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J. Biol. Chem., Vol. 282, Issue 14, 10210-10222, April 6, 2007

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Signaling through G₁₃ Switch Region I Is Essential for Protease-activated Receptor 1-mediated Human Platelet Shape Change, Aggregation, and Secretion^*

From the Department of Pharmacology, College of Medicine University of Illinois at Chicago, Chicago, Illinois 60612

This study investigated the involvement of G₁₃ switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the G₁₃ SRI (Myr-G₁₃SRI_pep) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G₁₃SRI_pep and Myr-G₁₃SRI_Random-pep were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G₁₃SRI_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 G₁₃ SRI with p115RhoGEF and a mechanism for Myr-G₁₃SRI_pep inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G₁₃SRI_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. [GenBank] It was also found that Myr-G₁₃SRI_pep inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G₁₃SRI_pep but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G₁₃SRI_pep effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of G₁₃ 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.

Received for publication, June 14, 2006 , and in revised form, February 7, 2007.

^* 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.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Illinois, 835 S. Wolcott Ave. (M/C 868), Chicago, IL 60612. Tel.: 312-996-4929; Fax: 312-996-4929; E-mail: gcl{at}uic.edu.

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