Rhog Regulates GPVI/FcRγ-Mediated Platelet Activation and Thrombus Formation

Abstract We investigated the mechanism of activation and functional role of a hitherto uncharacterized signaling molecule, RhoG, in platelets. RhoG is a ubiquitously expressed member of the Rho Family of GTPases. We demonstrated for the first time the expression [Fig 1A] and activation of RhoG [Fig 1B] in platelets. Platelet aggregation and dense-granule secretion in response to glycoprotein VI (GPVI) agonists, collagen-related peptide (CRP) and convulxin were significantly inhibited in RhoG-deficient platelets compared to wild type murine platelets [Fig 1C]. Integrin αIIbβ3 activation and α-granule secretion as measured by flow cytometry were also significantly inhibited in RhoG-deficient murine platelets downstream of GPVI agonists. In contrast, 2-MeSADP- and AYPGKF-induced platelet aggregation and secretion [Fig 1D] were minimally affected in RhoG deficient platelets, indicating that the function of RhoG in platelets is GPVI-specific.Figure 1(A): Increasing amounts of human platelet lysate (in μg) were separated by SDS-PAGE, Western blotted, and probed with anti-RhoG antibody. (B) RhoG activation was measured upon stimulation of washed human platelets with 5μg/ml CRP for various times. Washed platelets were lysed and active GTP-bound RhoG was determined by pull-down analysis using bacterially expressed GST-ELMO. (C) Washed platelets from RhoG -/- mice and RhoG +/+ littermates were stimulated with GPVI agonists, 2.5 μg/ml CRP and 100 ng/ml convulxin and (D) G protein coupled receptor agonists, 30 nM 2MeSADP and 100 μM AYPGKF for 3.5 min under stirring conditions. Platelet aggregation and ATP secretion were measured by aggregometry.Figure 1. (A): Increasing amounts of human platelet lysate (in μg) were separated by SDS-PAGE, Western blotted, and probed with anti-RhoG antibody. (B) RhoG activation was measured upon stimulation of washed human platelets with 5μg/ml CRP for various times. Washed platelets were lysed and active GTP-bound RhoG was determined by pull-down analysis using bacterially expressed GST-ELMO. (C) Washed platelets from RhoG -/- mice and RhoG +/+ littermates were stimulated with GPVI agonists, 2.5 μg/ml CRP and 100 ng/ml convulxin and (D) G protein coupled receptor agonists, 30 nM 2MeSADP and 100 μM AYPGKF for 3.5 min under stirring conditions. Platelet aggregation and ATP secretion were measured by aggregometry. CRP-induced phosphorylations of Syk, Akt and ERK, but not Src family kinases (SFKs), were significantly reduced in RhoG-deficient platelets compared to those of wild type [Fig 2A]. Consistently, CRP-induced RhoG activation was abolished by pan-SFK inhibitor but not by Syk or PI 3-kinase inhibitors [Fig 2B]. Interestingly, unlike CRP, platelet aggregation and Syk phosphorylation induced by fucoidan, a CLEC-2 agonist, were unaffected in RhoG deficient platelets [Fig 2C].Figure 2(A): Washed platelets from RhoG -/- mice and RhoG +/+ littermates were stimulated with 2.5 μg/ml CRP and at 37 °C for 2 min and probed with anti-phospho-Syk (Tyr525/526), anti-phospho-Src (Tyr416), anti-phospho-Akt (Ser473), anti-phospho-ERK, or anti-β-actin (lane loading control) antibodies by western blotting. (B): RhoG activation induced by 5μg/ml CRP for 60 sec was evaluated in the presence and absence of 10 μM PP2, 2 μM OXSI-2, or 100nM wortmannin. (C): Wild type and RhoG-deficient platelets were stimulated with 100 μg/ml fucoidan and probed with anti-phospho-Syk (Tyr525/526), anti-phospho-Akt (Ser473), or anti-β-actin (lane loading control) antibodies by western blotting.Figure 2. (A): Washed platelets from RhoG -/- mice and RhoG +/+ littermates were stimulated with 2.5 μg/ml CRP and at 37 °C for 2 min and probed with anti-phospho-Syk (Tyr525/526), anti-phospho-Src (Tyr416), anti-phospho-Akt (Ser473), anti-phospho-ERK, or anti-β-actin (lane loading control) antibodies by western blotting. (B): RhoG activation induced by 5μg/ml CRP for 60 sec was evaluated in the presence and absence of 10 μM PP2, 2 μM OXSI-2, or 100nM wortmannin. (C): Wild type and RhoG-deficient platelets were stimulated with 100 μg/ml fucoidan and probed with anti-phospho-Syk (Tyr525/526), anti-phospho-Akt (Ser473), or anti-β-actin (lane loading control) antibodies by western blotting. Finally, RhoG -/- mice had a significant delay in time to thrombotic occlusion in cremaster arterioles compared to wild type littermates [Fig 3A and 3B], indicating the important in vivo functional role of RhoG in platelets.Figure 3(A): Time required for occlusion of cremaster arterioles in RhoG +/+ and RhoG -/- mice was measured using microvascular thrombosis model with light/dye-induced injury. 5 mice of each genotype were used, and statistical analysis revealed a significant difference between the 2 genotypes of mice (*, P < .01). (B) Representative images of cremaster arterioles were taken from RhoG +/+ and RhoG -/- mice 30 min after the injury. As seen with the outline (arrows) of the thrombus formed, thrombus formation was inhibited in RhoG -/- mice.Figure 3. (A): Time required for occlusion of cremaster arterioles in RhoG +/+ and RhoG -/- mice was measured using microvascular thrombosis model with light/dye-induced injury. 5 mice of each genotype were used, and statistical analysis revealed a significant difference between the 2 genotypes of mice (*, P < .01). (B) Representative images of cremaster arterioles were taken from RhoG +/+ and RhoG -/- mice 30 min after the injury. As seen with the outline (arrows) of the thrombus formed, thrombus formation was inhibited in RhoG -/- mice. In conclusion, we show for the first time that RhoG is expressed and activated in platelets, plays an important role in GPVI/FcRγ-mediated platelet activation and is critical for thrombus formation in vivo. Disclosures: No relevant conflicts of interest to declare.