Published Online
on November 3, 2003

A more recent version of this article appeared on November 25, 2003

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Submitted on December 16, 2002
Revised on July 15, 2003
Accepted on July 15, 2003

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, and David R. Phillips PhD^*

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

^* To whom correspondence should be addressed. E-mail: david.r.phillips{at}mpi.com.

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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