Abstract 15058: RhoG Regulates GPVI/FcR?-Mediated Platelet Activation and Thrombus Formation
Rho GTPases play crucial role in the regulation of cellular activation. RhoG is a ubiquitously expressed member of the Rho Family of GTPases and is most similar to Rac1 and Cdc42 in sequence identity and function. Although RhoG is involved in the regulation of actin cytoskeletal rearrangements, nothing has been studied regarding the role of RhoG in platelet activation. Thus, we investigated the mechanism of activation and functional role of a hitherto uncharacterized signaling molecule, RhoG, in platelets. We demonstrated the expression and activation of RhoG in platelets. Platelet aggregation, integrin αIIbβ3 activation, and α- and dense-granule secretion in response to glycoprotein VI (GPVI) agonists collagen-related peptide (CRP) and convulxin were significantly inhibited in RhoG-deficient platelets. In contrast, 2-MeSADP- and AYPGKF-induced platelet aggregation and secretion were not affected in RhoG-deficient platelets, indicating that the function of RhoG in platelets is GPVI-specific. CRP-induced phosphorylation of Syk, PLCγ2, Akt, and ERK, but not Src family kinases (SFK), were significantly reduced in RhoG-deficient platelets. Consistently, using a pull-down assay, CRP-induced RhoG activation was abolished by pan-SFK inhibitor but not by Syk or PI 3-kinase inhibitors. Interestingly, unlike CRP, platelet aggregation and Syk phosphorylation induced by fucoidan, a CLEC-2 agonist, were unaffected in RhoG-deficient platelets. In contrast to the requirement of Rac1 in integrin αIIbβ3-mediated outside-in signaling, platelet spreading on immobilized fibrinogen and clot retraction were normal in RhoG-deficient platelets, suggesting that RhoG is not involved in regulation of Rac1 activation in platelets. Finally, using a microvascular thrombosis model with light/dye-induced injury, RhoG -/- mice had a significant delay in time to thrombotic occlusion in cremaster arterioles as compared to wild type littermates, indicating the important in vivo functional role of RhoG in platelets. Our data demonstrate for the first time that RhoG is expressed and activated in platelets. RhoG selectively regulates platelet functional responses downstream of GPVI/FcRγ-chain complex and is critical for thrombus formation in vivo.
- © 2013 by American Heart Association, Inc.