Cardiovascular Drugs and Therapy 1992;6:111-124 © Kluwer Academic Publishers, Boston. Printed in U.S.A.

Advances in Thrombolytic Therapy M. Verstraete Center for Thrombosk and Vascular Research, University of Leuven, Belgium

Summary. Alteplase and saruplase are more fibrin-specific thrombolytic drugs than anistreplase. These and the thrombolytic drugs of the first generation (streptokinase and urokinase) have shortcomings and limitations. The prolonged intravenous maintenance infusions have been replaced by a bolus injection, accelerated infusions, or the combined intravenous administration of thrombolytic agents. Numerous truncated alteplase or saruplase molecules have been constructed by deletion and domain substitution or hybrids made of the two molecules without gaining in thrombolytic potency. Recombinant staphylokinase and plasminogen activator from bat saliva have some interesting properties and are being investigated. Thrombus-targeted thrombolytic drugs were constructed using monoclonal antibodies against fibrin fragments or against epitopes of activated platelets. Fibrinspecific thrombolytic drugs require the concomitant use of a potent antithrombotic drug to prevent reocclusion. Whether hirudin or synthetic thrombin inhibitors are superior to heparin and whether novel antiplatelct agents, including monoclonal antibodies to platelet receptors and disintcgrins, are more effective than aspirin is under clinical investigation. The place of stable analogues of prostacyclin during thrombolytic treatment is still unsettled. Cardiovascular Drugs Ther 1992;6:111-124 Key Words. thrombolysis, alteplase, saruplase, streptokinase, urokinase, disintegrins, monoclonal antibodies, stable prostacyclin analogues, aspirin, ticlopidine, hirudin, argatroban

Bolus Intravenous Administration o f Thrombolytic Drugs Bolus infusion of streptokinase Clinical trials with streptokinase conducted in the last decade use a high-dose (1.5 megaunits streptokinase), brief-duration (60-90 minute infusion) drug regimen. Bolus treatment with 0.6 megaunits [1] or 1.5 megaunits of streptokinase administered over 10 minutes [2] was apparently associated with a high clinical success rate and a still acceptable incidence of side effects. A recent comparative study suggests that 0.75 megaunits of streptokinase administered over 30 minutes produce a similar reperfusion rate (indirect evidence) in patients with acute myocardial infarction as 1.5 megaunits over 1 hour [3].

2,000,000 units over 2-4 minutes in 47 patients (three groups of 16) with acute myocardial infarction. A reperfusion rate at 90 minutes was obtained in circa 50% of the patients without a difference between the two highest doses. No adverse effects were noted in this pilot study [4]. With a bolus dose of 2 million U urokinose, the coronary patency was 60% in 30 patients [5], and this dose was well tolerated [6]. With a higher total dose of urokinase (1.5 million U bolus and 1.5 million U over 90 minutes), the coronary patency was 66% in 47 patients at the end of the infusion.

Bolus administration of alteplase Because alteplase is rapidly cleared from the circulation with an initial half-life of only a few minutes, treatment with this thrombolytic drug is usually by infusion. The currently recommended dosage regimen of alteplase for the treatment of acute myocardial infarction is an intravenous infusion of 60, 20, and 20 mg hourly over 3 hours, with an initial bolus of 10% of the total dose. With this dosage regimen, the coronary recanalization rate at 90 minutes, which corresponds with the administration of 70 mg of alteplase, was 71% in 83 patients [7]. The biological half-lives of alteplase may be significantly different from their measured plasma halflives. Indeed, the thrombolytic effect of alteplase was found to be sustained beyond its time of clearance from the circulation in animal thrombosis models [8-11] and in patients with myocardial infarction [11]. Effective thrombolysis after bolus administration or a short infusion of alteplase has been reported in animals [8-10,12,13]. As the effect of bolus alteplase on coronary recanalization had not been investigated in patients, we compared in a pilot clinical study the efficacy in terms of angiographic coronary recanalization of alteplase given in a bolus of 50 mg (29 patients), 60 mg (28 patients), and 70 mg (25 patients) [14]. Because the highest angiographic recanalization rate (72%) was obtained with a 70-mg bolus of alteplase, the same

Bolus administration of urokinase

Address correspondence and reprint requests to Center for Thrombosis and Vascular Research, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B 3000 Leuven, Belgium.

Urokinase has been administered intravenously by rapid bolus injection of 500,000, 1,250,000, or

Plenary Session Lecture 4th International Symposium Geneva, April 22-25, 1991 111

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dose was given in a subsequent trial to a larger number of patients. The recanalization rate was only 48% (95% confidence interval, 37-60%) at 90 minutes in 60 patients; however, there was a very low reocclusion rate at 14-24 hours of 4% [15]. A single 70-mg bolus injection of alteplase seems to be less effective than 70 mg infused over 90 minutes, which is associated with a recanalization rate of 71% [7]. However, a treatment scheme of two boluses of 35 mg alteplase administered intravenously 30 minutes apart achieve a coronary patency rate of 87% in 30 patients [16]. Notwithstanding angiographic patency rates overestimates coronary reperfusion with 15 to 20% when compared with angiographic recanalization rates. The different results between the latter two trials are not easily explained.

mented reocclusion rate was 8% in this uncontrolled trial. A larger prospective trial in 216 patients with acute myocardial infarction revealed a 90-minute patency rate of 79% with the combined drug regimen (50 mg alteplase and 1,500,000 U streptokinase) vs. 64% with the standard dose of alteplase (100 mg over 3 hours) [21].

