Abstract 15673: CRISPR-Cas9 Gene Editing in Mice Reveals Novel Thrombotic Function of a Human SNP in STXBP5

Abstract

Introduction: Endothelial cells responds to vascular injury by exocytosis, releasing von Willebrand factor (vWF) into the blood stream. However, the regulation of endothelial vWF release remains poorly understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels. One top nonsynonymous single nucleotide polymorphism (SNP), rs1039084 (hg19 chr6:g.147635413A>G), encodes p. Asn436Ser substitution (STXBP5-N436S), and is associated with lower plasma vWF, higher bleeding score, and decreased venous thrombosis in humans. We recently discovered that STXBP5 inhibits endothelial vWF exocytosis and regulates thrombosis. However, the role of the STXBP5 genetic variants linked to vWF levels are not completely understood. Hypothesis: We hypothesized that STXBP5-N436S further inhibits endothelial exocytosis than wild type (STXBP5-WT). Methods: We overexpressed STXBP5-WT and STXBP5-N436S in cultured human endothelial cells and measured VWF release changes. Using CRISPR-Cas9 technique, we generated mice carrying the human rs1039084 SNP in Stxbp5 locus (Stxbp5-N437S mice). We conducted phenotypic analyses including endothelial exocytosis, hemostasis, and thrombosis in wild-type and Stxbp5-N437S mice. Results: In human endothelial cells, overexpression of STXBP5-N436S inhibits vWF exocytosis more potently than STXBP5-WT. Germline CRISPR-Cas9 gene editing efficiently and precisely knocked-in the human rs1039084 SNP in murine Stxbp5 locus, without causing detectable off-target genome cleavage. The baseline plasma vWF levels of Stxbp5-N437S mice are similar to WT mice, but Stxbp5-N437S mice showed impaired vWF exocytosis in response to epinephrine challenge. Moreover, Stxbp5-N437S mice have severe hemostasis defects displayed as prolonged bleeding time. Finally, Stxbp5-N437S mice have impaired mesenteric vessel thrombosis and carotid artery thrombosis. We are now studying the effects of the SNP upon STXBP5 structure and function. Conclusions: Our study validates the functional relevance of a candidate SNP identified by GWAS, and suggests that genetic variations within STXBP5 is a risk factor for thromboembolic disease.