Abstract 015: PAR4 Ala120Thr Variant Alters PAR4 Desensitization, Sensitivity to Platelet Antagonists and Risk of Large Vessel Stroke

Abstract

F2RL3 encodes protease-activated receptor 4 (PAR4) and harbors a A/G SNP (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP), AYPGKF, which does not occur in vivo. Platelet function studies from healthy human donors demonstrated that compared to rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation to low concentrations (<1 nM) of thrombin, increased Ca 2+ signaling, increased granule secretion and decreased PAR4 desensitization. The increased thrombin sensitivity of rs773902 AA platelets required both PAR4 and PAR1. Whereas receptor cleavage, dimerization, or lipid raft localization were not different between platelets from the two F2RL3 genotypes, rs773902 AA platelets required more than 3-fold amounts of PAR4-AP for receptor desensitization. PAR4-AP stimulation of PAR4-transfected COS-7 cells increased PAR4 levels on the cell surface. Thrombin-stimulated platelets from rs773902 AA donors displayed reduced inhibition by P2Y12 and PAR1 blockers, but not aspirin. Finally, the association of rs773902 alleles with stroke was assessed using the Stroke Genetics Network (SiGN) study. Patients carrying at least one copy of the rs773902 A allele had an increased risk of both large vessel stroke and diabetes in the SiGN dataset. In summary, both PAR4 and PAR1 are required for platelet activation to sub-nanomolar thrombin concentrations. Compared to rs773902 GG platelets, rs773902 AA platelets have a lower EC50 for thrombin and are relatively resistant to receptor desensitization, perhaps due to anterograde trafficking of internal PAR4 to the plasma membrane post-stimulation. The association of the rs773902 A variant with large vessel stroke, diabetes and partial resistance to PAR1 and P2Y12 inhibition must be considered for development of personalized anti-platelet agents.