Mechanisms of Signal Transduction
Signaling through Gα13 Switch Region I Is Essential for Protease-activated Receptor 1-mediated Human Platelet Shape Change, Aggregation, and Secretion*

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This study investigated the involvement of Gα13 switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Gα13 SRI (Myr-G13SRIpep) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G13SRIpep and Myr-G13SRIRandom-pep were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G13SRIpep 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 Gα13 SRI with p115RhoGEF and a mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G13SRIpep 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-G13SRIpep inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G13SRIpep but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G13SRIpep effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2) signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G13 signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.

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This work was supported in part by National Institutes of Health Grants HL-24530-23 and GM061454. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.