PB0830 - Inspecting underlying pathogenesis mechanisms of von Willebrand disease variants sited in A domains of the von Willebrand factor

Background: In our cohort of von Willebrand disease patients, we found five novel missense substitutions in the A1 linker (p.Cys1227Arg), the A1 domain (p.Leu1288Arg and p.Leu1340Arg), or the A2 domain (p.Val1524Gly and p.Val1530Gly) of von Willebrand factor (VWF).

Aims: We aimed to inspect the functional effects and underlying pathogenesis mechanisms of these newly identified mutations by gene expression studies.

Methods: The full-length wild-type (wt) or mutant VWF cDNA were expressed in human embryonic kidney 293 (HEK293)/T cells. Further, quantitative and qualitative assessments of the secreted VWF were performed. Structural impact and the intracellular trafficking of the VWF variants were assessed by homology modeling and immunofluorescence imaging, respectively.

Results: Homozygous expression of p.Cys1227Arg and p.Val1288Arg demonstrated a substantial reduction in VWF secretion (~16.59% and 31.38% of wt, respectively), defect in multimers, and diminished VWF binding to platelets (VWF:GPIb). Furthermore, we found an increased co-localization of the p.Cys1227Arg and p.Val1288Arg with the endoplasmic reticulum (ER) marker, compared with wt. The co-transfection of the p.Cys1227Arg and p.Leu1288Arg with the wt corrected secretion partially, but still showed diminished VWF:GPIb. The heterozygous expression of the variant p.Leu1340Arg showed a slight reduction in VWF secretion (83.29% of wt), impaired VWF:GPIb, but no effect on multimerization. The single expression of variants p.Val1524Gly and p.Val1530Gly showed a slight reduction in VWF secretion (83.47% and 86.07% of wt, respectively). Both single and co-expression of secreted Val1524Gly and p.Val1530Gly demonstrated multimers with triplet structures. Homology modeling showed easier access to the ADAMTS13 cleavage site by unfolding this domain by these variants.

Conclusion(s): Variants p.Cys1227Arg and p.Val1288Arg affect VWF multimerization and secretion, whereas the variant p.Leu1340Arg impaired the binding to platelets, with no impact on multimer elongation. Furthermore, we showed the gain of function variants p.Val1524Gly, and p.Val1530Gly in the A2 domain caused a structural alignment that results in the accessibility of the ADAMTs13 cleavage site.