Towards

Better Thrombolytic D. Collen,

H.R. Lijnen,

ARDIOVASCULAR diseases are a main cause of death and disability in Western C societies. They comprise three main categories, namely coronary artery disease leading to myocardial infarction or heart attacks, cerebrovascular disease causing strokes, and venous thrombosis predisposing to pulmonary embolism. It is well established that in myocardial or cerebral infarction, the triggering event of the acute episode is not the atherosclerotic lesion of the blood vessel wall, but more so the obstruction of the artery by a thrombus or blood clot. Thus, the commonly encountered vascular diseases, including myocardial infarction, cerebral infarction, and venous thromboembolism, have, as their immediate underlying cause, thrombosis of critically situated blood vessels with loss of blood flow to vital organs. PLACE OF THROMBOLYSIS IN CARDIOVASCULAR DISEASE

One approach to the treatment of an established thrombosis consists of pharmacological dissolution of the blood clot via the intravenous infusion of naturally occurring enzymes, called plasminogen activators, that activate an enzyme system known as the fibrinolytic system. The fibrinolytic system contains a proenzyme plasminogen, which by the action of plasminogen activators is converted to the active enzyme plasmin, which in turn digests fibrin to soluble degradation products. Inhibition of the fibrinolytic system occurs both at the level of the plasminogen activators, by plasminogen activator inhibitors (mainly PAI-l), and at the level of plasmin, mainly by ol,-antiplasmin. The biochemical properties of the components of the fibrinolytic system have been reviewed in more detail elsewhere.’ Currently, five thrombolytic agents are either approved for clinical use or under clinical investigation in patients with acute myocardial infarction. These include streptokinase, two-chain urokinase (tcu-PA), anisoylated plasminogen streptokinase activator complex (APSAC), recombinant tissue-type plasminogen activator (rt-PA), and recombinant single-chain urokinase-type plasminogen activator (rscu-PA, proProgress

in CardiovascularDiseases,

Vol XXXIV,

No 2 (September/October),

Therapy

and H.K. Gold

urokinase).’ Streptokinase, APSAC, and tcu-PA cause extensive systemic activation of the fibrinolytic system, which may result in degradation of several plasma proteins, including fibrinogen, factor V, and factor VIII. The physiological plasminogen activators, human tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA), activate plasminogen preferentially at the fibrin surface. Plasmin, associated with the fibrin surface, is protected from rapid inhibition by qantiplasmin and may thus efficiently degrade the fibrin of a thrombus.3 This fibrin-specific mechanism of action of t-PA and scu-PA has triggered great interest in the use of these agents as thrombolytic agents. Production of both t-PA4,’ and scu-PA6*’ by recombinant DNA technology has made these agents available for large-scale clinical use. In patients with acute myocardial infarction, reduction of infarct size, diminished loss of ventricular function, and decreased mortality have been demonstrated in patients with acute myocardial infarction following administration of streptokinase,*-lo acylated plasminogen streptokinase activator complex,” or rt-PA.‘*.13 This beneficial effect of thrombolysis is probably also true for the other plasminogen activators. A review of data from the literature on treatment of acute myocardial infarction with streptokinase and rt-PA, restricted to comparative studies and to uncontrolled studies with comparable design and endpoints, indicates that rt-PA is a more efficient agent than streptokinase for treatment of coronary thrombosis, producing both more rapid and more frequent recanalization of occluded coronary arteries.14 Thus, the predictions based on the biochemical

From the Center for Thrombosis and Vascular Research, Universi~ of Leuven, Leuven, Belgium; and the Cardiac Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA. Address reprint requests to D. Collen, MD, PhD, Center for Thrombosis and Vascular Research, University of Leuven, Campus Gasthuisberg 0 & N, Herestraat 49, B-3000 Leuven, Belgium. Copyright 0 1991 by W.B. Saunders Company 0033-062Ol9113402-0002$5.00/O 1991:

