Introduction

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The activation of platelets and the resultant aggregation have been shown to play an important role in the pathogenesis of cardiovascular, cerebrovascular and peripheral vascular diseases and in the therapy of acute coronary syndromes (Hennekens et al., 1989). Hence platelet adhesion and aggregation have been identified as promising targets for the development of antithrombotic drugs. GP IIb/IIIa antagonism exerts a strong antiplatelet effect, because this interference inhibits the final common pathway of platelet aggregation and is not dependent on a single activation pathway. In vivo studies in several thrombosis models have provided evidence that the blockade of GP IIb/IIIa receptors could protect animals from acute thrombosis induced by vascular injury and facilitate the lysis of preformed thrombi by thrombolytic agents (Coller, 1997). Clinical trials demonstrated benefits of GPIIb/IIIa receptor inhibitor therapy with different agents and in different clinical indications of acute coronary syndromes (Topol, 1997).

Despite the extensive testing of GP IIb/IIIa antagonists, it remains to be determined whether effective antithrombotic activity can be achieved without inducing unwanted hemorrhagic side effects. Moreover, comparative studies on the efficacy and tolerability of GP IIb/IIIa antagonists and other antiplatelet agents are lacking. In the clinical trials utilizing GP IIb/IIIa inhibitors, ASA was used in the control group. The effects of GP IIb/IIIa inhibitors were therefore additional to the effects of ASA. Clopidogrel, a ticlopidine-related thienopyridine, has also been shown to be more efficacious than, and at least as safe as, ASA in preventing the combined outcome cluster of myocardial infarction, ischemic stroke and peripheral arterial disease in patients with symptomatic atherosclerotic disease (Caprie, 1996). Clinical studies that compare the efficacy of GP IIb/IIIa antagonists and thienopyridines have not yet been done.

Species differences in the platelet inhibitory activity of GP IIb/IIIa antagonists have complicated their in vivo evaluation in experimental models of thrombosis. It is generally considered that rats and rabbits, in which most thrombosis models are developed, are not appropriate for testing GP IIb/IIIa antagonists designed for human use, because GP IIb/IIIa antagonism results in only a weak inhibition of platelet aggregation in these species, compared with the inhibitory effects on human platelets (Harfenist et al., 1988; Verhallen and Barth, 1991). Comparative studies in dogs and in guinea pigs revealed that GP IIb/IIIa antagonists possess a higher antithrombotic efficacy than ASA (Frederick et al., 1996; Nishiyama et al., 1995).

SR 121566, a novel nonpeptide antiplatelet agent with high affinity and specificity for the GP IIb/IIIa complex, exhibited potent in vitro antiaggregating activity in rabbit platelets (Savi et al., 1998; Hoffmann et al., 1997). We subsequently observed that SR 121566 and its prodrug SR 121787 exhibited antithrombotic efficacy in an arteriovenous shunt and in a venous stasis thrombosis model in rabbits (Hérault et al., 1998; Hoffmann et al., 1997). The present study was designed to characterize in more detail the effects of SR 121787 on acute arterial thrombosis in rabbits and to compare its efficacy and tolerability with that of ASA and clopidogrel. The ex vivo and in vivo antiplatelet effects after p.o. administration of SR 121787, ASA and clopidogrel were investigated. In addition, antithrombotic and thrombolysis-enhancing actions of these three compounds were compared, and bleeding side effects were examined.

	Materials and Methods

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Drugs and materials. The following drugs and chemicals were used in this study: ADP (Boehringer Mannheim, Mannheim, Germany), collagen (type I) (Sigma Chemical Co., St. Louis, MO), AA (Nu-Check Prep Inc., Elusian, MN), ASA (Synthelabo, Meudon-la-Foret, France), rt-PA (Boehringer Ingelheim, Karl Thomae, Biberach, Germany), heparin (Sigma, Saint-Quentin-Fallavier, France) and sodium pentobarbital, SR 121787 and clopidogrel (Sanofi Recherche, Toulouse, France). The dosages of clopidogrel and ASA that we used have been shown to be optimal in rabbits (Bernat et al., 1993; Herbert et al., 1993; Hoffmann et al., 1997).

Animals. Male New Zealand rabbits (3.0-3.5 kg) were obtained from Lago (Vonnas, France). The protocol of this study was approved by the Animal Care and Use Committee of Sanofi Recherche.