Combined Intravenous Administration of Alteplase and Single-Chain Urokinase (Saruplase)

Accelerated infusion of alteplase The dose of alteplase most often used is 100 mg administered over 3 hours (40, 20, 20 in successive hours). An accelerated administration of the same dose of 100 mg over 90 minutes (15 mg initial bolus, 50 mg over 30 minutes, and 35 mg over 60 minutes) increases the patency rate at 90 minutes from 75% (for the 3-hour infusion) to 91% (for the accelerated infusion) [17]. The higher patency rate of this accelerated infusion of alteplase has been confirmed with a slightly modified dose regimen (0.75 mg/kg/30 min followed by 0.5 mg/ kg/60 min), which resulted in a patency rate at 90 minutes of 84% in 63 patients, with a reocclusion rate of 5% at predischarge catheterization [18].

Because the intrinsic fibrin selectivity of alteplase and saruplase is mediated by different molecular mechanisms, their combined effect on clot dissolution might be more than additive. Combination of these drugs in animal models of thrombosis produced significantly greater lysis than could be explained on the basis of their additive effects [22]. In a pilot study in myocardial infarction, a relatively low dose of two-chain alteplase and single-chain urokinase-type plasminogen activator given together induced recanalization of the occluded coronary in almost all patients [23]. This clinical investigation was expanded using 20 mg of alteplase combined with 10, 15, or 20 mg of saruplase. In a study in 43 patients with acute myocardial infarction, successful reperfusion (TIMI grades 2 or 3) at 90 minutes was observed in 36% of the total study group [24]. It is obvious that the combination of the two drugs at the doses used is considerably less effective than either drug given alone in their presently recommended doses (100 mg alteplase and 80 mg saruplase).

Combined Intravenous Administration of Streptokinase and Alteplase

Combined Intravenous Administration of Alteplase and Urokinase

Streptokinase is associated with an activation of the systemic fibrinolytic system and the circulating plasmin digest fibrinogen and some other coagulation proteins; the r~eduction of fibrinogen of more than 70% decreases viscosity, which may be important to prevent intermittent and late reocclusion. Furthermore, streptokinase has a longer half-life (circa 30 minutes) than alteplase (initial half-life circa 6 minutes) [19], as the latter molecule is recognized by the liver primarily through its heavy chain. Combining alteplase with streptokinase provides the potential advantage of rapid coronary thrombolysis with alteplase while substituting a systemic fibrinolytic agent for maintenance infusion. With half the usual dose of alteplase and 1,500,000 U streptokinase, a patency rate at 90 minutes of 75% in 40 patients was obtained [20]. Somewhat disappointingly, the angiographically docu-

Also various combinations of alteplase with urokinase have been investigated both in the United States [18,25,26] as well as in Europe [27]. Although these combinations did not appear to improve significantly the reperfusion velocity or rate when compared with each agent used alone, an encouraging decreased incidence of reocclusion was noted when fibrin-specific alteplase and non-fibrin-specific urokinase were combined.

Accelerated Intravenous Infusion of Thrombolytic Drugs

Combined Intravenous Administration of Saruplase and Urokinase Saruplase (33-74 mg) plus urokinase (250,000 U) were given to patients with acute myocardial infarction

Advancesin ThrombolyticTherapy

[28-31]. No Clear improvement in reperfusion was obtained with the combination therapy compared with results in historical controls [32].

