Abstract Number: OC 43.1
Meeting: ISTH 2021 Congress
Background: Von Willebrand Factor (VWF) is a multimeric glycoprotein that recruits platelets to sites of blood vessel injury. Mutations in the VWF C domains can lead to intracellular retention and lower circulating VWF levels associated with the bleeding disorder type 1 von Willebrand disease (VWD). It is currently not well understood why some mutations lead to decreased VWF secretion, while others do not.
Aims: Objective: To identify every possible missense mutation in VWF that either increase or decrease its biosynthesis and secretion from cells.
Methods: We developed a VWF secretion assay in HEK293T cells using Fluorescence Activated Cell Sorting (FACS). Cells expressing VWF-eGFP variants with known secretion defects have increased mean fluorescence intensity (V1822G=157.72±5.40, C2693Y=184.06±2.10), compared to WT VWF-eGFP (69.22±1.87). Thus, eGFP retention is a consequence of impaired basal secretion of VWF. VWF variants may also exhibit impaired secretion in response to agonists.
Results: Cells treated with DDAVP show time-dependent decrease in eGFP signal for WT VWF-eGFP (83.7% MFI reduction) and C2693Y VWF-eGFP (46.7% MFI reduction), but minimal change in cells expressing V1822G VWF-eGFP (8.85% MFI reduction). We have also completed the assembly of the VWF mutagenesis library consisting of every possible amino acid substitution at each position of the VWF C-domains in full length human VWF. HEK293T cells expressing the VWF-eGFP mutagenesis library exhibited a broader distribution of MFI than cells expressing WT VWF-eGFP (fig. 1), confirming the presence of variants that affect basal secretion.
Conclusions: These data show that FACS is a useful method to screen for variants that impair basal and/or stimulated VWF secretion and provides the proof-of concept to perform an unbiased deep mutational scan of the VWF C domains. These data will provide a detailed structure/function analysis of VWF in mammalian cells and identify variants associated with type 1 and type 3 VWD.
To cite this abstract in AMA style:Sparring T, Singh K, Madarati H, Kretz C. Deep Mutational Scan of the VWF C Domains to Define Mutations Associated with VWD [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/deep-mutational-scan-of-the-vwf-c-domains-to-define-mutations-associated-with-vwd/. Accessed September 23, 2021.
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