficulties that have prevented wider use of this therapy. Since recent detailed reviews with full reference citation are available [4-9,11,12,704-1081, this section will be confined to questions of broad principle with only limited reference citation. The difficulties have included (1) controversy as to the biochemical properties required of the ideal therapeutic agent; (2) clinical toxicity of drugs; (3) hazards related to the secondary effects of the treatment; (4) an unsatisfactory basis for drug standardizations; (5) varying opinions as to the proper methods for laboratory control of the treatment; and (6) as is inevitable in clinical trials of this nature, uncertainty as to the significance of apparent clinical response in the assessment of treatment. Although this is a formidable list of problems, these ,vicissitudes, inevitable in a new field of therapeutic endeavor, are of a practical rather than a fundamental nature, and their existence in no way invalidates the conceptual validity of thrombolytic therapy. The mechanism Choice of Thrombolytic Agent. by which plasmin action is confined in uivo to the site of a thrombus or fibrinous deposit, through the mediation of plasminogen activator, has been described in an earlier section; as a consequence, thrombolytic therapy is based on the principle of inducing plasminogen activation at the local thrombus site. Since clinical thrombosis is an occurrence that, whatever its primary etiologic cause, represents, at least indirectly, failure of the plasminogen-plasmin system to lyse the developing thrombus, there is justification for the present therapeutic approach of utilizing exogenously introduced plasminogen activators in high dosage to produce, for prolonged periods, plasma thrombolytic states of an intensity never encountered under the most extreme physiologic or pathologic circumstances. This justification is supported by the findings of animal experiment [707-7031 and clinical trial [30,39,40,709,770]; the evidence for the feasibility of this approach in man [4,6] has been of crucial importance to the development of the field.

et al.

On the other hand, attempts to utilize proteolytic enzymes to induce thrombolytic states in man have been largely unsuccessful, as would be expected from physiologic considerations (see earlier section). Intravenously administered trypsin was discarded some years ago [6]; glycerol activated plasmin [ZO] has had only a limited trial in man [ 7771 with discouraging results; data on Aspergillin 0 (an enzyme derived from Aspergillin Oryzae) does not indicate therapeutic utility [772]; and Thrombin E (an acetylated thrombin derivative), although proposed [773] and tested as a thrombolytic agent in animals, has not for various reasons [708] been employed in man. Standardization of Agents. Streptokinase has been customarily standardized by the Christensen procedure [774] against a standard N.I.H. preparation, and plasmin is standardized against casein substrate using either the original [775] or a modified Remmert and Cohen method [83]. Although neither of these standardization procedures fulfills ideal criteria, their use has done much to facilitate comparison of preparations and understanding in the field. However, when commercial thrombolytic agents were released, their potency was expressed in arbitrary fibrinolytic units of the manufacturer’s own choosing, and the preparations were described as human fibrinolysin. Analysis of both preparations (Actase@ and Thrombolysin@) revealed that each was a streptokinase-plasmin mixture, that the manufacturer’s standardization procedure failed to provide adequate information as to the product’s composition, and that the potency of one manufacturer’s unit was seven to tenfold that of the other [776]. This combination of inappropriate nomenclature and unsuitable standardization confused many investigators and clinicians. Moreover since the in viuo activity of both preparations was due primarily to their streptokinase content [ 7 76,7 771, the recommendation to use these preparations in a fixed dosage, dependent upon the severity of the disease process, rather than in a variable dosage, dependent upon prior assay of the patient’s plasma streptokinase antibody titer [30,778,779], inevitably produced clinical disappointment. In our opinion [4-6,708], plasminogen activator-plasmin mixtures not only lack advantage over the use of plasminogen activators as thrombolytic agents, but also in certain circumstances might be hazardous. However it is AMERICAN