Ex vivo platelet aggregation. Rabbits were pretreated by gavage with SR 121787, clopidogrel, ASA or water. SR 121787 was dissolved in 0.1 N HCl and diluted with purified water. Clopidogrel was dissolved in water, and ASA was dissolved in carboxymethyl cellulose (0.6%). Two hours after p.o. pretreatment, a time-point that has been shown to optimal for the three compounds (Herbert et al., 1993; Hoffmann et al., 1997), blood samples obtained by venipuncture were collected into a 3.8% trisodium citrate solution (9/1 v/v). Platelet-rich plasma was obtained by centrifuging the blood sample at 80 × g for 20 min. Platelet aggregation was determined according to the method of Born (1961) on a dual-channel Chrono-Log aggregometer. Aggregation was induced by the addition of ADP, AA or collagen (final concentrations: 2.5 µM, 250 µM and 12 µg/ml, respectively). The extent of aggregation was estimated quantitatively by measuring the maximal curve height above the base-line level (ADP and AA) or the slope of the aggregation curve (collagen).

Collagen-induced thrombocytopenia. Collagen (4 mg/kg) or saline was infused for 3 min into the marginal ear veins of pentobarbital-anesthetized (30 mg/kg i.v.) rabbits 2 h after p.o. pretreatment with SR 121787, clopidogrel, ASA or water. At the indicated time-points, carotid artery blood was sampled into a 3.8% trisodium citrate solution (9/1 v/v), and the platelet count was determined immediately with a Baker instrument hematology counter.

Thrombus formation in the carotid artery. SR 121787, clopidogrel, ASA or water was administered p.o. 2 h before thrombosis induction, whereas heparin was injected i.v. 5 min before thrombosis induction. Thrombus formation was induced after creation of an endothelial lesion by electrical stimulation of the carotid artery according to a modified method of Hladovec (1971) as recently described (Herbert et al., 1996). Rabbits were anesthetized with pentobarbital (30 mg/kg i.v.). A segment of the left carotid artery (about 10 mm long) was exposed and dissected free of surrounding tissue. A small piece of insulating film (Parafilm M) and two stainless steel electrodes were positioned under the artery. Using a constant d.c. power supply (Apelex 3500), we stimulated the artery at 2.5 mA for 3 min. Blood flow was measured at a point distal to the site of the electrical stimulation with an electromagnetic flowmeter (Narcomatic, Roucaire, Velizy, France) at 5-min intervals over a 45-min period. After 45 min, the carotid arteries were opened longitudinally, and the thrombus, if apparent, was removed, blotted on filter paper and weighed.

Lysis of a thrombus in the jugular vein. The effect of SR 121787, clopidogrel and ASA on rt-PA-induced thrombolysis of a standard-sized, preformed thrombus in the external jugular vein of rabbits was investigated according to Collen et al. (1983) as recently described (Herbert et al., 1996). The jugular vein of pentobarbital-anesthetized rabbits (30 mg/kg i.v.) was isolated, and all tributaries at a distance of 4 cm from the main bifurcation of the external jugular and facial veins were ligated. A silk thread was then inserted through the vessel to anchor the thrombus and avoid embolization. Thrombus formation was induced by ligating the vein with two vessel clamps. After 30 min, the clamps were removed and the blood flow was restored. Thrombolysis was performed by infusion of rt-PA at 0.5 mg/kg for 4 h with a 10% bolus via the contralateral marginal ear vein. SR 121787, clopidogrel, ASA or saline was given as an i.v. bolus injection at the start of the infusion. At the end of the rt-PA infusion period, the thrombus was carefully removed from the vein and weighed.

Bleeding. Rabbits were anesthetized with sodium pentobarbital (30 mg/kg i.v.), and five standardized incisions were made through the ear with a scalpel blade (N°21, Swann-Norton) as described recently (Herbert et al., 1996). Care was taken to avoid any macroscopically visible vessel. The ear was immersed in a 500-ml saline bath at 37°C under continuous stirring. Blood loss was determined 10 min later by measurement of the hemoglobin content, using a spectrophotometric method, after the addition of a hemolyzing reagent (Zapoglobin, Coultronics, France). The antiplatelet drug or vehicle was administered by gavage 2 h before ear incision.

Statistical analysis. The results shown are mean ± S.E.M. Data were statistically analyzed with ANOVA followed by Dunnett's test. For the blood flow data, two-way ANOVA with a subsequent contrast analysis was used. Statistical significance was accepted at P < .05. ED₅₀ values ± 95% confidence intervals were calculated by fitting the logistic equation to the data by means of nonlinear regression.

