P13. Abstract Title: Deep Mutational Scan of VWF to Define Mutations that Enhance or Impair Secretion

Abstract

<h3>Rationale</h3> Von Willebrand Factor (VWF) is a multimeric glycoprotein that recruits platelets to sites of blood vessel injury and serves as a molecular chaperone for coagulation factor VIII (fVIII). Plasma VWF levels are a known risk factor for various thrombotic disorders including VTE, myocardial infarction, and stroke. GWAS studies have identified genetic variants in VWF that contribute to increased and decreased circulating VWF antigen levels. However, it is currently not known why some variants result in high VWF levels while others cause low VWF levels in circulation. We hypothesize that a subset of VWF variants impact its biosynthesis and secretion. <h3>Objective</h3> To identify every possible mutation in VWF that either increase or decrease its biosynthesis and secretion from cells. <h3>Results</h3> We have developed a protein secretion assay in HEK293T-Rex cells based on VWF- eGFP. Mutations in VWF that affect biosynthesis and secretion can be identified based on cell fluorescence intensity using Fluorescence Activated Cell Sorting (FACS). Initial studies have confirmed that cells expressing VWF mutants with known secretion defects have increased intracellular eGFP intensity (eGFP MFI: V1822G=157.72±5.40, C2693Y=184.06±2.10), relative to cells expressing WT VWF (eGFP MFI: WT=69.22±1.87) (p<0.001). This validates that eGFP specific cell retention is a consequence of impaired VWF basal secretion. Mutations of VWF may additionally cause inappropriate VWF secretion in response to physiological stimuli, and exacerbate imbalanced VWF plasma levels under various environmental influences. Cells treated with stimulus show time-dependent stimulation of VWF secretion in cells expressing WT (83.7% MFI reduction, p<0.01) and the C2693Y VWF (46.7% MFI reduction, p<0.01), but minimal response in V1822G VWF expressing cells (8.85% MFI reduction, p<0.05). This suggests that VWF variants may exhibit sensitized/reduced responses to stimulated VWF secretion. <h3>Conclusions</h3> These data provide the proof-of-concept to perform an unbiased deep mutational scan of VWF, to identify every possible mutation that can increase/decrease biosynthesis and secretion. Results of this project will provide a detailed structure/function analysis of VWF in mammalian cells and identify variants in VWF that can impair or enhance its secretion.