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properties of rt-PA have indeed translated into a significantly higher thrombolytic potency. A large body of experimental and clinical evidence suggests that clinical benefit of thrombolysis in patients with acute myocardial infarction is primarily determined by rapid restoration of blood flow in the occluded coronary artery. However, so far no study has shown a difference in improvement in left ventricular function or survival rates for either agent when rt-PA was compared with streptokinase, despite the evidence of a different efficacy for recanalization. The lack of a clear difference in mortality of streptokinase and rt-PA in the recent Jnternational Study Group trial may well have been due to the delayed subcutaneous administration of heparin.“@ Preliminary results of small randomized trials comparing streptokinase with rt-PA treatment, showing lower inhospital mortality rates with rt-PA treatment, are indeed consistent with the hypothesis that timely coronary artery reperfusion is the primary determinant for reduction of mortality.14 LIMITATIONS

OF CURRENT THERAPY

THROMBOLYTIC

Despite their widespread use, the currently available thrombolytic agents suffer from a number of significant limitations. Resistance to reperfusion occurs in 25% of patients despite the use of the most potent thrombolytic agents or combinations.14V’7*‘8 Stable coronary patency is not uniformly produced,‘4,17-‘9 whereas angiographically documented acute coronary reocclusion occurs in 5% to 25% of patients.“**’ The time to reperfusion is prolonged, with restoration of anterograde coronary flow requiring on average 45 minutes after initiation of therapy.22-25Significant bleeding may occur, with a frequency of intracerebral bleeding of approximately 0.5%.15 Furthermore, the fibrin-specificity of rt-PA and rscu-PA is not as pronounced in man as was anticipated from several animal models.26 Both rt-PA and rscu-PA have a very short plasma half-life as a result of rapid hepatic clearance, whereby their therapeutic use probably requires continuous intravenous infusion (which is time consuming) of relatively large amounts of material (50 to 100 mg) (which is expensive). Therefore, the quest on the one hand for thrombolytic agents with a higher

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thrombolytic potency, specific thrombolytic activity, and/or a better fibrin-selectivity and on the other for thrombolytic strategies that overcome resistance to clot lysis, accelerate recanalization, prevent reocclusion, and reduce the bleeding tendency continues. Thrombolytic therapy in its present form is based on the premise that dissolution of the fibrin component of a thrombus is necessary and sufficient for recanalization. However, the composition of the intraluminal thrombus formed in association with the rupture of an atherosclerotic plaque is heterogeneous, consisting of platelet-rich material contiguous to the area of plaque rupture and erythrocyte-rich material extending both proximally and distally. This suggests two alternative and potentially complementary targets for coronary thrombolysis: the erythrocyte-rich whole blood clot and the platelet-rich thrombus. Although the potential and limitations of fibrinolytic agents for the dissolution of whole blood clots are well known, the potential for pharmacological dispersion of platelet clumps and platelet-rich thrombus has not been fully explored. Furthermore, the mechanism of bleeding and strategies to reduce it need to be elucidated. STRUCTURAL PROPERTIES OF T-PA AND SCU-PA AND MECHANISMS OF FIBRIN-SPECIFIC THROMBOLYSIS

Structural Propertiesof t-PA

t-PA is a serine protease composed of one polypeptide chain containing 527 amino acids.4 Limited plasmic hydrolysis of the Ar~-Ile276 peptide bond converts the molecule to a twochain activator linked by one disulfide bond. The catalytic site of t-PA is composed of His3U, ASp3’l, and Ser4’“. The complete 2,530-base pair cDNA sequence of t-PA has been elucidated.4 The t-PA molecule contains four domains: (1) a 43-residue-long amino-terminal region (F-domain) that is homologous with the finger domains responsible for the fibrin affinity of fibronectin, (2) residues 44 to 91 (E-domain) that are homologous with human epidermal growth factor (residues 2 to 49), (3) two regions of 82 amino acids each (residues 92 to 173 and 180 to 261) (Kl and K2 domains) that share a high degree of homology with the five kringles of plasminogen, and (4) a serine protease part with the active site residues His, Asp, and Ser.

