Roytenberg, R.; Rorabaugh, B. R.; Yue, H.; Jividen, R.; Cameron, S.; Li, W.

Background: Platelet activation via G protein-coupled receptors (GPCRs) is central to arterial thrombosis. P2Y12 is a canonical Gi-coupled receptor mediating ADP-dependent platelet activation, yet the role of Regulator of G protein Signaling 6 (RGS6), a modulator of Gi signaling, in platelet function and thrombosis remains unclear. Objectives: To determine the role of RGS6 in platelet activation and arterial thrombosis and to define its impact on P2Y12/Gi signaling. Methods: Arterial thrombosis was assessed using a FeCl3-induced carotid artery injury model in wild-type (WT) and Rgs6-/- mice. Platelet aggregation was measured ex vivo. Signaling pathways were analyzed by Western blot in ADP-stimulated platelets. P2Y12/Gi signaling was further evaluated using a cAMP-responsive luciferase reporter assay in HEK293 cells. Results: Male Rgs6-/- mice exhibited significantly accelerated thrombosis compared with WT controls. Rgs6-/- platelets showed enhanced ADP-induced aggregation, whereas collagen-induced aggregation was unchanged. In ADP-stimulated platelets, RGS6 deficiency altered signaling kinetics, characterized by delayed Akt phosphorylation and reduced PKA and VASP phosphorylation. In a heterologous cAMP-luciferase assay, RGS6 attenuated P2Y12/Gi-mediated suppression of cAMP. Two-way ANOVA demonstrated significant effects of ADP and RGS6 expression on luciferase activity, with no interaction, indicating that RGS6 modulates signaling magnitude rather than agonist sensitivity. Pharmacologic inhibition of P2Y12 with clopidogrel abolished the genotype-dependent difference in thrombosis in vivo. Conclusions: RGS6 acts as a negative regulator of platelet P2Y12/Gi signaling and thrombus formation. Loss of RGS6 enhances ADP-dependent platelet activation and accelerates arterial thrombosis, establishing RGS6 as an endogenous brake on platelet activation.