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Fibrinolytic Mechanisms and Thrombolytic clearly essential, if plasminogen activatorplasmin mixtures continue to be used (or new ones developed), that they be adequately standardized in terms of the separate biochemical moieties contained in the preparation. This plea has recently been endorsed by a committee of the Protein Foundation [720]. Clinical Toxicity of Thrombolytic Agents. Clinichiefly manifest by pyrogenic cal toxicity, reactions, has been a persistent feature of clinical trials with streptokinase or streptokinase-plasmin mixtures. Since the pyrogenic effect has been variable with different batches of material, has lessened as purification procedures have improved [727,722], and in some instances, nonpyrogenic batches of streptokinase have been produced [30,39], it may tentatively be assumed that the pyrogenic batches contain impurities. Nevertheless the clinical toxicity of streptokinase or streptokinase-mixtures remains an important deterrent to extended clinical trial in dangerously ill patients. Fear of pyrogenic reactions has sometimes caused clinical investigators to recommend dosages which, from a biochemical viewpoint, must be regarded as inadequate; consequently some clinical trials with thrombolytic agents have been attempted at placebo dosage [ 7 771. Because of uncertainties in clinical assessment of treatment results, this defect has not always been apparent in the published manuscript. Elimination of pyrogenicity from streptokinase preparations has proved to be a difficult problem because no reliable animal test exists for its detection and because the occurrence of in individual patients, pyrogenic reactions, at any fixed dosage of the agent is inconsistent and unpredictable. Streptokinase is highly antigenic to man and because of previous streptococcal infection, the titer of plasma streptokinase antibody may vary a thousandfold in patients. Streptokinase antibody rapidly combines with streptokinase, whether the latter be infused alone or as a streptokinase-plasmin mixture [7X,777], and this antigen-antibody complex is both devoid of biochemical activity and rapidly cleared from the plasma [779]. Satisfactory, rapidly performed assays are available for the quantitation of streptokinase antibody, and for the calculation of effective individualized dosage [33,7 781. Unfortunately, these essential procedures have been omitted in many “clinical” trials, and the value of the trials correspondingly diminished, VOL.

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for it is improbable that the treated patients actually received effective therapy. Intensive streptokinase therapy invariably immunizes the patient; plasma streptokinase antibody levels usually peak at two or three months and, by six months, plasma antibody has usually dropped to a relatively low figure [30]. The infusion of streptokinase or streptokinase-plasmin mixtures has not produced, at least in the vast majority of patients, allergic i\lresponses from foreign protein reaction. though clinical experience has been reassuring and the dose of antigen infused relatively small [779], the possibility of allergic reaction, in these circumstances, cannot be ignored. Indeed a few case reports suggest that retreatment of patients may be complicated by a hypersensitivity reaction; however these studies were incomplete, and only presumptive evidence was offered to indicate that streptokinase, apart from an impurity in the preparation, was the responsible antigen. Secondary Hazards of the Treatment. A distinction has been previously drawn between thrombolysis or thrombolytic activity as a normal physiologic function and hyperplasminemia a state of pathologic plasma proteolysis. However when utilizing thrombolytic therapy, this distinction becomes blurred as it is not yet possible to induce intense plasma thrombolytic activity for therapeutic purposes without simultaneously producing a pathologic plasma proteolytic state with its concomitant coagulation defect. However clinical experience has demonstrated [30,39] that the controlled induction of pathologic plasma proteolysis need not be hazardous, provided that proper laboratory control is exercised [47,77,82]. An important consideration, when employing thrombolytic therapy, concerns the final state of infarcted tissue after the blood supply has been restored. Two groups of investigators [ 723-7251 using the dog, have observed the effects of lysing a thrombus obstructing a coronary artery. Both believed that the area of final infarction was reduced by such therapy, and neither noted any unusual histologic lesions in the treated animal. Moreover, in a small series of patients, suffering from acute myocardial infarction and treated with intensive thrombolytic therapy, evidence of any deleterious effects of the treatment was lacking [709,770]. An unresolved problem concerns the effects of thrombolysis in the cerebral circulation. In

748

Fibrinolytic Mechanisms and Thrombolytic Therapy--FZetc/zer et al.

an excellent study, the Mayo group [726] have shown that, under certain circumstances, the administration of anticoagulants to dogs suffering from obstructive cerebral vascular disease may lead to hemorrhage in the infarcted area. Although the clinical importance of these observations is not yet established, they convey the warning that from a functional viewpoint, infarction of the brain may differ in important respects from infarction in other organs. Laboratory Control and Clinical Trial. Since thrombolytic therapy involves the production and maintenance of a biochemically defined state of plasma thrombolytic activity for a stated period of time, suitable assays for its quantitation have been developed [30,77]. Although the methods for measuring plasminogen-plasmin system components and particularly plasma thrombolytic activity are undesirably complex [30,38] and unsuited to the routine laboratory, the use of these methods is clearly mandatory in clinical trials until either simpler methods are evolved or standard treatment schedules requiring less laboratory control are devised. At the present time, unless proof is furnished by the investigator that such a defined biochemical state has been achieved and maintained, it is impossible to decide whether a patient belongs to the control or treatment group of a trial. Considering that the application of scientific method to the evaluation of clinical observations is the most difficult in the entire area of clinical investigation (even under the most favorable circumstances of a well designed trial), claims of a favorable therapeutic response on the basis of empirical clinical appraisals, without proper laboratory measurement of thrombolytic activity, are not only unsuited for critical analysis but also serve to confuse the issue. CONCLUSIONS