Deletion, Domain Substitution, and Hybrids of Alteplase and Saruplase Considering that the best therapeutic regimen with thrombolytic agents of the first two generations failed to induce recanalization in approximately 20% of treated patients, a third generation of thrombolytic compounds is being designed. The aim is to improve the fibrin affinity or to prolong the half-life of novel agents, variants, or hybrids of alteplase or saruplase. A comprehensive review on novel strategies for the improvement of thrombolytic drugs has recently been published [33]. The fibrin selectivity of alteplase is mediated via its affinity for fibrin, which is supported by the finger domain and by the second kringle domain. Several approaches have been made to enhance the fibrin affinity of alteplase by alteration of the fibrin-binding domain. The rationale for this approach is based on the assumption that mutants of alteplase with enhanced fibrin affinity would constitute more fibrin-specific thrombolytic agents. Most recombinant variants, designed to mimic the high-affinity lysine-binding site of plasminogen, are unfortunately not endowed with a significantly improved thrombolytic potency [34,35]. Plasmin-resistant mutants of alteplase can be constructed by replacing the arginine of the plasminsensitive Arg275-Ile27~ peptide bond by glutamic acid or glycine. This compound has in the presence of fibrin a comparable thrombolytic potency as alteplase [33]. This indicates that single-chain alteplase does not have to be converted to a two-chain molecule to gain its full activity. Mutants of alteplase with altered catalytic efficiency in the presence of fibrin have been made by several groups [33] but did not result in animal models of thrombosis in a higher thrombolytic potency [35,36]. One of the characteristics of alteplase is its rapid clearance, which results in an initial half-life of about 3 minutes. This is due to two different uptake receptors in the liver: a mannose receptor, mainly in liver endothelial cells, and another unknown receptor in the parenchymal liver cells. Kringle 1 of alteplase contains a high mannose-type oligosaccharide and was deliberately deleted in a novel recombinant construction of the molecule, which was restricted to kringle 2 and the protease domain [37]. As this plasminogen activator (BM 06.022) was expressed in Escherichia coli cells, it lacks oligosaccharide side chains. Its half-life in rabbits was 18.9 -+ 1.5 minutes compared with 2.1 -- 0.1 minutes for alteplase. The 50% effective thrombolytic dose was 163 U/kg for the mutant and 871 U/

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kg for alteplase. At equipotent doses of two plasminogen activators have a similar fibrin specificity [38]. Substitution of a few selected amino acids in the amino-terminal domain of alteplase may yield mutants with significantly slower plasma clearance, possibly with better preservation of specific thrombolytic activity [33]. Plasminogen activators are inhibited in plasma by specific plasminogen activators (PAI), mainly by PAI-1, which rapidly inhibits alteplase. Mutants of alteplase resistant to inhibition by PAI-1 have been reconstructed. In view of the large excess of alteplase over PAI-1 during thrombolytic therapy, the resistance of alteplase mutants to PAI-1 may not constitute a major significant progress. Mutants and plasmin-resistant variants of saruplase have been constructed, but without gaining in thrombolytic potency [33]. A low molecular weight derivative of saruplase (molecular weight 33,000) with the same thrombolytic activity of the parent molecule may represent a useful alternative for large-scale production by recombinant DNA technology [39,40]. Recombinant chimeric proteins containing functional domains of alteplase and of other proteins have been constructed. For most of these chimeras, information on the in vivo thrombolytic properties is at present lacking.

Recombinant Staphylokinase, a Fibrin-Specific Thrombolytic Drug In plasma in the absence of fibrin, the plasminogenstaphylokinase complex is rapidly neutralized by ~2*antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by a~-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface [41]. In animal models of venous thrombosis, recombinant staphylokinase had a higher thrombolytic potency than streptokinase. The plasma clearance following a bolus injection of staphylokinase or steptokinase in hamsters or rabbits was comparably rapid (1.1-1.4 ml/min in hamsters and 14-15 ml/ min in rabbits) as a result of a short initial half-life (1.8-1.9 minutes in hamsters and 1.7-2.0 minutes in rabbits) [42]. No antibodies developed in animals treated with recombinant staphylokinase, whose thrombolytic potential warrants further investigation.

Plasminogen Activator from Bat Saliva The saliva of the vampire bat (Desmodus rotundus) contains a single-chain plasminogen activator with

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about 85% homology of alteplase but is missing the kringle 2 domain and a plasmin-sensitive cleavage site for conversion to a two-chain form [43]. A smaller molecular form additionally lacks the finger domain [44]. The fibrinolytic activity of the full-length form of the bat plasminogen activator is dramatically 200-fold more selective than alteplase towards fibrin-bound plasminogen [43,45]. As the molecule is lacking the plasmin-sensitive cleavage point, this naturally occurring molecule remains stable in the circulation. The protein is now being produced by recombinant technology in a eukaryotic cell line with a specific activity of about 300,000 U/mg as compared to alteplase in fibrin plates [46].

Thrombus- Targeted Thrombolytic Drugs Thrombi contain both fibrin- and platelet~rich material. Plasminogen activators may be targeted to a thrombus by conjugation with monoclonal antibodies directed against specific epitopes in fibrin or against surface proteins on platelets. Furthermore, bispecific monoclonal antibodies containing one site that recognizes the thrombus and one site that binds the plasminogen activator may be used to concentrate the therapeutic agent at the surface of the thrombus. One approach is to target the thrombolytic agent to a fibrin clot by conjugation with monoclonal antibodies that are fibrin specific and do not crossreact with fibrinogen [47]. Chemical conjugates of two-chain urokinase [47-49] or single-chain urokinase (saruplase) [50] with monoclonal antibodies directed against the NH 2 terminal of the B~-chain of fibrin were shown to have a three-fold enhanced thrombolytic potency in a plasma milieu in vitro [49]. A chemical conjugate between recombinant saruplase and a monoclonal antibody (MA-15C5) with a more than 1000-fold higher affinity for fragment Ddimer of human crosslinked fibrin than for fibrinogen has been prepared [51,52]. This conjugate had a 6.4fold higher fibrinolytic potency than saruplase in a human plasma milieu in vitro [52] and an eightfold higher thrombolytic efficiency with fourfold slower clearance than unconjugated saruplase in a rabbit jugular vein thrombosis model [53]. A two-chain derivative of this conjugate was prepared by treatment with thrombin, which enhanced its fibrinolytic potency at least 50-fold in a human plasma milieu, with a superior fibrin specificity to that of the former conjugate and of recombinant saruplase [54]. In addition, the latter conjugate is not inhibited by plasma protease inhibitors. The single-chain conjugate has a fourfold slower clearance than saruplase, as determined after bolus

