Background Von Willebrand factor (VWF) is a multimeric plasma protein that bridges the gap between vessel injury and platelet capture at high shear rates. Under high shear or tension, VWF can become activated upon the unfolding of its autoinhibitory module (AIM). AIM unfolding exposes the A1 domain, allowing for binding to platelet glycoprotein (GP)Ibα to initiate primary hemostasis. The characteristics of the AIM and its inhibitory properties within mouse VWF are unknown. Objectives To determine and characterize the autoinhibitory properties of mouse VWF. Methods Recombinant mouse VWF A1 fragments containing or lacking the flanking regions around the A1 domain were generated. We tested the ability of these fragments to bind to human or mouse GPIbα and platelets. We compared the unfolding of mouse AIM‐A1 to human AIM‐A1 by single‐molecule force spectroscopy. Results Recombinant mouse AIM‐A1 binds with higher affinity to GPIbα than its human counterpart. Recombinant mouse proteins lacking part of the AIM show increased binding to GPIbα. Activated A1 fragments lacking the AIM can effectively agglutinate platelets across the species barrier. Using single‐molecule force spectroscopy, we determined that the mouse AIM unfolds under forces similar to the human AIM. Additionally, the human AIM paired with mouse A1 largely recapitulates the behavior of human AIM‐A1. Conclusions Our results suggest that the regulation of VWF‐GPIbα binding has been specifically tuned to work optimally in different rheological architectures. Differences in the AIM sequence may contribute to the difference in VWF shear response between human and mice.

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 (GP)Ibα 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 impacts 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 to enhanced flexibility in the secondary GPIbα binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wildtype, but shares regions of solvent accessibility with both wildtype and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared to 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.