BACKGROUND: ADP is an important physiological agonist that induces integrin activation and platelet aggregation through its receptors P2Y(1) (Gα(q)-coupled) and P2Y(12) (Gα(i)-coupled). P2Y(12) plays a critical role in platelet activation and thrombosis. Adenosine-based P2Y(12) antagonists, 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2MeSAMP) and Cangrelor (AR-C69931MX) have been widely used to demonstrate the role of P2Y(12) in platelet function. Cangrelor is being evaluated in clinical trials of thrombotic diseases. However, a recent study reported that both 2MeSAMP and Cangrelor raise intra-platelet cAMP levels and inhibit platelet aggregation through a P2Y(12)-independent mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The present work, using P2Y(12) deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and Cangrelor inhibit platelet activation and thrombosis. 2MeSAMP and Cangrelor inhibited aggregation and ATP release of wild-type but not P2Y(12) deficient platelets. 2MeSAMP and Cangrelor neither raised intracellular cAMP concentrations nor induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in washed human or mouse platelets. Furthermore, unlike the activators (PGI(2) and forskolin) of the cAMP pathway, 2MeSAMP and Cangrelor failed to inhibit Ca(2+) mobilization, Akt phosphorylation, and Rap1b activation in P2Y(12) deficient platelets. Importantly, while injection of Cangrelor inhibited thrombus formation in a FeCl(3)-induced thrombosis model in wild-type mice, it failed to affect thrombus formation in P2Y(12) deficient mice. CONCLUSIONS: These data together demonstrate that 2MeSAMP and Cangrelor inhibit platelet function through the P2Y(12)-dependent mechanism both in vitro and in vivo.