Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain

•All represented type 2B VWD mutations share regions of enhanced dynamics and reduced mechanical stability.•Heterogeneity displayed by reported type 2B VWD mutants is detected in single-domain monomeric fragments. [Display omitted] Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain’s thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments. Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder due to a range of mutations in the autoinhibitory A1 domain of von Willebrand factor (VWF), resulting in enhanced binding of VWF to glycoprotein Ibα on platelets, enhanced consumption of platelets, cleavage of VWF, and reduction in large VWF multimers. Using detailed structure-function studies, Legan et al provide the mechanistic details that explain how various individual type 2B mutations affect the function of the autoinhibitory domain differently, providing insight to the variable clinical severity of type 2 VWD seen in patients.