Supplementary Materials1. genotypes (p0.02) and were inversely correlated with PFA-100 closure times (p LY2835219 distributor 0.04) and platelet volume (p 0.02). Leukocyte-depleted platelets contained abundant levels of the ~205 kD supervillin polypeptide. To assess functionality, mice lacking platelet supervillin were generated and back-crossed onto a C57BL/6 background. Compared to 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 LY2835219 distributor formation in the PFA-100? is associated with human variants and low expression, and Serpine2 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. platelet aggregation.9, 10 However, no GWAS has identified genes associated with shear stress-dependent platelet function. The Platelet Function Analyzer-100? (PFA-100) measures the time to platelet thrombus formation under a defined shear stress of 1500 sec?1 in whole blood.11 The assay requires platelet tethering to VWF, firm adhesion to collagen, platelet activation and secretion, and platelet aggregation mediated by VWF and fibrinogen. Abnormal assay results correlate with platelet hyperfunction and hypofunction associated with acute coronary syndromes12C15 and bleeding disorders, respectively.16, 17 The aim of this study was to identify genetic factors that influence platelet reactivity and thrombus formation LY2835219 distributor under high shear stress. We carried out a genome-wide screen with the PFA-100? to identify novel gene variants in AAs and EAs. We identified a novel platelet gene, (encoding the cytoskeletal regulatory protein, supervillin), whose expression negatively regulates platelet reactivity and thrombus formation in both humans and mice. Methods Subjects and platelet phenotyping The Platelet Genes and Physiology study was approved by the Institutional Review Boards of Baylor College of Medicine and Thomas Jefferson University, and informed consent was obtained from all volunteers. Healthy donors were recruited between 2000C2006 in Houston, Texas. Citrated whole blood was used to measure PFA-100? closure times in a collagen and ADP-impregnated cartridge (hereafter referred to as PFA-100ColA) within 30 min of phlebotomy. A platelet aggregation response of 10% was LY2835219 distributor considered as exposure to anti-platelet agents and reason for exclusion. Genotyping Genomic DNA was extracted from leukocyte buffy coats with the Qiagen DNA extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. DNA from AAs was genotyped on the Illumina Hum1M Beadarray. EA DNAs were genotyped on the Illumina Hum550k Beadarray. Due to the lower levels of linkage disequilibrium (LD) in African populations a denser SNP micorarray was selected to genotype the DNA samples from AA individuals to improve tagging of causal variants. Statistical analysis Quality control was performed before statistical analysis. Subjects were excluded for relatedness to other participants and for failing stringent genotyping quality control. Individual genotypes that failed quality control were also removed (see Supplemental Methods for details). PFA-100ColA closure times were natural log transformed and tested for associations using an additive model within a linear regression framework. All analyses of EA and AA samples were performed separately. The potential confounders, age, sex, VWF activity, plasma fibrinogen level and platelet CD41 level were tested for significance using forward selection and significant covariates were retained within the model (p-value 0.1). Principal Components Analysis (PCA) was also performed on the data using a subset of the SNP markers that are in linkage equilibrium (r2 0.3).18 To evaluate if there is inflation of the test statistic due to population substructure/admixture, lambda, was estimated when both no PCA components and when one through five PCA components were included in the linear regression model. Platelet gene expression analysis RNA from leukocyte-depleted platelets (LDP) was prepared for gene expression profiling in 29 healthy subjects. LDP was prepared using density centrifugation followed by CD45-positive cell depletion of platelet rich plasma (PRP).19 As controls, RNA from PRP and.