Heterogeneous Pathogenesis of A-Domain von Willebrand Factor Variants: Novel Insights into Defects in Trafficking, Multimer Remodeling, and Cellular Interactions

Introduction Missense substitutions within the A domains of von Willebrand factor (VWF) exhibit heterogeneous consequences that can range from impaired biosynthesis to defective binding functions. Clarifying how these alterations influence VWF is essential for understanding the diverse pathomolecular mechanisms underlying von Willebrand disease (VWD). In this study, we investigated the pathogenesis mechanisms of five newly identified A-domain mutations by protein expression and functional analyses. Method Wild-type (wt) and mutant full-length VWF cDNAs were expressed in HEK293/293T cells. VWF secretion and platelet interaction were measured by turbidometric assay, while multimerization was assessed by immunoblotting. In silico analyses supported structural interpretation. Intracellular trafficking and pseudo-Weibel–Palade body (WPB) biogenesis were examined by Zeiss ApoTome.2 microscopy and ZEN (blue edition) software. Recombinant proteins were tested for interaction with IL-8–stimulated neutrophils. Results Homozygous expression of C1227R and L1288R resulted in markedly reduced secretion (≤30% of wt) and loss of large multimers. C1227R showed ER retention, whereas L1288R exhibited increased colocalization with both PDI and the trans-Golgi marker TGN46, suggesting a trafficking defect with possible retrograde transport from the Golgi to the ER. For both variants, no increased colocalization with lysosomal markers (LAMP1/2) was detected, supporting degradation in the ER as the predominant route.Both variants showed a striking reduction in WPB formation. Co-expression with wt partially improved secretion and multimer patterns and increased the proportion of cells forming pseudo-WPBs, though they were less elongated. In contrast, L1340R, V1524G, and V1530G secreted at near-normal levels (~80% of wt) but displayed abnormal multimerization, impaired platelet binding, and pseudo-WPBs. Immunoblotting confirmed constitutive triplet patterns in V1524G and V1530G multimers upon secretion, consistent with enhanced ADAMTS13 susceptibility, as further supported by in silico analysis. Co-expression improved secretion, but susceptibility to ADAMTS13 digestion and the loss of large multimers persisted. Unexpectedly, recombinant L1340R, V1524G, and V1530G demonstrated increased interaction with IL-8–stimulated neutrophils compared with wt. Conclusion A-domain VWF variants cause heterogeneous defects, from ER retention with loss of secretion to unstable multimers prone to ADAMTS13 cleavage, all associated with impaired WPB morphology to varying degrees. Selected variants also increased VWF interaction with activated neutrophils, suggesting a role for aberrant VWF–neutrophil crosstalk in VWD pathogenesis. These findings refine the mechanistic spectrum of VWD, linking domain-specific misfolding to altered biosynthesis, multimer stability, and cellular interactions. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany