Abstract
BACKGROUND AND PURPOSE: Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated. EXPERIMENTAL APPROACH: The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca(2+) mobilization respectively. KEY RESULTS: When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca(2+) rises. Blockade of ADP P2Y(12) receptor with 2-methylthioadenosine 5'-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5'-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation. CONCLUSIONS AND IMPLICATIONS: These results suggest that PAR4 acts in parallel with the P2Y(12)/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.