AN ANTIBODY AGAINST THE EXOSITE OF THE CLONED THROMBIN RECEPTOR INHIBITS EXPERIMENTAL ARTERIAL THROMBOSIS IN THE AFRICAN-GREEN MONKEY

Background Thrombin inhibitors have been shown to be efficacious in an imal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-media ted fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet th rombin receptor in intravascular thrombus formation, a polyclonal anti body was raised against a peptide derived from the thrombin-binding ex osite region of the cloned human thrombin receptor. This antibody serv es as a selective inhibitor of the thrombin receptor for in vivo evalu ation. Methods and Results The immune IgG (IgG 9600) inhibited thrombi n-stimulated aggregation and secretion of human platelets. In contrast , it had no effect on platelet activation induced by other agonists in cluding ADP, collagen, or the thrombin receptor-derived peptide SFLLR- NH2. IgG 9600 also inhibited thrombin-induced aggregation of African G reen monkey (AGM) platelets. By Western blot analysis, the IgG identif ied a protein of approximate to 64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antib ody on arterial thrombosis was evaluated in an in vivo model of platel et-dependent cyclic flow reductions (CFRs) in the carotid artery of th e AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth mo nkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thr ombin was completely inhibited during the 120-minute postbolus observa tion period in all four animals. There was a twofold increase in bleed ing time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa r eceptor antagonist MK-0852 and the thrombin inhibitor recombinant hiru din also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effe ct on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. Conc lusions These results demonstrate that blockade of the platelet thromb in receptor can prevent arterial thrombosis in this animal model witho ut significantly altering hemostatic parameters and suggest that the t hrombin receptor is an attractive antithrombotic target.