Thrombolytic therapy, despite certain difficulties and problems, would appear to be a tool of considerable future promise. Many scientific gains have been made over the last few years and the evidence is sufficiently promising to suggest that the present problems may yield to further investigative effort. The main requirement is either for the discovery of a new plasminogen activator or further developmental work on the known plasminogen Recent progress in the further activators. purification of streptokinase is promising, al-

though this activator, because of its antigenic nature, can never constitute an ideal solution to the problem. Substantial progress has been made with the purification of human urokinase: from extensive biochemical [37], but still limited clinical studies [7271, it seems to possess highly desirable therapeutic properties. Moreover recent work on the isolation and purification of plasminogen activator from tissue [728] and from tissue culture [729] may lead to important developments. REFERENCES

1. MACFARLANE, R. G. and BIGGS,R. Fibrinolysis: its mechanism and significance. Blood, 3: 1167,1948. 2. ASTRUP, T. Fibrinolysis in the organism. Blood, 11: 781, 1956. 3. HALSE, T. Das fibrinolytische Potential. Enzymologie, Pathologic und Klinik. Die Medizinische Nr., 50/51: 1, 1958. 4. SHERRY, S., FLETCHER, A. P. and ALKJAERSIG,N. Fibrinolysis and fibrinolytic activity in man. Physiol. Rev., 39: 343, 1959. 5. FLETCHER, A. P. and SHERRY, S. Thrombolytic (fibrinolytic) therapy for coronary heart disease. Circulation, 22: 619, 1960. 6. SHERRY, S. and FLETCHER, A. P. Proteolytic enzymes: a therapeutic evaluation. Clin. Pharmacol. & The@., 1: 202: 1960. 7. CLIFFTON, E. E. Historical review of clinical use of enzymes. In: Enzymes in Health and Disease, D. 212. Edited bv Greenberg, D. M. and Harper, h. A. Springfielh, III., 1960:~Charles C Thomas. 8. CELANDER, D. R., MESSER, D. and GUEST, M. M. The fibrinolytic system: a review of its therapeutic significance and control with particular emphasis on its inhibition by epsilon aminocaproic acid. Texas R@. Biol. &? Med., 19: 16, 1961. 9. SAWYER, W. D., ALKJAERSIG,N., FLETCHER, A. P. and SHERRY, S. Thrombolytic therapy: basic and therapeutic considerations. Arch. Znt. Med., 107: 274, 1961. 10. MARTIN, G. J. (Ed.). Symposium on proteolytic enzymes and their clinical application. Ann. New York Acad. SC., 68(l): 1957. 11. (a) Symposium on clinical effects of fibrinolytic activity. Angiology, 10: 243, 1959. (6) Thrombolytic activity and related phenomena. Thromb. et Diath. Haemorrh., vol. 6 (supp. I), 1961. 12. Symposium on fibrinolysis. Am. J. Cardiol., 6: 367, 1960. 13. SHULMAN, S., ALKJAERSIG, N. and SHERRY,S. Physicochemical studies on human plasminogen (profibrinolysin) and plasmin (fibrinolysin). J. Biol. Chem., 233: 91, 1958. 14. KLINE, D. L. The purification and crystallization of plasminogen (profibrinolysin). J. Biol. Chem., 204: 949, 1953. 15. KLINE, D. L. and FISHMAN,.I. B. Improved procedure for the isolation of human plasminogen. J. Biol. Chew., 236: 3232, 1961. AMERICAN

