Poster Q1 - Single-Molecule Visualization of VWF Stem Conformation: Impact of D4 Domain Histidine Residue and C Domain Pathogenic Mutations

Introduction: Von Willebrand factor (VWF), a plasma multimeric glycoprotein, is crucial for primary hemostasis. The D4-C6 domains of VWF form a dimeric stem structure, and evidence suggests that the conformation of VWF, particularly the openness of this stem structure, influences its thrombogenic potential. Both pH and Ca2+ ions play critical roles in stem formation. Previous research has shown that histidine (His) residues in the VWF propeptide act as pH sensors to facilitate multimerization. This study investigates the role of His Residue 2009 in the VWF-D4 domain in regulating VWF conformation. Additionally, mutations R2313H and R2384W in the VWF C1 and C2 domains, respectively, can lead to von Willebrand Disease (VWD), a common bleeding disorder. However, the molecular mechanisms by which these mutations cause disease are still unclear. We conducted an atomic force microscopy (AFM) study to directly visualize the stem conformation in the VWF variants at the single-molecule level, providing important insights into the molecular mechanisms of VWD.

Materials and

Methods: Mutations H2009A, R2313H, and R2384W, as well as the D’CK∆D4 (lacking the D4 domain) variant, were introduced into a full-length VWF expression vector using site-directed mutagenesis and expressed in HEK293T cells as dimers. The VWF dimer was purified by Ni-NTA chromatography, followed by size-exclusion chromatography. Protein function was confirmed through static and flow-based assays. The structure of the variants was analyzed using AFM. For AFM sample preparation, 20 μL of 5 μg/mL VWF mutations were adsorbed onto a mica substrate coated with poly-L-lysine. After 30 seconds, the slides were gently rinsed with ultrapure water and air-dried. AFM images (256 × 256 pixels) were recorded in Quantitative Imaging (QI) mode in air, using a NanoWizard AFM (Bruker) and SNL-10 cantilevers. From the AFM images, the length of the stem was measured for each single VWF dimer particle and was scored from 0 to 1, with 0 representing a fully open stem and 1 as a fully closed stem conformation.

Results, Conclusions, and Discussions: All variants expressed at levels similar to wild-type VWF (wtVWF) and showed normal multimer formation, as well as collagen and GPIIbIIIa receptor binding based on the static and flow-based binding assays. Interestingly, the H2009A and R2384W variants exhibited gain-of-function activity under high shear stress. AFM experiments confirmed that the D4 domain is crucial for modulating the stem structure: 95% of D’CK∆D4 were in the fully open state (i.e., the length of stem scored as 0), and 80% of the H2009A variant was also in the fully open state, indicating that H2009A renders the stem more "open" than wtVWF, of which 50% are in the fully open state. Additionally, mutations in the C1 or C2 domains also regulate stem conformation. Specifically, 85% of the R2384W variants were in the fully open state, indicating that R2384W renders the stem more "open," while only 44% of the R2313H variants were in the fully open state, indicating that R2313H renders the stem more "closed" (Figure 1).

In conclusion, using AFM, we directly visualized VWF's stem conformation at the single-molecule level. Histidine residue H2009 and mutation R2384 in the VWF D4 and C2 domains, respectively, are crucial modulators of the VWF stem conformation. Mutations in these residues resulted in a more open stem conformation, leading to a gain-of-function phenotype and potentially contributing to VWD pathology.

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