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The assembly of the t-PA gene is an example of the “exon shuffling” principle. This is suggested by the findings that the different structural domains on the heavy chain (F, E, Kl, K2) are encoded by a single exon or by two adjacent exons.27 Because of the striking correlation between the intron-exon distribution of the gene and the putative domain structure of the protein, it was suggested that these domains would be autonomous, structural, and/or functional entities (“modules”).27X28 t-PA is a poor enzyme in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen. This has been explained by an increased affinity of fibrin-bound t-PA for plasminogen (lower K,,,) without significantly influencing the catalytic rate constant (k,,) of the enzyme.29 The kinetic data of Hoylaerts et a129 support a mechanism by which t-PA and plasminogen adsorb to a fibrin clot in a sequential and ordered way, yielding a ternary complex. Fibrin essentially increases the local plasminogen concentration by creating an additional interaction between t-PA and its substrate. Therefore, the high affinity of t-PA for plasminogen in the presence of fibrin allows efficient activation on the fibrin clot, whereas no efficient plasminogen activation by t-PA occurs in plasma. Although different kinetic constants for plasminogen activation have been reported,30 most investigators agree that fibrin stimulates plasminogen activation by t-PA by at least two orders of magnitude. Functional domains responsible for the fibrinbinding and for the catalytic activity of t-PA have been localized in the t-PA mo1ecule.31,32 The purified B-chain activates plasminogen with kinetic constants similar to those of intact t-PA, but fibrin does not stimulate the activation of plasminogen by the B-chain. The purified A-chain binds to fibrin with an affinity similar to that of intact t-PA but does not activate plasminogen. Van Zonneveld et a133have proposed that initial binding of t-PA to fibrin would be governed by the finger domain and that following partial degradation of fibrin, newly exposed carboxy-terminal lysine residues produce enhanced binding of t-PA via kringle 2. Therefore, early fibrin digestion by plasmin could accelerate fibrinolysis by increasing the binding of both t-PA*’ and plasminogen.34

Structural Properties of scu-PA

scu-PA is a single-chain glycoprotein containing 411 amino acids with 24 cysteine residues.6,35 Hydrolysis of the Lys158-Ile15Qpeptide bond by plasmin converts the molecule to urokinase, a tcu-PA linked by one disulfide bridge. The scu-PA molecule contains three domains: a cysteine-rich amino-terminal region (residues 5 to 49) that is homologous with epidermal growth factor and also occurs in t-PA (residues 44 to 91) a kringle region comprising amino acid residues 50-136 that also occurs five times in plasminogen and twice in t-PA, and a serine protease part with the active site residues His, Asp, and Ser in positions 204, 255, and 356 respectively, that constitutes the carboxy-terminal region of the molecule. scu-PA has a very low reactivity towards low-molecular-weight synthetic substrates or active site inhibitors that are very reactive towards tcu-PA.36,37 Kinetic analysis has shown that the conversion of scu-PA to tcu-PA and plasminogen to plasmin can quantitatively be described by a sequence of three reactions each of which obeys Michaelis-Menten kinetics.38 In a first reaction, scu-PA directly activates plasminogen to plasmin then plasmin converts scu-PA to tcu-PA and in the third reaction plasminogen is activated by tcu-PA. From these results it was concluded that scu-PA has intrinsic plasminogen activating potential, which was eventually estimated to be 0.5% of that of tcu-PA.39 A low intrinsic plasminogen-activating potential of scu-PA was confirmed by other investigators,40 whereas some investigators have claimed that scu-PA has no enzymatic activity and that it is a genuine proenzyme. Mechanisms

of Fibrin-Specific

Thrombolysis

Plasmin, the proteolytic enzyme of the fibrinolytic system, has a low substrate specificity. In purified systems it will degrade fibrinogen almost as well as fibrin and, when circulating freely in the blood, it will degrade a number of plasma proteins including fibrinogen and the blood coagulation factors V and VIII. However, plasmin generated in blood will rapidly be neutralized by q-antiplasmin via interaction with both its lysine-binding sites and its active center. Extensive systemic activation of the fibrinolytic system will result in q-antiplasmin

