Antiplatelet action of R‐99224, an active metabolite of a novel thienopyridine‐type G i ‐linked P2T antagonist, CS‐747

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. R‐99224 produced a concentration‐dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03 – 1 μg ml −1 ), which was relatively specific to ADP compared to collagen and thrombin. R‐99224 (0.1 – 3 μg ml −1 ) also elicited a similar inhibition of ADP‐induced aggregation in rat platelets. The inhibition by R‐99224 (10 μg 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. R‐99224 (0.1 – 100 μ M ) decreased binding of [ 3 H]‐2‐methylthio‐ADP ([ 3 H]‐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 μ M (1.4 μg ml −1 ), but complete inhibition was not achieved even at the highest concentration used (100 μ M ). R‐99224 (10 μ M ) in combination with ARL‐66096 (0.3 μ M ), an ATP analogue‐type G i ‐linked P2T receptor antagonist, produced no additional inhibition of [ 3 H]‐2‐MeS‐ADP binding. In contrast, [ 3 H]‐2‐MeS‐ADP binding was completely abolished by R‐99224 (10 μ M ) in combination with A3P5PS (300 μ M ), a selective P2Y 1 antagonist, suggesting that R‐99224 selectively binds to the G i ‐linked P2T receptor. R‐99224 (0.01 – 3 μg ml −1 ) inhibited ADP‐induced [ 125 I]‐fibrinogen binding to human platelets in a concentration‐dependent manner. R‐99224 (0.1 – 1 μg 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 μ M )‐induced Ca 2+ mobilization. These findings suggest that R‐99224 is a selective and irreversible antagonist of G i ‐linked P2T receptors and that R‐99224 is a responsible molecule for in vivo actions of CS‐747. British Journal of Pharmacology (2001) 132 , 47–54; doi: 10.1038/sj.bjp.0703761