PB0346 - