OC 09.1 - A Common Variant in GRK5 Reveals Novel Targets for its Regulation of Platelet Function

Background: The variant rs10886430 in G protein-coupled receptor kinase 5 (GRK5) is associated with decreased GRK5 mRNA expression, increased platelet reactivity, and a greater risk of cardiovascular disease (CVD). Recently, GWAS summary statistics from the largest meta-analysis of CVD suggest that GRK5-mediated thrombin signaling are promising targets for therapeutic intervention. However, how GRK5 regulates GPCR signaling in platelets is poorly understood.

Aims: We hypothesize that GRK5 regulates platelet activity via thrombin receptor PAR1 and can be targeted to regulate platelet function in CVD.

Methods: GRK5 regulation in PAR1/PAR4 signaling was measured using 1) a GRK5-specific inhibitor, 2) A>G mutant induced Pluripotent Stem Cell (iPSC) line. Furthermore, Grk5 was deleted from a humanized PAR4 mouse model in mPar4 KO (hPAR4+/mPar4-/-/Grk5-/-).

Results: Our data show that A>G expressing iMKs show increased integrin activation in response to PAR1 agonist but not to PAR4 agonist. Pharmacological inhibition of GRK5 increases markers of platelet activation such as calcium mobilization, integrin activation, and P-selectin surface expression to PAR1 agonist but not to PAR4 agonist. We observed that both GRK5 and beta-arrestin-2, but not beta-arrestin-1, form a complex with activated PAR1. To assess the functional consequences of GRK5 regulation on human PAR4, Grk5 was deleted from hPAR4+/mPar4-/- mice. There was no difference in aggregation between hPAR4+/mPar4-/-/Grk5+/+ platelets and hPAR4+/mPar4-/-/Grk5-/- platelets to PAR4 agonist. Lastly, GRK5 is autophosphorylated in resting platelets and becomes dephosphorylated upon thrombin stimulation, and the availability of GRK5 to activated PAR1 is further regulated by binding to calmodulin.

Conclusion(s): We show for the first time that 1) GRK5 preferentially regulates PAR1- over PAR4-mediated signaling; 2) beta-arrestin-2 and GRK5 work in concert to downregulate PAR1 signaling; and 3) GRK5 itself is regulated by two mechanisms. Our findings create an avenue for designing superior therapeutics for the large subset of the population that carries the intronic variant in GRK5.