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.

Figure 1

Chemical structures of CS-747 and R-99224.

Figure 2

In vitro effects of R-99224 (0.03 – 1 μg ml⁻¹) on platelet aggregation induced by ADP (A), collagen (B) and thrombin (C) in washed human platelets. Results are expressed as the mean±s.e.mean (n=5 – 6). ^*P<0.05, ^**P<0.01 vs each control.

Figure 3

In vitro effects of R-99224 (0.1 – 3 μg ml⁻¹) on platelet aggregation induced by ADP (A), collagen (B) and thrombin (C) in washed rat platelets. Results are expressed as the mean±s.e.mean (n=5 – 6). ^*P<0.05, ^**P<0.01 vs each control.

Figure 4

Ex vivo effect of single intravenous administration of R-99224 (0.1 – 3 mg kg⁻¹) on ADP (0.3 – 30 μM)-induced platelet aggregation in rats. R-99224 was intravenously administered to rats 1 h before the blood collection. Results are presented as the mean±s.e.mean (n=6).

Figure 5

In vitro duration of antiaggregatory effects: (A) Rat platelet-rich plasma was preincubated with R-99224 (10 μg ml⁻¹) or PGE₁ (1 μM) for 1 min, and then ADP (10 μM)-induced platelet aggregation was measured before and after washing the platelets; (B) ADP (10 μM)-induced ex vivo aggregation of platelets from vehicle- or CS-747 (3 mg kg⁻¹, p.o.)-treated rats was measured before and after washing the platelets. Results are presented as the mean±s.e.mean (n=6). ^**P<0.01 vs each control.

Figure 6

In vitro effect of R-99224 (0.1 – 100 μM) on [³H]-2-MeS-ADP binding to washed human platelets. Results are expressed as the mean±s.e.mean (n=6).

Figure 7

(A) In vitro effect of R-99224 (10 μM) combined with ARL-66096 (0.3 μM) on [³H]-2-MeS-ADP binding to washed human platelets; (B) In vitro effects of A3P5PS (300 μM) combined with R-99224 (10 μM) or ARL-66096 (0.3 μM) on [³H]-2-MeS-ADP binding to washed human platelets. Results are expressed as the mean±s.e.mean (n=6). ^**P<0.01, NS, not significant.

Figure 8

In vitro effect of R-99224 (0.01 – 3 μg ml⁻¹) on [¹²⁵I]-fibrinogen binding to washed human platelets. Results are expressed as the mean±s.e.mean (n=6). ^**P<0.01 vs control.

Figure 9

In vitro effect of R-99224 (0.1 – 1 μg ml⁻¹) on ADP (10 μM)-induced cyclic AMP decrease in PGE₁ (10 μM)-stimulated human platelets. ADP was added to the reaction mixture 3 min after PGE₁ stimulation. Results are expressed as the mean±s.e.mean (n=6). ^**P<0.01 vs control.

Figure 10

In vitro effect of R-99224 (0.03 – 1 μg ml⁻¹) on ADP (10 μM)-induced increase in intracelular Ca²⁺ concentration in washed human platelets. Results are expressed as the mean±s.e.mean (n=6). There are no statistically significant differences at any points.