Differential effects of P2Y1 versus P2Y12 receptor antagonism on thrombosis and bleeding in rabbits

Abstract

Purpose: This study investigated the effects of two platelet puringeric receptors on arterial thrombosis and provoked bleeding in rabbits, using the selective P2Y1 antagonist BMS-884775 (1-(5-Chlorothiazolo[5,4-b]pyridin-2-yl)-3-(2-(5-fluoro-4-(4-fluorophenyl)-7-hydroxy-1'-neopentylspiro[indoline-3,4'-piperidin]-1-yl)phenyl)urea) and the selective P2Y12 antagonist clopidogrel. BMS-884775 is a small-molecule, reversible and potent receptor antagonist of human P2Y1 receptors with IC50 of 0.1 nM in a human platelet P2Y1 calcium flux assay. It has greater than 10000-fold selectivity over human P2Y12 and P2Y2 receptors. Methods: Antithrombotic activity was evaluated by thrombus weight reduction in a rabbit model of electrically-induced carotid artery thrombosis. Bleeding time (BT) was measured for up to 20 min following cuticle incision. Ex vivo inhibition of peak platelet aggregation to ADP, arachidonic acid and collagen were determined in citrated platelet-rich plasma by optical aggregometry. BMS-884775 was infused i.v. starting 30 min before and maintained throughout the experiment. Clopidogrel was dosed orally for 3 days in rabbits, and experiments were conducted at 2 to 3 hr after the last dose. Results: BMS-884775 at 0.01+0.04, 0.03+0.12, 0.1+0.04 and 0.3+1.2 mg/kg+mg/kg/h IV reduced thrombus formation by 33±2, 56±3, 77±2 and 93±4%, respectively, and increased BT by 1.2±0.1, 1.9±0.1, 2.9±0.2 and 4.6±0.3-fold (n=5-6 per group). Likewise, clopidogrel at 1, 3 and 10 mg/kg/d p.o. inhibited thrombus formation by 21±7, 60±4 and 80±3%, respectively, and increased BT by 1.8±0.04, 2.4±0.1 and 6.2±0.3-fold (n=6 per group). At about 80% antithrombotic efficacy, BMS-884775 increased BT by about 3-fold, which was approximately 50% less than that of clopidogrel. BMS-884775 at 0.3+1.2 mg/kg+mg/kg/h IV inhibited platelet aggregation response to ADP completely but not to arachidonic acid and collagen, supporting its selectivity for platelet ADP receptors. Conclusion: In rabbits, BMS-884775 was similar to clopidogrel in preventing arterial thrombosis. However at antithrombotic doses, BMS-884775 produced less of BT increases when compared to clopidogrel. This preclinical study supports that P2Y1 receptor antagonism may represent a promising drug target for the development of new antiplatelet therapies.