Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis

Background - This study was designed to determine whether (1) P2Y(12) antagonism synergizes with other antithrombotics and ( 2) anticoagulants ( thrombin inhibitors) affect the antithrombotic activity elicited by P2Y(12) antagonism. Methods and Results - Thrombosis was achieved by perfusion of human and murine blood through type III collagen coated capillaries at arterial shear rate. CT50547, a direct-acting P2Y(12) antagonist, inhibited thrombosis in PPACK- but not heparin-anticoagulated human blood. In contrast, CT50547 inhibited thrombosis in aspirin-treated individuals independently of the anticoagulant. Thrombin and TXA(2) also synergized with P2Y(12) in the absence of anticoagulation, because combined treatment of aspirin or C921-78 (a factor Xa inhibitor) with CT50547 or 2-MeSAMP (a P2Y(12) antagonist) inhibited the thrombotic process, whereas all treatments failed to inhibit thrombosis when used individually. Synergism was also observed ex vivo when P2Y(12)-deficient (P2Y(12)(-/-)) mice were administered aspirin or coagulation inhibitors (C921-78 and bivalirudin). Finally, using intravital microscopy, we found that both C921-78 and bivalirudin abrogated the thrombotic process in P2Y(12)(+/-) mice, whereas each showed only partial efficacy in P2Y(12)(+/+) animals. Conclusions - Our study indicates that ( 1) thrombin inhibitors and aspirin have a demonstrable synergy of antithrombotic activity with P2Y(12) antagonism and ( 2) the in vitro analysis of the antithrombotic activity of P2Y(12) antagonists is affected by the anticoagulant used for blood collection. This suggests that the antithrombotic potential of P2Y(12) antagonists in vitro may be overestimated in anticoagulated samples of blood and best achieved in vivo by the inclusion of aspirin and/or a thrombin inhibitor.