injection from the plasma disappearance after bolus injection and an eightfold slower clearance as determined from the steady-state plasma levels [54,55]. The single-chain conjugation increased the fibrinolytic potency about ninefold in a rabbit model with jugular vein clots prepared from human plasma, whereas the two-chain urokinase conjugate obtained after plasmin treatment had a fourfold increase in potency [53,56]. The fibrin selectivity of the saruplase conjugate was superior to that of saruplase. While conjugation per se reduces the thrombolytic potency, its targeting property against the thrombus overcomes the negative effect of conjugation. Experiments in rabbits cannot be extrapolated to humans because the monoclonal antibodies used do not interact with rabbit fibrin and fibrinogen, whereas in humans interaction of the conjugate with circulating fibrinogen or fibrin(ogen) degradation products may interfere with its thrombolytic potency. It is reconforting, however, that human fibrin fragment D-dimer does not influence plasminogen activation by the conjugate in purified systems [51]. Chemical conjugates have also been made between single-chain alteplase and a monoclonal antibody specific for the NH2-terminal part of the B~-chain of fibrin (MA-59D8) [49,55]. This resulted in a 3.2- to 4.5-fold enhancement of clot lysis in human plasma in vitro and a 2.8-9.6 times higher potency than alteplase in a rabbit thrombosis model, without causing fibrinogenolysis. Schnee and coworkers have engineered a recombinant version of the alteplase MA-59D8 conjugate [57]. The MA-59D8 heavy-chain gene was cloned and combined in an expression vector with sequence coding for a portion of the 2b constant region and for the B-chain of alteplase, which contains the catalytic site. This construct was transfected into cloned cells derived from the MA-59D8 hybridoma that had lost the capacity to express the heavy chain. The chimeric proteins indeed had antifibrin antibody activity and retained plasminogen activating potential [57]. Another approach consists of the production of bifunctional antibodies that contain a fibrin-specific monoclonal antibody and an alteplase-specific monoclonal antibody. Such duplex antibodies have been obtained by chemical coupling [58,59] or by recombinant DNA technology [60], and were indeed shown to concentrate alteplase at a fibrin matrix. Monoclonal antibodies that recognize epitopes on the surface of activated platelets, but not of resting platelets, might represent another targeting vector for thrombolytic agents towards platelet-rich thrombi. Bode et al. [61] have chemically coupled two-chain t-PA to a monoclonal antibody that selectively binds to platelet membrane glycoprotein IIb/ IIIa with remarkable in vitro enhancement of clot lysis. Also saruplase was chemically conjugated to a monoclonal antibody (MA-TSPI-1) directed against human thrombospondin, a platelet a-granule glyco-

Adva~cesin ThrombolyticTherapy

protein that is expressed on the surface of stimulated platelets [62].

Concomitant Treatment with Alteplase and High-Dose Intravenous Heparin There is experimental and clinical evidence that thrombolytic therapy with any thrombolytic agent is associated with activation of the coagulation system and that this activation can largely be blocked by heparin, provided the blood levels of heparin are high enough [63]. Clinical trials with alteplase and saruplase have generally been combined with early intravenous heparin. In a trial in patients receiving a 3-hour infusion of alteplase, immediate intravenous heparin did not appear to improve the patency rate assessed at 90 minutes after the initiation of thrombolytic treatment [64]. The dosages of heparin used may have been too low, and too few patients were involved in this trial to judge the effectiveness. Three other trials revealed a significantly higher patency rate at 18 hours [65], 60 hours [66], and 81 hours [67] in patients given alteplase plus immediate intravenous heparin compared to alteplase without heparin. Also a significantly higher patency rate was obtained when saruplase and intravenous heparin were given concomitantly than saruplase and placebo [68]. The latter four trials are concordant in their conclusion that a higher patency rate after alteplase or saruplase is maintained in the presence than in the absence of coadministered intravenous heparin. The important role of intravenous heparin is also evident from the levels of fibrinopeptide A (a sensitive marker of circulating thrombin activity), which are high during treatment with alteplase or streptokinase in the absence of heparin and fall as soon as h eparin is added [69-72]. The intravenous heparin infusion can be discontinued 24 hours after alteplase therapy and replaced with an oral antiplatelet regimen without any adverse effects on reinfarction and left ventricular function [73]. Whether the observed higher patency rate is due to potentiation by heparin of the lytic effect of alteplase or saruplase, or inhibition by the anticoagulant of thrombus accretion could not be differentiated in the experimental animals used so far. This problem has now been circumvented, and it was shown that unfractionated heparin and a low-molecular-weight heparin (enoxaparin) do not influence the lysis of thrombi by alteplase; the apparent enhancement of thrombolysis in the presence of heparin is due to diminished clot accretion [74]. In vitro experiments also demonstrate that unfractionated heparin and enoxaparin do not limit the fibrin selectivity of alteplase by augmenting systemic plasmin generation.

