The severe von Willebrand Disease variant p.M771V leads to impaired anterograde trafficking of Von Willebrand factor in patient-derived and base-edited ECFCs.

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of VWF. The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients.

Objective: This study aims to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex-vivo cell models.

Methods: ECFCs were isolated from venous blood of VWD patients from the Willebrand in the Netherlands (WiN) cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord-blood derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy.

Results: Two unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired proVWF processing into mature VWF with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum (ER); this was confirmed in various populations of base edited CB-ECFCs harboring the p.M771V variant.

Conclusion: The severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modelling system, show that ER retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771.