title: Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type G(i)-linked P2T antagonist, CS-747.
abstract: 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 microg ml(-1)), which was relatively specific to ADP compared to collagen and thrombin. 3. R-99224 (0.1 - 3 microg ml(-1)) also elicited a similar inhibition of ADP-induced aggregation in rat platelets. The inhibition by R-99224 (10 microg 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 microM) 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 microM (1.4 microg ml(-1)), but complete inhibition was not achieved even at the highest concentration used (100 microM). 5. R-99224 (10 microM) in combination with ARL-66096 (0.3 microM), 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 microM) in combination with A3P5PS (300 microM), a selective P2Y(1) antagonist, suggesting that R-99224 selectively binds to the G(i)-linked P2T receptor. 6. R-99224 (0.01 - 3 microg ml(-1)) inhibited ADP-induced [(125)I]-fibrinogen binding to human platelets in a concentration-dependent manner. R-99224 (0.1 - 1 microg 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 microM)-induced Ca(2+) mobilization. 7. 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.
authors.affiliation: Pharmacology and Molecular Biology Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Sugidachi
authors.forename: F
authors.initials: F
authors.lastname: Asai
authors.forename: K
authors.initials: K
authors.lastname: Yoneda
authors.forename: R
authors.initials: R
authors.lastname: Iwamura
authors.forename: T
authors.initials: T
authors.lastname: Ogawa
authors.forename: K
authors.initials: K
authors.lastname: Otsuguro
authors.forename: H
authors.initials: H
authors.lastname: Koike
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000097:blood; D003521:Cyclopropanes / Q000627:therapeutic use*; D005340:Fibrinogen / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology*; D010880:Piperidines / Q000627:therapeutic use*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D000068799:Prasugrel Hydrochloride; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D013873:Thionucleotides / Q000378:metabolism; D013876:Thiophenes / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C092108:2-propylthio-D-beta,gamma-difluoromethylene ATP; D003521:Cyclopropanes; D008565:Membrane Proteins; C548576:P2RY12 protein, human; C553058:P2ry12 protein, rat; D010879:Piperazines; D010880:Piperidines; D010975:Platelet Aggregation Inhibitors; D058919:Purinergic P2 Receptor Antagonists; C426068:R-99224; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D013873:Thionucleotides; D013876:Thiophenes; C002480:methylthio-ADP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005340:Fibrinogen; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000068799:Prasugrel Hydrochloride; D002118:Calcium