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(Circulation. 2003;108:2697.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Anticoagulants (Thrombin Inhibitors) and Aspirin Synergize With P2Y₁₂ Receptor Antagonism in Thrombosis

Patrick André, PhD; Thomas LaRocca, BA; Suzanne M. Delaney, PhD; Pei Hua Lin, BS; Diana Vincent, BS; Uma Sinha, PhD; Pamela B. Conley, PhD; David R. Phillips, PhD

From Millennium Pharmaceuticals, Inc, South San Francisco, Calif.

Correspondence to David R. Phillips, Millennium Pharmaceuticals, Inc, 256 E Grand Ave, South San Francisco, CA 94080. E-mail david.r.phillips{at}mpi.com

Received December 16, 2002; de novo received June 6, 2003; revision received July 15, 2003; accepted July 15, 2003.

Background— This study was designed to determine whether (1) P2Y₁₂ antagonism synergizes with other antithrombotics and (2) anticoagulants (thrombin inhibitors) affect the antithrombotic activity elicited by P2Y₁₂ 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 P2Y₁₂ 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 TXA₂ also synergized with P2Y₁₂ in the absence of anticoagulation, because combined treatment of aspirin or C921-78 (a factor Xa inhibitor) with CT50547 or 2-MeSAMP (a P2Y₁₂ antagonist) inhibited the thrombotic process, whereas all treatments failed to inhibit thrombosis when used individually. Synergism was also observed ex vivo when P2Y₁₂-deficient (P2Y₁₂^-/-) 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 P2Y₁₂^+/- mice, whereas each showed only partial efficacy in P2Y₁₂^+/+ animals.

Conclusions— Our study indicates that (1) thrombin inhibitors and aspirin have a demonstrable synergy of antithrombotic activity with P2Y₁₂ antagonism and (2) the in vitro analysis of the antithrombotic activity of P2Y₁₂ antagonists is affected by the anticoagulant used for blood collection. This suggests that the antithrombotic potential of P2Y₁₂ 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.

Key Words: anticoagulants • thrombosis • receptors, purinergic P2 • synergism

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