JOURNAL

OF MEDICINE

Fibrinolytic Mechanisms and Thrombolytic Therapy--Fletcher 16. DAVIES, M. C. and ENGLERT, M. E. Physical properties of highly purified human plasminogen. J. Biol. Chem., 235: 1011, 1960. 17. ALKJAERSIG,N. Thrombolytic activity and related phenomena. Chemistry of fibrinolysis. Thromb. et Diath. Haemorrh., 6 (supp. 1): 129, 1962. 18. ALKJAERSIG,N., FLETCHER, A. P. and SHERRY, S. The activation of human plasminogen. II. A kinetic study of activation with trypsin, urokinase and streptokinase. J. Biol. Chem., 233: 86, 1958. 19. WHITE, W. and MOZEN, M. Personal communication, 1962. 20. ALKJAERSIG,N., FLETCHER, A. P. and SHERRY, S. The activation of human plasminogen. I. Spontaneous activation in glycerol. J. Biol. Chem., 233: 81, 1958. 21. CHRISTENSEN, L. R. The activation of plasminogen by chloroform. J. Gen. Physiol., 30: 149, 1946. 22. NIE~IAROWSKI, S. and PROW-WARTELLE, 0. Role du facteur contact (facteur Hageman) dans la hbrinolyse. Thromb. et Diath. Haemorrh., 3: 593, 1959. 23. IATRIDIS,G. G. and FERGUSON,J. H. Active Hageman factor: a plasma lysokinase of the human fibrinolytic system. J. Clin. Invest., 41: 1277,1962. 24. JACOBSSEN,K. Studies on the trypsin and plasmin inhibitors in human blood serum. Scandinau. J. Clin. & Lab. Invest., vol. 7 (supp.), 1955. 25. NORMAN, P. S. Studies of the plasmin system. II. Inhibition of plasmin by serum or plasma. J. Exper. Med., 108: 53, 1958. 26. NORMAN, P. S. and HILL, B. M. Studies of the plasmin system. III. Physical properties of the two plasmin inhibitors in plasma. J. Exper. Med., 108: 639, 1958. 27. SHULMAN, N. R. Studies on the inhibition of proteolytic enzymes by serum. I. The mechanism of the inhibition of trypsin, plasmin and chymotrypsin by serum using fibrin tagged with I’s’ as a substrate. J. Exfer. Med., 95: 571, 1952. 28. JOHNSON,S. A. and SCHNEIDER,C. L. The existence of antifibrinolysin activity in platelets. Science, 117: 229,1953. 29. ALKJAERSIG, N. The antifibrinolytic activity of platelets. In: Blood Platelets. In: Henry Ford Hospital International Symposium. Boston, 1961. Little, Brown & Co. 30. FLETCHER, A. P., ALKJAERSIG,N. and SHERRY, S. The maintenance of a sustained thrombolytic state in man. I. Induction and effects. J. Clin. Invest., 38: 1096, 1959. 31. ALKJAERSIG,N., FLETCHER, A. P. and SHERRY, S. The mechanism of clot dissolution by plasmin. J. Clin. Invest.,38: 1086, 1959. 32. SHERRY, S., LINDEMEYER,R. I., FLETCHER, A. P. and ALKJAERSIG, N. Studies on enhanced fibrinolytic activity in man. J. Clin. Invest., 38: 810, 1959. 33. FLETCHER, A. P., ALKJAERSIG,N. and SHERRY, S. Influence of clot composition on thrombolysis in plasma. Fed. Proc., 21: 63, 1962. 34. MOSESSON, M. W. The preparation of human fibrinogen free of plasminogen. Biochim. et biophys. acta, 57: 204, 1962. 35. BERGSTROM,K. and WALLEN, P. Removal of conVOL.

33,

NOVEMBER

1962

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46. 47.

48.

49.

50.

51. 52.

53.

et al.