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consumption, excess free-plasmin generation, and secondary hemostatic breakdown. In contrast, plasmin that is generated at the fibrin surface has both its lysine-binding sites and active center occupied and thus is only very slowly inactivated by cu,-antiplasmin.3.42 Clotspecific thrombolysis will, in view of these interactions, require plasminogen activation at or in the vicinity of the fibrin clot. The two physiological plasminogen activators, t-PA and scu-PA, exert clot-specificity in a plasma environment; however, via entirely different molecular mechanisms.’ t-PA is relatively, but not totally, inactive in the absence of fibrin. It binds specifically to fibrin, thereafter acquiring a high affinity for plasminogen, resulting in preferential plasminogen activation on the fibrin clot. In a plasma milieu, relatively fibrin-specific clot lysis can be obtained within a rather narrow concentration range with scu-PA. In the absence of fibrin, no significant plasminogen activation occurs. Conversion of scu-PA to tcu-PA in the vicinity of a fibrin clot apparently constitutes a primary positive-feedback mechanism for clot lysis.43,” Binding of plasminogen to fibrin or predigestion of fibrin was found to result in relatively minor additional acceleration of fibrinolysis.43 The molecular interactions that regulate the fibrin-specific activation of the fibrinolytic system by scu-PA remain to be further detailed. Because bothM, 54,000 scu-PA and scu-PA-32k (obtained by hydrolysis of the G1u’43-leu’44 peptide bond45) induce very similar fibrin-specific clot lysisTti it appears that the structures responsible for the fibrin specificity of scu-PA are independent of the NH,-terminal portion of the protein. NEW APPROACHES, TO THROMBOLYTIC THERAPY

Present research in this area is carried out along several lines, including (1) attempts to improve the thrombolytic potency and specific thrombolytic activity of fibrinolytic agents, (2) attempts to interfere with platelet-rich thrombus, and (3) attempts to reduce or reverse the bleeding tendency. The aims of this research is to overcome resistance to recanalization, to accelerate thrombolysis, to avoid reocclusion, and to reduce bleeding.

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Attempts to improve the Thrombolytic Potency and Specific Thrombolytic Activity of Fibrinolytic Agents These attempts have focused on (1) mutants of rt-PA, (2) mutants of rscu-PA, (3) recombinant chimeras of t-PA and scu-PA, and (4) complexes between fibrin-specific antibodies and plasminogen activators. Mutants of r&PA. Three main approaches have been used for the development of rt-PA mutants with higher fibrin affinity, longer in vivo half-life, or improved fibrin specificity. These include mutations of specific amino acids, deletion mutants of functional domains, and glycosylation variants. For further information on this subject, please refer to the scientific literature.47”9 Larsen et a15” have studied the pharmacokinetic properties in rats of recombinant deletion mutants lacking the finger domain (rt-PA-AF), the growth factor domain (rt-PA-BE), or both domains together (rt-PA-AFE) and found that deletion of the finger domain reduces the clearance approximately lo-fold. Glycosylation variants of rt-PA-AFE, either with the glycosylated Asn”’ residue mutagenized to Gln (rt-PAAFElX), or with the three known glycosylated Asn residues replaced by Gln (rt-PA-AFE3X) were constructed. Following infusion over 4 hours in rabbits, t-PA-related antigen disappeared from plasma with an initial t,,Z of 25 minutes for rt-PA-AFE, 42 minutes for rt-PAAFElX and 14 minutes for rt-PA-AFE3X, as compared with 4 minutes for natural t-PA.‘l The thrombolytic potency (percent clot lysis per milligram per kilogram compound administered) and fibrin specificity of these mutants in rabbits with jugular vein thrombosis were found to be comparable to those of natural t-PA?l However, the specific thrombolytic activity (percent clot lysis per microgram per milliliter steady-state plasma concentration) was markedly reduced. rt-PA-AFE3X also had a markedly reduced plasma clearance of 21 to 36 mL/min as compared with 520 mL/min for natural t-PA in dogs.5Z On bolus injection of rt-PA-AFE3X in dogs with copper-coil-induced coronary artery thrombosis, this mutant was shown to be a more potent thrombolytic agent than wild-type t-PA.52 In a similar approach, two recombinant variants of t-PA lacking the finger-