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Recombinant Hirudin and Synthetic Hirudin Analogues Although a powerful anticoagulant, heparin is not the ideal protection against thrombus accretion. When a thrombus forms, thrombin is bound to fibrin and incorporated into the growing thrombus. Both free thrombin in the circulation and fibrin-bound thrombin stimulate new thrombin generation by activating factors V and VIII, and by activating platelets. Free and bound thrombin cleave fibrinogen to form fibrin. Free thrombin is readily inhibited by the circulating antithrombin III, particularly when this natural protein is potentiated by heparin, but fibrin-bound thrombin is only inhibited to a minor extent by the antithrombin III-heparin complex. Fibrin-bound thrombin thus continues to activate platelets and fibrinogen in the presence of circulating heparin. In contrast, hirudin, a natural protein produced by leeches, binds to the noncatalytic site in thrombin and also inactivates fibrin-bound thrombin because hirudin does not need to complex with antithrombin III and, being a small molecule, can penetrate the thrombus [75]. In an animal model of thrombosis and in contrast to heparin, the antithrombotic activity of hirudin is sustained beyond its plasma clearance, presumably because of the inactivation of thrombus-bound thrombin [74]. Several in vitro and in vivo animal studies suggest that hirudin would have a clear advantage over heparin [74-76], and this hypothesis is presently being tested in patients. Hirudin, which is now being produced in large quantities by recombinant technology, has another advantage; while heparin used alone at pharmacological concentrations increases platelet adhesion to fibrin and extracellular matrix, hirudin does not. This may be important, particularly after PTCA [77]. After intravenous infusion in volunteers, plasma concentrations of hirudin decrease with a half-life of approximately 0.5 hours in the early phase and about 3.7 hours in the late phase [75]. In order to prolong its action, recombinant hirudin was covalently bound to polyethylene glycol (PEG). This coupling prolongs the in vivo activity of hirudin without loss of activity and selectivity [78]. The region in the hirudin molecule that inhibits thrombin-catalyzed fibrinogen cleavage is located at the C-terminal end (HIR 54-65). The carboxyterminal dodecapeptide Hir 53-64 comprises a limited domain of hirudin, still showing maximal anticoagulant activity (e.g., hirugen and hirullin P18, a 61amino-acid hirudin fragment [79]). A 20-mer peptide hybrid (hirulog) has been synthesized, combining the antithrombin activities of D-phenyl-prolyl-arginyl (DPhe-Pro-Arg) and the dodecapeptide of hirudin [80]. This hybrid peptide exhibits greater antithrombin efficacy in a baboon model than the present molecules, approaching the potency of D-Phe-Pro-Arg chloro-

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methylketone without its toxicity [81]. A synthetic decapeptide (hirudin 55-65, MDL 28050) was shown to be an effective antithrombotic agent in experimental thrombotic models in mice and rats [82].

Synthetic Thrombin Inhibitors Argatroban (MCI-9038) is a synthetic thrombin inhibitor that blocks the active site of thrombin and is, like hirudin, independent of antithrombin III for its activity. This molecule is more effective than heparin in reducing platelet deposition at the site of balloon injury produced ex vivo [83] in a canine coronary artery stenosis model [84] and in a rabbit femoral arterial eversion graft model [85]. This molecule accelerates the reperfusion in animal models and lowers the reocclusion rates when compared to standard thrombolytic therapy alone [86-88]. In human volunteers, steady-state plasma concentrations were achieved after l-hour intravenous infusion of 1 ~g/kg/min. The elimination half-life was 24 -- 4 minutes. The bleeding time remained unchanged, which opens the possibility of coadministration with aspirin [88]. MD-805 is another potent and synthetic selective inhibitor of thrombin that also acts independently of antithrombin III; this compound has been tested in patients with progressing cerebral thrombosis with good tolerability and apparent success [89].

P-PACK The "fibrinogen-like" sequence D-phenylalanyl-Lprolyl-L-arginyl chloromethylketone (P-PACK) is a synthetic irreversible thrombin inhibitor; it is, in fact, an imitation of fibrinopeptide A. This compound markedly reduces or prevents thrombus growth in experimental thrombosis models [90]. In a baboon arteriovenous model, P-PACK was shown to reduce reocclusion following thrombolysis [91]. The brief local treatment of implanted vascular grafts with high doses of this compound also prevents subsequent thrombus formation without risk of hemorrhage [92]. Compounds have been synthesized in which the arginine of P-PACK has been replaced by the boronic acid analog of arginine, boroArg; their advantage is the potential oral bioavailability [93]. Platelet inhibition

As noted above, rethrombosis is partly related to the adequacy of thrombolysis and the residual minimal diameter of the infarct-related artery, and also to the intensity of antiplatelet therapy. The anchored residual thrombus produces not only a persistent stenosis but alters the local rheology of blood flow. High shear increases the local concentration of adenosine diphosphate, an inducer of platelet aggregation, and forces more platelets to the periphery of the artery [94,95].