749

taminating plasminogen from purified bovine fibrinogen. Arkiu Kemi, 17: 503, 1961. ALKJAERSIG,N. The activation of plasminogen in vitro and in vivo. In: International Symposium on Anticoagulants and Fibrinolysins. Philadelphia, 1961. Lea & Febiger. SAWYER, W. D., ALKJAERSIG,N., FLETCHER,A. P. and SHERRY, S. A comparison of the fibrinolytic effects of plasminogen and fibrinogenolytic activators and proteolytic enzymes in plasma. Thromb. et Diath. Haemorrh., 5: 149. 1960. SAWYER, W. D., FLETCHER,A. P., ALKJAERSIG,N. and SHERRY, S. Studies on the thrombolytic activity of human plasma. J. Clin. Inuest., 39: 426, 1960. JOHNSON,A. J. and MCCARTY, W. R. The lysis of artificially induced intravascular clots in man by intravenous infusions of streptokinase. J. Clin. Invest., 38: 1627, 1959. DEUTSCH, E. and FISCHER, M. Die Wirkung intraveniis applizierter Streptokinase auf Fibrinolyse und Blutgerinnung. Thromb. et Diath. Haemorrh., 4: 482, 1960. FLETCHER, A. P., ALKJAERSIG,N. and SHERRY, S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (L‘fibrinolytic”) states. I. The significance of fibrinogen proteolysis and circulating fibrinogen breakdown products. J. Clin. Invest., 41: 896, 1962. AMBRU~, C. M. and MARKUS, G. Plasmin-antiplasmin complex as a reservoir of fibrinolytic enzyme. Am. J. Physiol., 199: 491, 1960. AMBRUS,C. M., BACK, N. and AMBRUS,J. L. On the mechanism of thrombolysis by plasmin. Circulation Res., 5: 161, 1962. ASTRUP, T. and STERNDORFF,I. Fibrinolysokinase activity in animal and human tissue. Acta physiol. scandinav., 37: 40, 1956. ALBRECHTSEN,0. K. The fibrinolytic activity of animal tissues. Acta physiol. scandinav., 39: 284, 1957. ALBRECHTSEN,0. K. The fibrinolytic activity of human tissues. Brit. J. Haemat., 3: 284, 1957. RATNOFF, 0. D. Studies on a proteolytic enzyme in human plasma. IV. The rate of lysis of plasma clots in normal and diseased individuals, with particular reference to hepatic disease. Bull. Johns Hopkins Hosp., 83: 29, 1949. KWAAN, H. C., MCFADZEAN, A. J. S. and COOK, J. Plasma fibrinolytic activity in cirrhosis of the liver. Lance& 1: 132, 1956. KWAAN, H. C., MCFADZEAN, A. J. S. and COOK, J. On plasma fibrinolytic activity in cryptogenic splenomegaly. Scottish M. J., 2: 137, 1957. WEINER, M., REDISCH, W. and STEELE, J. M. Occurrence of fibrinolytic activity following administration of nicotinic acid. Proc. Sot. Exper. Biol. & Med., 98: 755, 1958. BIEDERMAN,O., MOORE, D. and FLETCHER, A. P. In preparation, 1962. RATNOFF, 0. D. and CONLEY, C. L. Studies in afibrinogenemia. II. Defibrinating effect on dog plasma of intravenous injection of thromboplastic material. Bull. Johns Hopkins Ho@., 88: 414,195l. LEWIS, J. H., FERGUSON,E. E. and SCHOENFELD,C.

750

54. 55.

56.

57.

58.

59.

60.

61.

62. 63. 64.

65.

66.

67.

68.

69.

70.

