RGS10 shapes the hemostatic response to injury through its differential effects on intracellular signaling by platelet agonists.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5\u27-diphosphate (ADP) and thromboxane A2 (TxA2). Hemostatic thrombi formed in RGS10-/- mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA2, both of which evoked considerably greater maximum responses in RGS10-/- platelets with enhanced Gq- and Gi-mediated signaling. Shape change, which is G13-mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5\u27-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation

Regulators Of G Protein Signaling Modulate Platelet Function To Impact Normal Physiology And The Hemostatic Response

G protein-coupled receptors (GPCRs) are critical mediators of platelet activation whose signaling is limited in part by members of the regulator of G protein signaling (RGS) family. To better understand how individual RGS proteins impact the optimal balance between activation and inhibition, the most abundant RGS in platelets, RGS10 and RGS18, were deleted both individually and simultaneously in mice. Loss of RGS10 causes increased platelet activation and accumulation following hemostatic injury, due to an expansion of the P-selectin(─) shell, driven by thromboxane A2 and ADP, rather than the P-selectin(+) core, driven by thrombin. Loss of RGS18 results in milder increases in GPCR signaling, primarily thrombin-dependent, and causes moderate thrombocytopenia, due to decreased platelet production. Loss of both RGS10 and RGS18 results in dramatically increased platelet activation and accumulation in vivo, with an expansion of both the P-selectin(+) core and the P-selectin(─) shell, and uninhibited growth that increases the occurrence of vascular occlusion. Furthermore, dual deletion of RGS10 and RGS18 results in reduced platelet survival due to premature activation in circulation and subsequent clearance. Additionally, in efforts to explore RGS-mediated regulation of Gq signaling in platelets, we induced a homozygous RGS-insensitive G188S mutation in mouse Gq(alpha) [Gq(alpha) G188S]. Unexpectedly, Gq(alpha) G188S mice had dramatically reduced platelet accumulation in vivo, which was due to decreased Gq signaling via disrupted PLC(beta) interactions. Structural and computational analyses revealed substantial overlap between RGS and effector binding interfaces, but provided candidate mutations predicted to specifically disrupt RGS interactions. Finally, to corroborate our results with mouse models, we sought to identify predicted loss-of-function RGS10 and RGS18 variants in human patients and analyze their platelet function. We identified 16 variants in 101 patients and have plans to recall them to analyze platelet reactivity and RGS expression levels. Viable candidates will also be selected for in vivo hemostatic analysis using iPSC-derived megakaryocytes and a humanized mouse model. Overall, these studies demonstrate how RGS-mediated regulation of platelet GPCRs is important for platelet production, survival and hemostatic reactivity. Furthermore, it suggests that a delicate equilibrium between negative and positive platelet activation regulators is necessary to promote rapid responsivity while restraining unwarranted signaling