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like, growth factor-like, and first kringle domains (amino acids 6 through 173) were shown to have a 20-fold reduced clearance and a somewhat higher thrombolytic potency than rt-PA following bolus injection in dogs with copper-coil-induced coronary artery thrombosis.53 In an effort to further characterize the biochemical and functional properties of recombinant domain deletion/duplication variants of t-PA, five cDNAs were expressed encoding rt-PA-AFE, rt-PA-AKlVK2, rt-PA-AEKlVK2, rt-PA-AFKlVK2, and rt-PA-AFEK1VK2?4 Replacement in rt-PA of K, by a second copy of K,, which is known to contain a lysine-binding site, significantly enhanced its affinity for lysine, however with maintenance of its affinity for intact fibrin. Deletion of the finger and growth factor domains resulted in a decreased fibrin affinity and fibrinolytic potency in a plasma milieu, but these properties were partially restored by replacement of K, by K2.54The thrombolytic potency and specific thrombolytic activity of these variants were determined in a hamster pulmonary embolism mode1.55 Deletion of the finger and growth factor domains in rt-PA (rt-PA-AFE) was not associated with marked alteration of the thrombolytic potency but was associated with a significant reduction of the specific thrombolytic activity. Furthermore, substitution of the kringle 1 domain by a second copy of the kringle 2 domain in rt-PA and in rt-PA-AFE did not significantly alter the thrombolytic potency. None of the variants had a higher specific thrombolytic activity than wildtype rt-PA. Plasma clearances were reduced loto 20-fold for the rt-PA mutants lacking the F and E domains and three to fivefold for the rt-PA variant with a duplicated K2 domain. A prolonged half-life in animals of rt-PA mutants with deletion of the F and/or E domains has also been obtained by other investigators?’ Taken together, these studies indicate that rt-PA mutants can be produced with significantly reduced plasma clearance, which may, however, be associated with a reduced specific thrombolytic activity, whereby these molecules do not have a significantly improved thrombolytic potency. Mutants of rscu-PA. Because clot lysis with scu-PA is associated with a higher degree of

fibrin specificity as compared with tcu-PA, attempts were made, using recombinant DNA technology, to prevent its conversion.58 Such plasmin-resistant mutants, produced by replacement of the lysine at position 158 by other amino acids, were found to have an approximately fivefold reduced thrombolytic potency in rabbits with jugular vein thrombosis.59 These findings support the hypothesis that scu-PA has intrinsic plasminogen-activating potential in vivo. However, the residual thrombolytic potency of such mutants may be too low to be used in humans. A low M, derivative (residues 144-411) of human scu-PA was purified from cell cultures4’ or was obtained by recombinant DNA technol~gy.~~In a rabbit jugular vein thrombosis model, comparable thrombolysis was obtained with rscu-PA-32k as compared with low-molecularweight two-chain urokinase,& whereas thrombolysis with rscu-PA-32k was associated with much less extensive systemic fibrinogen breakdown than with two-chain urokinase. The functional properties of rscu-PA-32k, expressed with high efficiency, were found to be similar to those of its previously characterized natural counterpart.45 Therefore, rscu-PA-32k may represent a useful alternative for large-scale production by recombinant DNA technology. Recombinant

chimeras

of t-PA and scu-PA.

The rationale for the construction of recombinant chimeric molecules between t-PA and scu-PA is based on two observations. First, the structures in t-PA responsible for its fibrin affinity are apparently localized in the A-chain (finger domain and second kringle). Second, the fibrin specificity of scu-PA is not dependent on the NH,-terminal 143 amino acids but is only preserved if the Lys’58-Ile159 peptide bond is intact. Chimeric proteins consisting of parts of the A-chain of t-PA (amino acids Ser’ through Arg275) and containing scu-PA-32k (amino acids Leu’” through Leu4”) might thus combine the mechanisms of fibrin selectivity of both molecules. The authors have produced and characterized two such recombinant chimeric plasminogen activators, one consisting of the NH,terminal 1 through 263 amino acids of t-PA, fused to the COOH-terminal 144 through 411 amino acids of SCU-PA,60and the other consist-