Fibrinolytic Mechanisms and Thrombolytic Therapy--Fletcher Studies concerning the turnover of fibrinogen Ii3i in the dog. J. Lab. & Clin. Med., 58: 247, 1961. HARRISON,C. V. Experimental pulmonary arteriosclerosis. J. Path. & Bact., 60: 289, 1948. HEARD, B. E. An experimental study of thickening of the pulmonary arteries of rabbits produced by the organization of fibrin. J. Path. & Bact., 64: 13, 1952. SPINGATE, C. S., FECHNER, R. E., SCOTT, R. C., JYSTAD, G. R. and O’NEAL, R. M. Disappearance of clot-emboli in rabbits. Arch. Path., 73: 407, 1962. KWAAN, H. C., Lo, R. and MCFADZEAN, A. J. S. On the production of plasma fibrinolytic activity in vivo by serotonin (5-hydroxy-tryptamine) creatinine sulphate. Clin. SC., 16: 255, 1957. KWAAN, H. C., Lo, R. and MCFADZEAN, A. J. S. On the production of plasma fibrinolytic activity within veins. Clin. SC., 16: 241, 1957. KWAAN, H. C., Lo, R. and MCFADZEAN, A. J. S. On the inhibition of plasma fibrinolytic activity by exercised ischaemic muscles. C&z. SC., 17: 361, 1958. KWAAN, H. C., Lo, R. and MCFADZEAN, A. J. S. On the lysis of thrombi experimentally produced within veins. Brit. J. Haemat., 4: 51, 1958. ASTRUP, T., ALBRECHTSEN, 0. K., CLASSEN,M. and RASMUSSEN,J. Thromboplastic and fibrinolytic activity of the human aorta. Circulation Res., 7: 969,1959. HUME, R. Fibrinolysis in myocardial infarction. Brit. Heart J., 20: 15, 1958. NESTEL, P. J. Fibrinolytic activity of the blood in intermittent claudication. Lancet, 2: 373, 1959. LACKNER, H. and MERSKEY, C. Variation in fibrinolytic activity after acute myocardial infarction and after the administration of oral anticoagulant drugs and intravenous heparin. Brit. J. Haemat., 6: 402, 1960. MERSKEY, C., GORDON,H., LACKNER,H., SCHRIRE, V., KAPLAN, J., SOUGIN-MIBASHAN,R., NOSSEL, H. L. and MOODIE, A. Blood coagulation and fibrinolysis in relation to coronary heart disease. Brit. M. J., 1: 219, 1960. JAMES,D. C. O., DRYSDALE, J., BILLIMORIA,J. D., WHEATLEY, D., GAVEY, C. J. and MACLAGAN, N. F. Lipemia and blood coagulation defects in relation to ischemic heart disease. Luncet, 2: 799, 1961. SHYRNIOTIS,F. E., FLETCHER, A. P., ALKJAERSIC, N. and SHERRY, S. Urokinase excretion in health and its alteration in certain disease states. Thromb. et Diath. Haemorrh., 3: 257, 1959. RIGGENBACH,N. and VON KAULLA, K. V. Urokinase excretion in patients with carcinoma. Cancer, 14: 889, 1961. NILSSON, I. M., KROOK, H., STERNBY, N. H., SODERBERG, E. and SODERSTROM,N. Severe thrombotic disease in a young man with bone marrow and skeletal changes and with a high content of an inhibitor in the fibrinolytic system. Acta med. scandinau., 169: 323, 1961. GREIG, H. B. W. and RUNDE, I. A. Studies on the inhibition of fibrinolysis by lipids. Lamet,2: 461, 1957.

et al.

71. MITCHELL, J. R. A. and BRIERS,S. EtTect of cholesterol, cholesterol esters and neutral fats on fibrinolysis. Lam-et, 2: 435, 1959. 72. MERIGANS, J. G., FARQUHAR, J. W., WILLIAMS, J. H. and SOKOLOW,M. Effects of chylomicrons on the fibrinolytic activity of normal human plasma in rsitro. Circulation Res., 7: 205, 1959. 73. KWAAN, H. C. and MCFADZEAN, A. J. S. Inhibition of fibrinolysis in viva by feeding cholesterol. iVature, London, 179: 260, 1957. 74. BANG, N. U. and CLIFFTON, E. E. The effect of alimentary hyperlipemia on thrombolysis in vivo. Thromb. et Diath. Hemorrh., 4: 149, 1960. 75. ALKJAERSIG,N., FLETCHER, A. P. and SHERRY, S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (‘
JOURNAL

OF

MEDICINE

Fibrinolytic Mechanisms and Thrombolytic

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

VOL.

