Project Abstract Plasma concentrations of von Willebrand Factor (VWF), a central component of primary hemostasis, vary five- fold in human populations and are controlled by the balance of cellular secretion rates and half-life in the circulation. The critical role of plasma VWF concentration in hemostasis is demonstrated by the increased risk for the bleeding disorder von Willebrand disease (VWD) in individuals with low plasma VWF and the heightened risk for venous thromboembolic disease, myocardial infarction, and ischemic stroke in individuals with high plasma VWF. Patients with bleeding phenotypes and low concentrations of plasma VWF are diagnosed Type I VWD, the most common inherited bleeding disorder. This proposal refers to VWF coding changes as cis variants. To date, the accepted understanding of the functional consequences of VWF cis variation originates from low- throughput, laboratory-based investigations of patient-derived mutations. But the vast majority of potential VWF missense variants have never been characterized, which leads to uncertainty when clinicians identify new variants of unknown significance in their patients with bleeding phenotypes. In healthy individuals, plasma VWF levels are highly heritable (64.5%) and mostly determined by variation outside the VWF locus. For example, common blood type–linked variants in the ABO gene determine ~20% of the variation in plasma VWF. Genome- wide association studies (GWAS) have identified multiple genetic loci outside of the VWF locus where common variants are associated with altered plasma VWF levels, implicating a strong role in VWD. The impact of these trans variants on plasma VWF levels is further implicated by the fact that 30% of the population of individuals with moderately low VWF (30-50 I.U./dL) and bleeding have no identifiable mutation in the VWF gene. Therefore, a large gap in the understanding of VWF biology that drives bleeding or thrombosis risk is the role of cis and trans genetic variants. The objective of this proposal is to comprehensively characterize VWF cis variation and to identify a network of genes harboring trans genetic variants that are critical for VWF secretion. To achieve this objective, Aim I will use high-throughput techniques to validate and expand a deep mutational scan of VWF and generate a comprehensive map of cis missense variants that alter VWF secretion. Aim II will identify trans genes critical for VWF secretion in a reporter cell line and in a cultured endothelial cell system using lentiviral delivered CRISPR/Cas9. Both aims will take advantage of access to whole genome sequencing data from several hundred patients with VWD Type I in the NHLBI-funded Zimmerman Program on VWF Biology. The combined experience of the assembled group of investigators will leverage the group’s previous history of collaborative science and capitalize on complementary expertise with deep mutational scanning, CRISPR screening, and VWF biology. Results from the propose...