Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion.

This study investigated the involvement of Galpha(13) switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Galpha(13) SRI (Myr-G(13)SRI(pep)) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G(13)SRI(pep) and Myr-G(13)SRI(Random-pep) were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G(13)SRI(pep) specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Galpha(13) SRI with p115RhoGEF and a mechanism for Myr-G(13)SRI(pep) inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G(13)SRI(pep) specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G(13)SRI(pep) inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G(13)SRI(pep) but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G(13)SRI(pep) effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Galpha(13) SRI with p115RhoGEF is required for G(13)-mediated RhoA activation in platelets; 2) signaling through the G(13) pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G(13) signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.