PB0403 - A Common Variant in GRK5 Reveals a PAR1-Specific Regulation in Platelets

Background: Two separate genome-wide association studies have identified the variant rs10886430 in the G protein-coupled receptor kinase 5 (GRK5) gene with a minor allele frequency of ~14% in Caucasians. Both studies show that the minor allele (G) is associated with decreased GRK5 mRNA, increased platelet reactivity, and a greater risk of cardiovascular disease. However, there is no consensus regarding the role of GRK5 in regulating thrombin receptor PAR1 or PAR4 signaling.

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

Methods: GRK5 regulation in PAR1/PAR4 was measured using 1) a GRK5-specific inhibitor, 2) A>G mutant megakaryoblastic cells, and 3) a mass-spec analysis. Furthermore, GRK5 was deleted from a humanized PAR4 mouse model in mPar4 KO background (hPAR4+/mPar4-/-/GRK5-/-), and the impact on PAR4-mediated platelet activation was measured by flow and aggregation.

Results: GRK5 protein expression is decreased by 80% in A>G mutant Meg-01 cells compared to WT control cells. Pharmacological inhibition of GRK5 increases markers of platelet activation such as integrin activation and P-selectin exposure in response to PAR1-agonist peptide (PAR1-AP) but not to PAR4-AP. Furthermore, this inhibition increases Ca2+ mobilization in response to PAR1-AP but not to PAR4-AP. Consistent with the results of our functional assays, mass spectrometry analysis using thrombin-activated platelets revealed an interaction between GRK5 and PAR1 but not PAR4. In contrast to the increased platelet aggregation observed in mPar4+/+/GRK5-/- platelets, there was no difference between hPAR4+/mPar4-/-/GRK5-/- and hPAR4+/mPar4-/-/GRK5+/+ platelets in response to PAR4-AP.

Conclusion(s): Our data show that 1) this GRK5 variant regulates GRK5 at the transcriptional and translational levels, and 2) GRK5 preferentially regulates PAR1 over human PAR4. Our findings will enable novel therapeutics for cardiovascular risk groups especially those individuals who carry the intronic variant in GRK5 and are more likely to experience thrombotic events.