Platelet deposition renders residual mural thrombi more thrombogenic and, in experimental in vivo models, vasoconstriction directly relates to the log of platelet deposition [96,97]. It is remarkable that in experimental studies antiplatelet agents with no vasodilatatory effects reduce both vasoconstriction and platelet deposition [96], while vasodilators, such as nifedipine or verapamil, do not result in decreased platelet deposition [97]. It has been noted that thrombi trailing distally beyond the area of residual stenosis at early coronarography (90 minutes after the start of thrombolytic treatment) resolve in about 50% of patients within the following 24 hours, which is most probably related to the continued administration of lytic drugs. However, a further reduction of residual stenosis and improvement in the minimal lumen diameter has been shown at hospital discharge angiography [98-100]. It is remarkable that the median values for minimal luminal diameter, percent diameter, and area of obstruction of the coronary lesion were not found to be different at hospital discharge in patients randomized to combined treatment consisting of alteplase, intravenous heparin, and aspirin from those receiving only the last two drugs [101]. Late changes in the size of coronary lesions can be attributed to resorption of hemorrhage, dissolution of residual thrombus due to endogenous thrombolysis, and altered vasomotor tone; it is reasonable to assume that the prevention of rethrombus by aspirin and heparin contribute to these effects. Other authors maintain that anticoagulation can contribute to the reduction of residual stenosis of the infarct-related artery at angiography repeated after 1 month [102]. Aspirin is the antiplatelet drug most widely used in conjunction with thrombolytic drugs. This drug is also of proven benefit in patients with unstable angina, myocardial infarction, cerebrovascular disease, and after coronary or peripheral artery bypass surgery. This is remarkable because aspirin does not prevent platelet adhesion to exposed subendothelial structures, nor does it prevent platelet aggregation induced by thrombin. The proper dose to fully inhibit the synthesis of platelet thromboxane while leaving the endothelial synthesis of prostacyclin largely untouched is still a matter of debate, but 75 mg daily seems, most probably, to be enough [103]. A thromboxane synthase inhibitor presents some major advantages when compared to aspirin [104]. Despite its interesting properties, the first clinical trials with thromboxane synthase inhibitors have been disappointing [105], to some extent due to the fact that PGH.~, accumulating during thromboxane synthase inhibition, can itself interact with the platelet endoperoxide receptor and thereby activate platelets. The more recently developed thromboxane receptor antagonists specificially impede the action of both thromboxane and PGH 2 on their receptor, while leaving the normal pattern of thromboxane and prosta-

Advances in Thrombolytic Therapy

glandin formation unaltered. These drugs give a more reproducible and pronounced inhibition of platelet function and prolong the bleeding time more than thromboxane synthase inhibitors. Preliminary clinical trials seem to indicate that thromboxane receptor antagonism may be more effective antiplatelet therapy than thromboxane synthase inhibitors [106]; some are very long acting and, moreover, are endowed with an anti-ischemic effect [107,108]. However, possible drawbacks of this class of compounds are represented by their competitive nature, which could lead to their displacement from receptors by exceedingly high levels of TXA2 and PGH2 generated at localized sites of platelet activation. Furthermore, as compared to thromboxane synthase inhibitors, thromboxane receptor blockers do not increase the endogenous production of platelet-inhibitory prostaglandins. Finally, like aspirin and other cyclooxygenase inhibitors and thromboxane synthase inhibitors, thromboxane receptor blockers also do not affect platelet activation induced by thromboxane-independent agonists, such as high-dose collagen, thrombin, and, to some extent, ADP [109]. Molecules with the dual activity of inhibiting

thromboxane synthase and blocking the receptors for thromboxane and endoperoxides have several advantages, one being that accumulating endoperoxide substrate in the platelets may be donated to the endothelial prostacyclin synthetase at the site of platelet-vascular interaction. Such an agent is ridogrel, which reduces elevated levels of markers of in vivo platelet activation in patients [110]. Ridogrel was also shown to enhance alteplase-induced reperfusion in canine coronary arteries [111-113] and in patients with acute myocardial infarction [114]. The landmark progress made by molecular cloning of a functional thrombin receptor in h~man platelets and vascular endothelial cells [115] will open new perspectives in a selective inhibition of thrombin action on platelets, sparing its role in the activation of coagulation components.

