Deep molecular modeling and mechanistic insights into type 2A von Willebrand disease with von Willebrand factor A2 domain mutations.

Background: Type 2A von Willebrand disease (VWD) is characterized by impaired platelet adhesion due to the selective loss of high-molecular-weight von Willebrand factor (VWF) multimers. Objectives: To investigate A2 domain variants underlying type 2A VWD and elucidate associated disease mechanisms through comprehensive VWF assays and structural modeling. Methods: Sixty-five patients with 15 different VWF variants were investigated using full VWD phenotypic assays, underlying disease mechanisms, and genetic testing. Deep structural modeling (molecular dynamics, scaled molecular dynamics, and molecular docking) was employed to elucidate the effects of variants on the A2 domain. Results: Laboratory findings confirmed impaired VWF function, with reduced VWF glycoprotein Ib binding activity/VWF antigen (VWF:Ag) and VWF collagen binding/VWF:Ag ratios and increased ristocetin-induced platelet agglutination, consistent with multimer loss. Factor VIII clotting activity and VWF:Ag levels were <50 IU/dL in 45% and 58% of cases, respectively. Variants generally showed intact VWF synthesis; most patients had normal intraplatelet VWF:Ag levels; 40% had isolated reductions in intraplatelet VWF activity; 44% had reduced intraplatelet VWF:Ag and activity; and 16% were normal. VWF propeptide was normal in 80% of patients, while 89% showed elevated VWF propeptide/VWF:Ag, indicating increased clearance. Structural analyses showed that A2 variants maintained overall compactness under early shear stress, with α6 helix rigidification playing a key role in modulating interactions with the ADAMTS-13 spacer domain. All variants modestly increased solvent accessibility at the cleavage site, even in the absence of an external force. Conclusion: This integrated clinical, biochemical, and structural study reveals that A2 domain variants in type 2A VWD contribute to disease through multiple mechanisms, including impaired multimerization, altered susceptibility to proteolysis, and increased clearance.