DAUMET, P. and FAYET, H. Syndrome h&norrhagiques mortels avec incoagulabilite totale par defibrination et avec fibrinolyse. I. Au tours des exereses pulmonaires. Reu. hemat., 7: 30, 1952. SCHNEIDER,C. L. Fibrination and Defibrination. Physiologie und Pathologie der Blutgerinnung in der Gestations-periode. Stuttgart, 1957. Schattauer-Verlag. SCHNEIDER, C. L. Etiology of fibrinogenemia: fibrination defibrination. Ann. New York Acad. SC., 75: 634, 1959. RATNOFF, 0. D., PRITCHARD, J. A. and COLOPY, J. A. Hemorrhagic states during pregnancy. New England J. Med., 253: 63, 1955. MARGOLIUS, A., JACKSON, D. P. and RATNOFF, 0. D. Circulating anticoagulants: a study of 40 cases and a review of the literature. Medicine, 40: 145, 1961. GRAHAM, J. H., EMERSON, C. P. and ANGLEM, T. J. Postoperative hypofibrinogenemia: diffuse intravascular thrombosis after fibrinogen administration. New England J. Med., 257: 101, 1957. MCNICOL, G. P., FLETCHER,A. P., ALKJAERSIG,N. and SHERRY, S. Impairment of hemostasis in the urinary tract: the role of urokinase. J. Lab. @ Clin. Med., 58: 34, 1961. McNrco~, G. P., FLETCHER,A. P., ALKJAERSIC,N. and SHERRY, S. Use of epsilon aminocaproic acid in management of postoperative hematuria. J. Ural., 86: 829, 1961. NAEYE, R. L. Thrombotic state after a hemorrhagic diathesis, a possible complication of therapy with epsilon aminocaproic acid. Blood, 19: 694, 1962. CELANDER, D. R. and GUEST, M. M. The biochemistry and physiology of urokinase. Am. J. Cardiol., 6: 409, 1960. NILSSON, I. M., SJOERDSMA,A. and WALDENSTROM, .I. Antifibrinolytic activity and metabolism of e-aminocaproic acid in man. Lancet, 1: 1322, 1960. MCNICOL, G. P., FLETCHER,A. P., ALKJAERSIG,N. and SHERRY, S. Plasma amino acid chromatography with ion exchange loaded paper: assay of e-aminocaproic acid. J. Lab. B Clin. Med., 59: 7, 1962. MCNICOL, G. P., FLETCHER,A. P., ALKJAERSIG,N. and SHERRY, S. The absorption, distribution and excretion of e-aminocaproic acid following oral or intravenous administration to man. J. Lab. & Clin. Med., 59: 15, 1962. SACK, E., SPAET, T., GENTILE, R. and HUDSON,P. Reduction of postprostatectomy bleeding by epsilon ‘aminocaproic acid. New England J. Med., 266: 541, 1962. SHERRY, S., TITCHENER, A., GOTTESMAN, L., WASSERMAN,P. and TROLL, W. The enzymatic dissolution of experimental arterial thrombi in the dog by trypsin, chymotrypsin and plasminogen activators. J. Clin. Invest., 33: 1303, 1954. TILLETT, W. S., JOHNSON,A. J. and MCCARTY, W. R. The intravenous infusion of the streptococcal fibrinolytic principle (streptokinase) into patients. J. Clin. Invest., 34: 169, 1955. 33,

NOVEMBER

1962

Therapy---Fletcher et al.

751

103. BACK, N., AMBRUS, J. L., SIMPSON, C. L. and SHULMAN,S. Study on the effect of streptokinaseactivated plasmin (fibrinolysin) on clots in various stages of organization. J. Clin. Znuest., 37: 864, 1958. 104. SHERRY, S., FLETCHER, A. P. and ALKJAERSIC,N. Developments in fibrinolytic therapy for thromboembolic disease. Ann. Int. Med., 50: 560, 1959. 105. SHERRY, S. and FLETCHER, A. P. Thrombolytic therapy. Am. Heart J., 61: 575, 1961. (Editorial.) 106. SHERRY, S. Status of therapy: critique and outlook for the future. In: Proceedings of the International Conference on Thrombolytic Activity and Related Phenomena, Princeton, New Jersey. Thromb. et Diath. Haemorrh., 6 (supp. 1): 1962. 107. SHERRY, S., FLETCHER, A. P. and ALKJAERSIC,N. Rationale and limitations of the use of thrombolytic (fibrinolytic) agents in the treatment of thromboembolic disease. In: Progress in Hematology, vol. 3. Edited by Tocantins, L. M. New York, 1962. Grune & Stratton, Inc. 108. FLETCHER, A. P. Developments in thrombolytic therapy. In: Proceedings of the Seventh Congress International Therapeutic Union, Geneva, 1961. In press. 109. FLETCHER, A. P., SHERRY, S., ALKJAERSIG, N., SMYRNIOTIS,F. E. and JICK, S. The maintenance of a sustained thrombolytic state in man. II. Clinical observations on patients with myocardial infarction and other thrombo-embolic disorders. J. Clin. Invest., 38: 1111, 1959. 110. FLETCHER, A. P., ALKJAERSIC, N., SMYRNIOTIS, F. E. and SHERRY, S. The treatment of patients suffering from early myocardial infarction by massive and prolonged streptokinase therapy. Tr. A. Am. Physicians, 71: 287, 1958. 111. JOHNSON,A. J. and MCCARTY, W. R. Some aspects of the mechanism of thrombolysis. Thromb. et Diath. Haemorrh., 5: 391, 1961. 112. MARIN, H. M., STEFANINI, M., SOARDI, F. and MUELLER, L. Fibrinolysis. v. The thrombolytic and anticoagulant activity of mold fibrinolysin (Aspergillin 0) in viva. J. Lab. @ Clin. Med., 58: 47, 1961. 113. SEEKERS,W. H., LANDABURU,R. H. and JOHNSON, J. F. Thrombin-E as a fibrinolytic enzyme, Science, 131: 726, 1960. 114. CHRISTENSEN,L. R. Methods for measuring the activity of components of the streptococcal fibrinolytic system, and streptococcal desoxyribonuclease. J. Clin. Znuest., 28: 163, 1949. 115. REMMERT,L. F. and COHEN, P. Partial purification and properties of a proteolytic enzyme of human serum. J. Bi?l. Chew., 181: 431, 1949. 116. FLETCHER, A. P., ALKJAERSIC,N. and SHERRY, S. The assay of thrombolytic (“fibrinolytic”) mixtures intended for therapeutic use. J. Lab. @ Clin. Med., 57: 620, 1961. 117. FLETCHER, A. P., ALKJAERSIG,N., SAWYER, W. D. and SHERRY, S. Evaluation of human fibrinolysin (Actase). Lack of fibrinolytic activity after intravenous administration in man. J. A. M. A., 172: 912, 1960. 118. JOHNSON, A. J., FLETCHER, A. P., MCCARTY,