C o m b i n e d A n t i t h r o m b o t i c Prevention w i t h A s p i r i n a n d Ticlopidine Among the numerous compounds decreasing platelet function, aspirin and ticlopidine clearly emerge as the most effective in clinical conditions where the arterial component is pathogenetically relevant. Aspirin acetylates cyclooxygenase in platelets, endothelial cells, and other cells, and render aspirinated platelets less sensitive to their activation by several agonists, such as ADP, adrenaline, and low concentrations of collagen, but not to thrombin or higher concentrations of collagen. Ticlopidine seems to inhibit fibrinogen binding to platelet ADP receptors and would therefore inhibit platelet aggregation induced by most aggregating agents. Clopidogrel, an analogue of ticlopidine,

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also involves an irreversible inhibition of platelet aggregation, specifically affecting ADP-dependent activation of glycoprotein IIb/IIIa. On the basis of these distinct mechanisms of action, a synergism of aspirin and ticlopidine, and its analogue (clopidogrel) is a not unreasonable hypothesis [116,117]. Both drugs increase the bleeding time, an effect that becomes much larger with their combined use. Whether the theoretical antithrombotic benefit would outweigh the risk for hemorrhage can only be resolved in a clinical trial. A suitable test model would be coronary or peripheral artery balloon angioplasty, which is associated with sudden exposure of subendothelial collagen. Either drug used alone offers an inadequate antithrombotic protection in this particular condition, which might be offered by their combined use.

Monoclonal A n t i b o d i e s to Platelet Receptors Monoclonal antibodies directed against the platelet glycoprotein receptor IIb/IIIa can prevent the aggregation of platelets, irrespective of the pathway of activation [86,118-120]. When compared to heparin, 70% of platelet deposition can be prevented in injured rabbit aortas mounted in a perfusion chamber. This means that 70% of platelet deposition is GPIIb/IIIa dependent and 30% is due to platelet subendothelium adhesion. Murine monoclonal antibodies to GPIIb/IIIa have been shown to shorten the recanalization time, even of platelet-rich thrombi [84], and to prevent reocclusion in thrombotic models in animals [121]. In patients with stable angina, 7E3 Fab produced marked inhibition of platelet function at single doses ->0.25 mg/kg, which can be sustained by continuous infusion [122]. Unfortunately, murine monoclonals against GPIIb/IIIa can induce murine anti-murine antibodies and thrombocytopenia [86]. Also a purified peptide-specific monoclonal antibody inhibiting von Willebrand factor binding to GPIIb/IIIa, without interacting with other adhesive proteins containing the sequence Arg-Gly-Asp, inhibits the deposition of platelets on human atherosclerotic wall [123].

Disintegrins Platelet aggregation is dependent on the interaction of the platelet membrane glycoprotein (GP) IIb/IIIa complex with macromolecular plasma-adhesive glycoproteins, including fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Platelet receptors to these four molecules belong to the superfamily termed integrins, which are calcium-dependent heterodimers composed of two subunits, one being common to all of them (the ~-subunit). The other two superfamilies of receptors are the immunoglobulin gene superfamily and the selectins [124].

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The relative importance of the integrin receptor is less well known; under conditions of high shear stress, the binding of von Willebrand factor seems to predominate. Binding studies to GPIIb/IIIa have identified a distinct + + amino-acid sequence present in fibrinogen and the other three integrins, arginine-glycylaspartyl (RGD). Tigramin, a protein extracted from the snake venom Trimeresurus gramineus, was the first natural compound shown to bind the glycoprotein IIb/IIIa complex [125,126]. This group of compounds, termed disintegrin, inhibit platelet aggregation induced by a large spectrum of agonists, including ADP, collagen, thrombin, and sodium arachidonate. Sequence analysis revealed that it is a cysteine-rich, single-chain polypeptide with an Arg-Gly-Asp (RGD) sequence near its carboxy-terminal end [127]. So far, over 10 trigamin-like polypeptides (a.o. albolabrin, bitistatin, echistatin, applaggin, kristin, etc.) have been reported, and they all have similar properties and highly homologous sequences [128]. These disintegrins are 100-2000 times more potent than cyclic RGD peptides [129]. Kristin remarkedly accelerates reperfusion with alteplase, even complete resolution of experimental coronary thrombi in the dog could be obtained [130]. More recently, cyclic and noncyclic synthetic peptidomimetics have been synthesized that are three orders of magnitude more potent than RGDs and, like the latter, inhibit the binding of all ligands to the glycoproteins IIb/IIIa of stimulated or nonstimulated platelets [131].

Stable Analogues o f Prostacyclin The inherent instability of prostacyclin (PGI 2) limits the potential therapeutic usefulness of the compound, despite its important biological profile. Consequently, intense efforts have been focused on modifying the prostacyclin molecule to obtain synthetic analogues with greater chemical and metabolic stability, and comparable physiological activity. Iloprost was the first described stable analogue of prostacyclin. This compound protects ischemic myocardium in experimental models of coronary artery reperfusion [132,133]. The combination of iloprost with alteplase did not improve immediate or follow-up coronary artery patency or left ventricular functional recovery compared to alteplase alone [134]. The combination of cicaprost and aspirin exerts significant and synergistic effects in two models of arterial thrombosis [135], which confirms in vitro findings [136] and has implications for multiple mechanisms for downregulation of the cAMP pathway. Beraprost, another stable prostacyclin analogue used in combination with alteplase, does not potentiate thrombolysis in dogs but prevents reocclusion [137]. Another stable analogue is taprostene, which in patients with myocardial infarction results in a higher

patency rate than in patients receiving saruplase alone (the two groups were also on IV heparin and aspirin). At a dose of over 12.5 ng/kg/min, taprostene prevents reocclusion in the first 48 hours in patients with myocardial infarction [138].

