YD-3, a novel inhibitor of protease-induced platelet activation

1. In the present study, the antiplatelet effects and mechanisms of a new synthetic compound YD-3 1-benzyl-3(ethoxycarbonylphenyl)-indazole] were examined. 2. YD-3 inhibited the aggregation of washed rabbit platelets caused by thrombin (IC(50)=28.3 microM), but had no or little inhibitory effect on that induced by arachidonic acid, collagen, platelet-activating factor (PAF) or U46619. YD-3 also suppressed generation of inositol phosphates caused by thrombin. On the other hand, thrombin-induced fibrin formation was not affected by YD-3, indicating YD-3 does not inhibit the proteolytic activity of thrombin. 3. In washed human platelets, however, YD-3 had only mild inhibitory effect on the low concentration (0.05 u ml(-1)) of thrombin-induced human platelet aggregation, and did not affect that induced by higher concentrations (> or =0.1 u ml(-1)) of thrombin or SFLLRN, the protease-activated receptor 1 (PAR1) agonist peptide. By contrast, YD-3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC(50) values of 38.1 microM and 5.7 microM, respectively. 4. YD-3, at 100 microM, had no effect on ristocetin-induced glycoprotein Ib (GPIb)-dependent aggregation of human platelets. In addition, platelets treated with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated the inhibition of YD-3 on thrombin-induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD-3. 5. In SFLLRN-desensitized human platelets, high concentration of thrombin (1 u ml(-1)) could still elicit intracellular Ca(2+) mobilization, and the rise of Ca(2+)](i) was prevented by either leupeptin or YD-3. 6. Our results suggest that YD-3 inhibits a non-PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human platelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.