Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type Gi-linked P2T antagonist, CS-747

1 CS-747 is a novel thienopyridine-type platelet ADP inhibitor which lacks in vitro activity. This study examined pharmacological profiles of R-99224, a hepatic metabolite of CS-747. 2 R-99224 produced a concentration-dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03-1 mug ml(-1)), which was relatively specific to ADP compared to collagen and thrombin. 3 R-99224 (0.1-3 mug ml(-1)) also elicited a similar inhibition of ADP-induced aggregation in rat platelets. The inhibition by R-99224 (10 mug ml(-1)) persisted even after platelets were washed three times. Intravenous injection of R-99224 (0.1-3 mg kg(-1)) to rats resulted in a dose-dependent inhibition of ex vivo ADP-induced platelet aggregation. 4 R-99224 (0.1-100 muM) decreased binding of [H-3]-2-methylthio-ADP ([H-3]-2-MeS-ADP), a stable ligand for platelet ADP receptors, to washed human platelets. The inhibition by R-99224 reached a plateau at a concentration of 3 muM (1.4 mug ml(-1)), but complete inhibition was not achieved even at the highest concentration used (100 muM). 5 R-99224 (10 muM) in combination with ARL-66096 (0.3 muM), an ATP analogue-type Gi-linked P2T receptor antagonist, produced no additional inhibition of [H-3]-2-MeS-ADP binding. In contrast, [H-3]-2-MeS-ADP binding was completely abolished by R-99224 (10 muM) in combination with A3P5PS (300 muM), a selective P2Y(1) antagonist, suggesting that R-99224 selectively binds to the G(i)-linked P2T receptor. 6 R-99224 (0.01-3 mug ml(-1)) inhibited ADP-induced [I-125]-fibrinogen binding to human platelets in a concentration-dependent manner. R-99224 (0.1-1 mug ml(-1)) also inhibited the ADP-induced decrease in cyclic AMP levels in PGE(1)-stimulated platelets, whereas the agent did not affect ADP (10 muM)-induced Ca2+ mobilization. 7 These findings suggest that R-99224 is a selective and irreversible antagonist of Gi-linked P2T receptors and that R-99224 is a responsible molecule for in vivo actions of CS-747.