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ing of amino acids 1 through 274 of t-PA and 138 through 411 of scu-PA.6l The purified molecules were obtained in the single-chain form with a specific activity and plasminogen-activating potential comparable to that of scu-PA. In addition, as compared with SCU-PA, the chimerit molecules had acquired affinity for fibrin, which was less pronounced than that of t-PA. In a rabbit jugular vein thrombosis model, the thrombolytic properties and fibrin specificity of the chimeras was very similar but not superior to those of SCU-PA.~~ Several other chimeric plasminogen activators consisting of various portions of t-PA and urokinase-type plasminogen activator (u-PA) have been constructed and partially characterized.63 Most investigators have obtained chimeras that have maintained the enzymatic properties of u-PA or of t-PA, confirming that the catalytic domains of both enzymes are functionally autonomous. However, the fibrin affinity of these chimeras is usually lower than that of wild-type t-PA, possibly due to the fact that the fibrin binding domains in the NH,-terminal region of t-PA are not folded correctly in the chimeras. However, these findings confirm that the truncated scu-PA molecule can be used as an acceptor for protein structures with specific targeting functions toward the thrombus and also indicate that combination of a partial fibrin affinity of t-PA with the enzymatic properties of scu-PA has not materially improved the thrombolytic potency of the molecule. The authors have recently developed a chimera consisting of the two kringle domains of t-PA and the serine protease domain of scu-PA (t-PA-AFElscu-PA-e), which has little fibrin affinity in vitro@ but has a lo-fold delayed in vivo clearance with relatively maintained specific thrombolytic activity, resulting in a markedly enhanced thrombolytic potency in venous thrombosis models in animals.65 The lack of correlation between the in vitro properties of such t-PA/u-PA chimeras, their pharmacokinetics, and their specific thrombolytic activity indicates that, at least for this type of molecule, extrapolation of biochemical properties to potential clinical use is not warranted. However, such extrapolations have formed the basis for intense research into improved thrombolytic agents by the authorse65 and other investigators.-

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Complexes between fibrin-specific antibodies and plasminogen activators. The fibrin specificity of thrombolytic agents may be improved by targeting of the agent to a fibrin clot by conjugation with monoclonal antibodies that are fibrin specific and do not cross-react with fibrinogen.69;70 Chemical conjugates of tcu-PA7’ or scu-PA7’ with monoclonal antibodies directed against the NH,-terminal of the BPchain of fibrin were shown to have an enhanced thrombolytic potency. We have prepared a chemical conjugate between scu-PA and a monoclonal antibody (MA-15C5) with a more than l,OOO-fold higher affinity for fragment D-dimer of human cross-linked fibrin than for fibrinogen.73 This conjugate had a significantly higher thrombolytic potency and slower clearance than conjugated scu-PA in a rabbit jugular vein thrombosis model74 and in a baboon femoral vein thrombosis model.75 Chemical conjugates have also been made between single chain t-PA and a monoclonal antibody specific for the NH,-terminal part of the BP-chain of fibrin.76,77This resulted in a 3.2to 4.5-fold enhancement of clot lysis in human plasma in vitro and a 2.8 to 9.6 times higher potency than t-PA in a rabbit thrombosis model without causing fibrinogenolysis. Another approach consists of the production of bifunctional antibodies that contain a fibrinspecific monoclonal antibody and a t-PAspecific monoclonal antibody78S79 that concentrate t-PA at a fibrin matrix.78.79 Therefore, antibody targeting with fibrinspecific monoclonal antibodies appears to have the potential to increase the concentration of pIasminogen activator in the vicinity of a thrombus, thereby leading to enhanced clot lysis. Attempts to Integere With Platelet-Rich Thrombus An intraluminal thrombus formed in association with the rupture of an atheromatous plaque is heterogeneous in composition, consisting predominantly of aggregated platelets at the site of the rupture and various amounts of erythrocyterich zones and platelet-rich zones. These zones constitute two alternative and potentially complementary targets for coronary thrombolysis. Furthermore, the infusion of thrombolytic agents is associated with a procoagulant effect, as

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evidenced by the generation of fibrinopeptide A in blood, and a platelet-activating effect, as suggested by the production of proaggregatory thrornboxane A,. Finally, after successful thrombolysis, both the traumatized segment of the vessel wall and the residual thrombus, which is rich in adsorbed thrombin, constitute a powerful stimulus for recurrent thrombosis. In view of the complexity of the pathogenesis of coronaryartery thrombosis, it is therefore not surprising that a single therapeutic approach, consisting of the short-term activation of the fibrinolytic system in vivo, cannot achieve maximal and persistent coronary recanalization. The efficacy of both aspirin and heparin in accelerating coronary thrombolysis, overcoming resistance to lysis, and preventing reocclusion is limited. Even with concomitant administration of heparin and aspirin, thrombolytic therapy does not produce maximal stable coronary artery recanalization in patients with evolving myocardial infarction. This is not surprising in view of aspirin’s unselective inhibition of the synthesis of both proaggregatory and antiaggregatory prostaglandins and of the relative inefficacy of heparin in inhibiting clot-associated thrombin activity. Therefore, the quest continues for improved thrombolytic strategies with the conjunctive use of more potent and more selective antiplatelet and anticoagulant agents. One approach to increased efficacy and speed of coronary artery recanalization might consist of the pharmacological dispersion of plateletrich thrombus in combination with fibrindissolving therapy. Alternatively, specific interference with platelet deposition during fibrinoIysis might accelerate thrombolysis and prevent reocclusion (see next section). Complexes between antiplatelet antibodies and plasminogen activators. Bode et al@-’have chem-

