Human Genome-Wide Association and Mouse Knockout Approaches Identify Platelet Supervillin as an Inhibitor of Thrombus Formation Under Shear Stress

Abstract

Background— High shear force critically regulates platelet adhesion and thrombus formation during ischemic vascular events. To identify genetic factors that influence platelet thrombus formation under high shear stress, we performed a genome-wide association study and confirmatory experiments in human and animal platelets. Methods and Results— Closure times in the shear-dependent platelet function analyzer (PFA)–100 were measured on healthy, nondiabetic European Americans (n=125) and blacks (n=116). A genome-wide association ( P <5×10 −8 ) was identified with 2 single-nucleotide polymorphisms within the SVIL gene (chromosome 10p11.23) in African Americans but not European Americans. Microarray analyses of human platelet RNA demonstrated the presence of SVIL isoform 1 (supervillin) but not muscle-specific isoforms 2 and 3 (archvillin, SmAV). SVIL mRNA levels were associated with SVIL genotypes ( P ≤0.02) and were inversely correlated with PFA-100 closure times ( P <0.04) and platelet volume ( P <0.02). Leukocyte-depleted platelets contained abundant levels of the ≈205-kDa supervillin polypeptide. To assess functionality, mice lacking platelet supervillin were generated and back-crossed onto a C57BL/6 background. Compared with controls, murine platelets lacking supervillin were larger by flow cytometry and confocal microscopy and exhibited enhanced platelet thrombus formation under high-shear but not low-shear conditions. Conclusions— We show for the first time that (1) platelets contain supervillin; (2) platelet thrombus formation in the PFA-100 is associated with human SVIL variants and low SVIL expression; and (3) murine platelets lacking supervillin exhibit enhanced platelet thrombus formation at high shear stress. These data are consistent with an inhibitory role for supervillin in platelet adhesion and arterial thrombosis.