OC 59.2 - Histidine Residues in the Von Willebrand Factor D4 Domain Alter VWF Function in Response to Shear Stress

Background: The D4 and C-terminal domains of Von Willebrand Factor (VWF) have been shown to assemble into a dimeric bouquet and stem structure. It has been hypothesised that an “open” stem may render the molecule more thrombotic. The VWF-D4 domain, pH, and Ca2+ ions have been shown to mediate stem formation, with deletion of the VWF-D4 domain resulting in a fully open stem. Previously, it was demonstrated that Histidine residues in the propeptide act as pH sensors to aid multimerisation. We, therefore, investigated the role of Histidine residues in the VWF-D4 domain.

Aims: To investigate the functional role of Histidine residues in the VWF-D4 domain.

Methods: A panel of eleven VWF-D4 Histidine to Alanine mutants was generated in a full-length VWF expression vector using site-directed mutagenesis and subsequently expressed in HEK293T cells. VWF protein expression was measured using ELISA, and protein function was investigated using static and flow-based assays. The variants’ biophysical properties were analysed using optical tweezer (OT) pulling experiments using dimeric VWF fragments.

Results: All Histidine variants expressed similarly to wild-type VWF (wtVWF) and demonstrated normal multimers, collagen, and GPIIbIIIa binding. Interestingly, two variants, His1957Ala and His2169Ala, showed reduced binding to gain-of-function GPIbα, while His1989Ala demonstrated increased binding to GPIbα. Flow-based assays revealed normal VWF-mediated platelet capture to collagen for all variants at 1500s-1, however, several variants had significantly reduced function at 5000s-1, most notably His1957Ala, which virtually failed to form thrombi and did not form rolling VWF-platelet aggregates. Conversely, the His1989Ala and His2009Ala variants revealed gain-of-function activity under high shear stress. OT experiments confirmed that the D4 domain is essential for modulating the stem structure, and moreover, the His2009Ala variant renders the stem more “open”.

Conclusion(s): Histidine residues in the VWF-D4 domain are important determinants of VWF function in response to shear stress.