ically coupled tcu-PA to a monoclonal antibody that selectively binds to platelet membrane glycoprotein IIb/IIIa. This conjugate indeed targeted tcu-PA to a platelet-rich clot, resulting in markedly enhanced clot lysis in human plasma in vitro. Chemical conjugates of scu-PA with monoclonal antiplatelet antibodies directed against thrombospondin or against Iigandinduced binding sites (LIBS) on glycoprotein IIb/IIIa were also found to have a somewhat

enhanced thrombolytic pulmonary embolism.‘l

potency in hamsters with

Animal models for the investigation of resistance to clot lysis and reocclusion. In order to

investigate approaches to overcome the resistance of clots to lysis and to prevent reocclusion, the authors have developed two coronary artery thrombosis models in the dog and two femoral artery thrombosis models in the rabbit. The first model consists of a whole blood cIot (WBC) in the left anterior descending (LAD) coronary artery in the setting of a high-grade stenosis in the dog (LAD-model)“’ or of a WBC in the femoral artery of the rabbit (WBC-model),“3 which predispose to reocclusion with plateletrich material. These models are useful for the investigation of attempts to prevent reocclusion following recanalization. The second model consists of a platelet-rich (PR) arterial thrombus that occurs spontaneously in an everted circumflex (CX) coronary arterial segment in the dog (CX-model)K4 or in an everted femoral arterial segment in the rabbit (PR-model).83 These models are suitable for the investigation of the sensitivity of preformed PR occlusive thrombi to recanalization strategies. Prevention of reocclusion. Neither full heparinization nor extensive fibrinogen depletion prevented reocclusion after thrombolysis with rt-PA in the canine LAD model.*’ However, accelerated recanalization and prevention of reocclusion was obtained in the canine LAD model with the intravenous infusion of a saturating dose of F(ab’), fragments of a monoclonal antibody (7E3) directed against the platelet GPIIb/IIIa receptor.” Thus 7E3-F(ab’), fragments or alternative agents interfering with the GPIIb/IIIa receptor-mediated pathways of platelet acti- vation may constitute useful adjunctive agents to thrombolytic therapy. However, the marked prolongation of the bleeding time observed at the high dose of 7E3-F(ab’), used in the dog studies will require further careful dose-response studies before its potential use in humans.86 The effects of the synthetic competitive thrombin inhibitor Argatroban (Genentech Inc, South San Francisco, CA) on thrombolysis with rt-PA was also investigated. In both the canine LAD” and rabbit WBC models,” the frequency of persistent reflow was significantly higher with

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Argatroban than with heparin. Argatroban in combination with rt-PA prolonged the template bleeding time somewhat more than heparin combined with rt-PA, however, all bleeding times normalized within 1 hour after the end of the infusion. Provided the stability of arterial patency can be confirmed in chronic experiments, infusion of Argatroban in combination with rt-PA may offer promise for improved, effective, and safe pharmacological recanalization of occluded coronary arteries in patients with acute myocardial infarction. Lysfi of platelet-rich clot. From the results obtained in the canine CX-model and in the rabbit PR-model it appeared that platelet-rich thrombus is indeed resistant to dispersion with rt-PA.83,84 In the canine CX-modeJs4 potent antiplatelet agents such as 7E3-F(ab’), do not consistently disperse occlusive platelet-rich thrombus. However, 7E3-F(ab’), markedly potentiated recanalization in the canine CXmodel, even with a reduced dose of rt-PA and abolished reocclusion. The mechanism of the enhancing effect of rt-PA and 7E3-F(ab’), on the lysis of platelet-rich thrombus is not clear. If rt-PA would disperse the thrombus by dissolution of the fibrin strands that hold the platelet clumps together, one would not expect such great resistance to thrombolysis with rt-PA boluses alone, at doses that efficiently dissolve a erythrocyte-rich clot.85*86Possibly inhibition of the accretion of new platelets on the thrombus surface may shift the dynamic balance of thrombosis and thrombolysis towards the latter. Prevention of arterial graft occlusion. Intraarterial infusion of Argatroban for 1 hour maintained femoral arterial graft patency during an observation period of 3 hours in the rabbit PR-model, whereas heparin at an equivalent anticoagulant dose was ineffective.89 This study showed not only that the specific thrombin inhibitor was more potent than the standard anticoagulant but also that “passivation” of the vessel wall (ie, loss of thrombogenicity) may occur relatively rapidly. Attempts to Reduce or Reverse the Bleeding Time Prolongation Associated With Dwombolysis