752

Fibrinolytic

Mechanisms

and Thrombolytic

201, 1957.

120. 121.

FLETCHER, A. P., ALKJAERSIG,N. and SHERRY, S. The clearance of heterologous protein from the circulation of normal and immunized man. J. Clin. Inuest.,37: 1306, 1958. SURGENOR, D. M. Fibrinolysis and fibrinolysin products. J. A. M. A., 180: 536, 1962. FLETCHER, A. P. and JOHNSON, A. J. Methods employed for purification of streptokinase. Pm.

125.

126.

Sac. Exper. Biol. f?? Med., 94: 233, 1957. 122.

et al.

FREIMAN, A. H., BANG, N. U., CLIFFTON, E. E. and LADUE, J. S. Fibrinolytic (plasmin) therapy of experimental coronary thrombi with alteration of the evolution of myocardial infarction. Circulation, 19: 7, 1959.

W. R. and TILLET~, W. S. The intravascular use of streptokinase. Ann. New York Acad. SC., 68: 119.

Therapy--Fletcher

NYDICX, I., RUEGSEGGER,P., BOUVIER, C., HUTTER, R. V., ABARQUEZ, R., CLIFFTON, E. E. and LADUE, J. S. Salvage of heart muscle by fibrinolytic therapy after experimental coronary occlusion. Am. Heart J., 61: 93, 1961. WHISNANT, J. P., MILLIKAN, C. H., SAYRE, G. P. and WAXIM, K. G. Effect of anticoagulants on experimental cerebral infarction. Circulation, 20:

A. J., FLETCHER, A. P., MCCARTY, W. R. and TILLETT, W. S. Effects in patients of intravenous infusions of purified streptokinase preparations. Pm. Sot. Exper. Biol. & Med., 94:

56, 1959. 127. FLETCHER, A. P., ALKJAERSIG,N. and SHERRY, S.

254,1957.

128.

JOHNSON,

123. AGRESS, C.

M., JACOBS, H. I., BINDER, M. J., CLARK, W. G., KAPLAN, L., LEDERER, M. and GLASSNER, H. F. Intravenous trypsin in experimental myocardial infarction. Circulation Res.,

2: 397, 1954. 124. RUEGSEGGER, P.,

NYDICK,

I.,

HUTI’ER,

R.

C.,

129.

Unpublished observations. BACHMANN,F. W., FLETCHER, A. P. and SHERRY, S. Partial purification of plasminogen activator from pig heart. Fed, Pm., 21: 64, 1962. PAINTER, R. H. The plasminogen activator from kidney tissue cultures. In: International Symposium on Anticoagulants and Fibrinolysins. Philadelphia, 1961. Lea & Febiger.

AMERICAN

JOURNAL

OF MEDICINE