Molecular mechanism of thromboxane A(2)-induced platelet aggregation - Essential role for P2T(AC) and alpha(2A) receptors

Thromboxane A, is a positive feedback Lipid mediator produced following pla telet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TP al pha, and G(i)-coupled TP beta subtype have been shown in human platelets. A DP-induced platelet aggregation requires concomitant signaling hom two P2 r eceptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i) respectively. me investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy- 9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through cc-acti vation of TP alpha and TP beta receptor subtypes. Here we report that secre tion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inh ibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregati on. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobili zation or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 rece ptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine sub type 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) rece ptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated plate let aggregation, which was further inhibited by yohimbine, an alpha(2A)-adr energic receptor antagonist Furthermore, inhibition of U46619-induced plate let aggregation by Ro 31-8220 was relieved by activation of the G(i) pathwa y by selective activation of either the P2T(AC) receptor or the alpha(2A)-a drenersc receptor. We conclude that whereas thromboxane A(2) causes intrace llular calcium mobilization and shape change independently, thromboxane A(2 )-induced inhibition of adenylyl cyclase and platelet aggregation depends e xclusively upon secretion of other agonists that stimulate G(i)-coupled rec eptors.