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

Jin-Sheng Huang,Lanlan Dong,Tohru Kozasa,Guy C Le Breton

doi:10.1074/jbc.m605678200

Abstract

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.

Highlights

The first thrombin receptor was cloned and sequenced by Coughlin and co-workers in 1991 [3]
The results indicated that both PAR1 and PAR4 receptor activation caused an increase in intraplatelet calcium levels, the kinetics of this calcium mobilization appeared to be different for each receptor type [11]
It is known that platelets possess multiple G protein signaling pathways that contribute to the different platelet functional responses, the relative participation of these individual pathways in platelet shape change, aggregation, and secretion is not well characterized

Summary

Introduction

The first thrombin receptor was cloned and sequenced by Coughlin and co-workers in 1991 [3]. B and C, treatment of human platelets or HEK cells with Myr-G13SRIpep blocked TRAP1-induced RhoA activation, whereas treatment with Myr-G13SRIRandom-pep was without apparent effect. The experiments determined the involvement of this signaling pathway in the PAR1-mediated human platelet shape change response.

Results

Conclusion