	Results

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Antiaggregating effects. Ex vivo platelet aggregation induced by ADP, AA or collagen was inhibited in a dose-dependent manner 2 h after the p.o. SR 121787 treatment of rabbits (fig. 1). The ED₅₀ values for ADP-, AA- and collagen-induced aggregation were 2.3 ± 0.3, 6.1 ± 0.9 and <2.5 mg/kg, respectively. Oral pretreatment with 100 mg/kg clopidogrel inhibited ADP- and collagen-induced aggregation by 68% and 80%, respectively, whereas AA-induced aggregation was not significantly influenced. Oral ASA pretreatment at 100 mg/kg inhibited the aggregating effects of AA and collagen (93% and 97%, respectively) without any influence on ADP-induced platelet aggregation.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effects of SR 121787, clopidogrel and ASA on platelet aggregation ex vivo. Single doses of SR 121787 (black columns), clopidogrel (hatched columns) or ASA (white columns) were administered by gavage. Ex vivo platelet aggregation was induced 2 h later by 2.5 µM ADP, 250 µM AA or 12 µg/ml collagen (final concentrations) as described under "Materials and Methods." Each point represents the mean ± S.E.M. of five animals (* P < .05 vs. control).

Inhibition of collagen-induced thrombocytopenia. Infusion of saline into a marginal ear vein for 3 min did not significantly change the number of circulating platelets (fig. 2). Infusion of collagen (4 mg/kg) induced a drop in the circulating platelet count within 5 min, and platelet count remained at this low level during the 30-min observation period. Oral pretreatment with SR 121787 at a dose of 10 mg/kg 2 h before collagen infusion inhibited the thrombocytopenic effect of collagen slightly but nonsignificantly; a complete elimination of the collagen effect was observed after pretreatment with SR 121787 at 20 mg/kg (fig. 2A). Clopidogrel pretreatment at 100 mg/kg exhibited an inhibitory action on the drop in platelet count after collagen infusion that attained significance level at 20 and 30 min after collagen infusion, whereas pretreatment with ASA at 100 mg/kg did not influence collagen-induced thrombocytopenia (fig. 2B).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   A) Inhibition of collagen-induced thrombocytopenia by SR 121787. Animals were pretreated by gavage with SR 121787 (10 or 20 mg/kg) or saline. Two hours later, collagen (4 mg/kg) was injected into a marginal ear vein. A second control group was not pretreated and was injected with saline. Each point represents the mean ± S.E.M. of 5 to 7 animals (* P < .05 vs. collagen-treated controls). B) Effects of clopidogrel and ASA on collagen-induced thrombocytopenia. Animals were pretreated by gavage with clopidogrel (100 mg/kg), ASA (100 mg/kg) or saline. Two hours later, collagen (4 mg/kg) was injected into a marginal ear vein. A second control group was not pretreated and was injected with saline. Each point represents the mean ± S.E.M. of 5 to 7 animals (* P < .05 vs. collagen-treated controls).

Antithrombotic efficacy in a carotid artery lesion model. Under control conditions, the thrombogenic subendothelial surface that was exposed after electrical stimulation of the carotid artery initiated the formation of an occlusive thrombus within 23.3 ± 2.4 min, i.e., blood flow was 100% inhibited (fig. 3A). Oral SR 121787 pretreatment at 5 mg/kg 2 h before electrical stimulation did not affect the reduction in blood flow. However, blood flow reduction was inhibited in a dose-dependent manner by pretreatment with SR 121787 at 10 or 20 mg/kg p.o. (fig. 3A). At the highest dose (20 mg/kg), initial blood flow was reduced by 32% at 45 min. The ED₅₀ value was 16.0 ± 0.3 mg/kg.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A) Effects of SR 121787 on thrombus formation in the carotid artery. The indicated doses of SR 121787 were administered by gavage 2 h before electrical stimulation as described under "Materials and Methods." Results are expressed as percent reduction of initial carotid blood flow. Each point represents the mean ± S.E.M. of 10 to 16 animals (* P < .05 vs. control). B) Effects of clopidogrel and ASA on thrombus formation in the carotid artery. Clopidogrel or ASA was administered p.o. at 100 mg/kg 2 h before electrical stimulation as described under "Materials and Methods." Results are expressed as percent reduction of initial carotid blood flow. Each point represents the mean ± S.E.M. of 10 to 15 animals (* P < .05 vs. control).

Lysis of a preformed thrombus in the jugular vein. Infusion of rt-PA at the threshold dose of 0.5 mg/kg did not significantly reduce the weight of the preformed thrombus in the jugular vein, compared with thrombi from control animals that were not treated with rt-PA (fig. 4). SR 121787 potentiated rt-PA thrombolysis (0.5 mg/kg) in a dose-dependent manner at a dose range between 1 and 6 mg/kg. The rt-PA thrombolysis-enhancing effects of clopidogrel and ASA (100 mg/kg) in this rabbit model were marginal and did not attain statistical significance (fig. 4). SR 121787, clopidogrel and ASA per se exhibited no thrombolytic activity.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effects of SR 121787, clopidogrel and ASA on rt-PA thrombolysis of a preformed thrombus in the jugular vein. After clot formation as described under "Materials and Methods," blood flow was restored, and rt-PA (0.5 mg/kg with a 10% bolus) or saline was infused for 4 h. Antiplatelet agents were given at the indicated doses as i.v. bolus injections at the start of the infusions. Each column represents the mean ± S.E.M. of seven rabbits except the control group, in which there were 22 animals (* P < .05 vs. rt-PA-treated animals).