Glycosaminoglycan Extracted from Sea Cucumber A depolymerized fragment of the glycosaminoglycan extracted from a sea cucumber (Strichopusjaponicus) was found to have anticoagulant properties. The compound mainly prevents the activation of prothrombin by in~ibition of the generation of the prothrombinase complex [139].

Recombinant-Activated Protein C Protein C is an endogenous vitamin K-dependent anticoagulant that is activated by the catalytic complex of thrombin and thrombomodulin, a protein released by normal endothelium. Activated protein C acts by inhibiting thrombin formation by means of enzymatic cleavage and destruction of the activated coagulation factors V and VIII, thus providing a negativefeedback regulation of coagulation. Purified plasmaderived activated protein C has been shown to be a safe and potent antithrombotic agent in venous [140] and arterial [141] models of thrombosis. Recombinant activated protein C is now available [142]; this material was evaluated in a nonhuman primate thrombosis model [143].

Selective Inhibition of Activated Coagulation Factor X A highly selective polypeptide inhibitor of activated coagulation factor X has been isolated from salivary gland extracts of the leech Haementuria officinalis. The compound is termed antistasin and is a 199amino-acid protein inducing a reversible, slow-tight binding of factor Xa [144]. A second polypeptide inhibitor was isolated from extracts of the tick Ornithodoros moubata [145,146]. The latter is a single-chain acidic polypeptide composed of 60 amino acids, also inhibiting factor Xa in a reversible, slow-tight binding. Both agents are highly selective for factor X, without inhibiting thrombin. Both proteins are now available as a recombinant isoform and are effective in the thrombotic prevention in rabbits [147]. Specific factor Xa inhibition with these agents was shown to enhance alteplase-induced reperfusion and to prevent acute reocclusion in the canine copper-coil model of arterial thrombosis [147].

Advatwes i~ Thrombolytic Therapy

Endothelium Relaxing and Nitric Oxide

Factor

In the area of endothelium-vascular smooth muscle interaction, the most attention in the past few years has been given to the production and release by endothelial cells of a substance termed endotheliumderived relaxing factor (EDRF). Acetyl choline and other exogenous substances cause the release of EDRF, which is transferred to vascular smooth muscle cells, causing vasodilatation. Whether E D R F is identical to nitric oxide (NO) or a closely related nitrosothrol derivative continues to be challeged [148,149]. E D R F is negatively charged and is a selective relaxant of vascular smooth muscle cells, whereas nitric oxide is uncharged and will relax a wide variety of nonvascular smooth muscle types. Nitrates generate E D R F and nitric oxide, and activate soluble guanylate cyclase, and thus elevate cyclic guanosine monophosphate (GMP) levels, causing vasodilatation and inhibition of platelet aggregation. These two effects are attenuated in atherosclerosis and other situations associated with either loss of endothelium or deficient formation of E D R F [150]. Molsidomine and its metabolite SIN-l, a donor of nitric oxide, are potent vasodilators and inhibit platelet adhesion and aggregation [151]. Furthermore, nitricoxide-generating drugs potentiate the activity of thrombolytic agents in experimental conditions [152] and in volunteers [153].

Angiotensin-Converting-Enzyme Inhibitors Infarction

Early After Myocardial

Several clinical studies have shown that converting enzyme inhibition can improve symptomless left ventricular dysfunction and possibly prevent congestive heart failure when treatment is started 1 week after myocardial infarction or later. In a double-blind study, 100 patients with Q-wave myocardial infarction, but without clinical heart failure, were randomly allocated to captopril (50 mg) or placebo starting 24-48 hours after the onset of symptoms [154]. Left ventricular volumes measured regularly during 3 months of treatment revealed a significant difference; furthermore, at 3 months a significant intergroup difference also emerged in the change in ejection fraction from baseline. In the ISIS-4 pilot study, 81 patients with suspected acute myocardial infarction were randomized at a mean of 12.7 hours from pain onset to receive 4 weeks of captopril or isosorbide mononitrate [155]. Both drugs reduced systolic blood pressure, but only captopril resulted in a marked and persistent increase in cardiac output. This appeared to be related to a substantial and sustained reduction in systemic vascular resistance that may help in the myocardial remod-

119

eling process. In a similar American pilot study, adjunctive early treatment with captopril to alteplase prevented the increase in end-diastolic volume observed in the first 7 days in the placebo group [156].

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Advances in Thrombolytic Therapy

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