Bleeding constitutes the main side effect of thrombolytic therapy. Although it occurs most

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frequently in association with vascular puncture or other invasive procedures, it may occur unexpectedly, even in carefully selected patients. Spontaneous bleeding does not appear to correlate strongly with the extent of systemic fibrinolytic activation and fibrinogen breakdown nor with any other demographic or clinical characteristic of the patient. The template bleeding time as an index of bleeding tendency. In a study of 52 consecutive patients with acute myocardial infarction treated with rt-PA, in which spontaneous bleeding was observed in 13 patients, a transient prolongation of the template bleeding time was found to predict bleeding with a specificity and sensitivity of 69%. Therefore, the template bleeding time constitutes the first recognized parameter that significantly correlates with spontaneous bleeding in association with thrombolytic therapy. Furthermore, this study showed that aspirin intake is associated with an increased incidence of spontaneous bleeding in association with thrombolytic therapy.% Strategies to reverse bleeding time prolongation.

The interactive effect of aspirin and rt-PA on bleeding time prolongation and bleeding was confirmed in experimental animal studies in rabbits and dogs.91,92Injection of plasminogen activator inhibitor-l in rabbits or of the plasmin inhibitor aprotinin in dogs immediately reversed the bleeding time prolongation and arrested the bleeding at incision sites. The combination of aspirin and streptokinase in rabbits was also found to be associated with a marked increase in bleeding time that could be reduced by administration of 1-desamino-8-D-arginine vasopressin (DDAV).93 Clozel et al% have reported that thrombolysis with rt-PA in rabbits could be blocked by administration of aprotinin, which also could prevent the increase in bleeding time secondary to rt-PA injection. Thus, although the mechanism of the prolongation of the bleeding time remains to be elucidated, the reversibility of this phenomenon argues against degradation of platelet membrane receptors. The phenomenon is suggestive of a plasminmediated derangement of platelet function that cannot be detected by ex vivo platelet aggregation. Therefore, the template bleeding time may constitute a useful quantitative index of the hemorrhagic diathesis occurring in some pa-

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tients during thrombolytic therapy with rt-PA and may represent a useful parameter for the investigation of the mechanisms of spontaneous bleeding. CONCLUSIONS

The beneficial effects of thrombolytic therapy in acute ischemic coronary syndromes, and particularly in acute myocardial infarction, are now well established. However, the limited efficacy and potentially life-threatening side effects of currently available thrombolytic agents remain a major problem. The present limitations of thrombolytic therapy can be explained on the basis of the heterogeneity of coronary arterial thrombus, consisting of both erythrocyterich and platelet-rich zones, and knowledge of the mechanism of fibrin-dissolution and platelet disaggregation. This unified concept suggests alternative and complementary pharmacological approaches to coronary artery recanalization that require evaluation with specific rele-

vant in vivo animal models. Available evidence suggests that the efficacy of coronary thrombolysis may be augmented either by improvement of the potency and specificity of fibrin-dissolving agents, by dispersion of aggregated platelets, or by a combination of both. Although it is clear that compounds with antithrombin or antiplatelet properties may enhance and sustain the action of thrombolytic agents, their optimal use and potential hemorrhagic side effects remain to be further explored. Continued investigations along several new research lines, some of which have been reviewed previously, will provide new insights and promote progress towards the development of the ideal thrombolytic therapy, characterized by maximized stable coronary arterial thrombolysis with minimized bleeding. The authors anticipate that optimized thrombolytic therapy eventually will consist of administration of potent specific plasminogen activators in combination with conjunctive targeted anticoagulant and/or antiplatelet agents.

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