Hemostasis. The effects of p.o. pretreatment with SR 121787, clopidogrel and ASA on blood loss from the incised rabbit ear are summarized in table 2. SR 121787, at p.o. doses up to 15 mg/kg administered 2 h before measurement of bleeding, did not cause a significant increase in blood loss. A significant bleeding effect was found at 20 mg/kg SR 121787 (16-fold increase in blood loss). For each dose tested, bleeding was not observed at sites other than the confined site of measurements of bleeding time. Clopidogrel and ASA, at 100 mg/kg administered 2 h before the bleeding test, did not influence the blood loss from the incised ear.

	Discussion

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Main findings. The principal findings of this study are 1) that SR 121787 demonstrated antiplatelet, antithrombotic and thrombolysis-enhancing actions after p.o. administration to rabbits, 2) that SR 121787 exerted these actions with a high potency and efficacy compared with clopidogrel or ASA and 3) that no significant bleeding effect of SR 121787 was observed at a dose that resembled the ED₅₀ in the arterial thrombosis model.

Antiplatelet effects. It is generally considered that rats and rabbits, in which most thrombosis models are developed, are not appropriate for testing GP IIb/IIIa antagonists designed for human use, because GP IIb/IIIa antagonism results in only a weak inhibition of platelet aggregation in these species (Harfenist et al., 1988; Verhallen and Barth, 1991). It has been shown, however, that SR 121566, the active moiety of the orally active prodrug SR 121787, exhibited a potent in vitro antiaggregating effect on rabbit platelets (Hoffman et al., 1997). Thienopyridines, on the other hand, exhibit a lower antiplatelet efficacy in rabbits than in rats, because ADP plays a less important role in thrombogenesis in rabbits (Defreyn et al., 1991). However, rats cannot be used for testing GP IIb/IIIa antagonists. Therefore, the rabbit seems an acceptable compromise, especially if one takes into account that the rabbit is one of the most frequently used species in thrombosis research.

Antithrombotic actions. An endothelial injury model was used to examine the antithrombotic efficacy of SR 121787 in arterial-type acute thrombosis. Endothelial injury initiates platelet activation via subendothelial connective tissue structures and stimulates tissue factor-dependent generation of thrombin via proteolytic activation of the serine proteases that comprise the coagulation cascade (Harker, 1997). In this study, it was shown that SR 121787 inhibited thrombus formation in a dose-dependent manner, as evidenced by an inhibition of reduction in blood flow and by a decrease in thrombus weight. The antithrombotic ED₅₀ was comparable to effective doses in the collagen-induced thrombocytopenia model.

Thrombolysis-enhancing activity. Thrombolysis has become a standard treatment for patients with acute myocardial infarction. However, post-thrombolytic reocclusion represents a significant problem. Experimental and clinical data implicate platelet-rich thrombus formation as a major factor in the failure to achieve successful thrombolysis and in abrupt closure of luminal patency (Collen, 1990). In the present rabbit rt-PA thrombolysis model, SR 121787 showed a dose-dependent effect with a maximal efficacy of 54% in enhancing rt-PA thrombolysis. Comparable data have been published for the GP IIb/IIIa antagonist DMP728 in a canine model of femoral artery thrombosis, in which DMP728 enhanced the thrombolytic effect of streptokinase (Mousa et al., 1994b). In vitro, DMP728 dispersed a preformed platelet-rich clot and potentiated the effects of different thrombolytic drugs (Mousa et al., 1994b). In the current rabbit rt-PA thrombolysis model, clopidogrel and ASA revealed lower efficacy. The potential of clopidogrel to enhance rt-PA- and streptokinase-induced thrombolysis was previously described in dogs (Yao et al., 1994) and rabbits (Bernat et al., 1993). In the canine coronary artery thrombosis model, clopidogrel was more effective than aspirin in enhancing rt-PA thrombolysis.

Hemorrhagic side effects. Because of the key role of the GP IIb/IIIa complex in hemostasis, it remains to be determined whether effective antiaggregatory and antithrombotic activity in humans can be achieved by blocking this receptor without causing hemorrhagic problems (Raddatz and Gante, 1995). In the present study in rabbits, SR 121787 caused a slight but non-significant prolongation of bleeding time (1.7-fold) at a dose (15 mg/kg) that resembles the antithrombotic ED₅₀. However, there is considerable controversy about the correlation between bleeding models and blood loss in a clinical situation (Lind, 1991), so the relevance of the present observations for the clinical experience remains to be established.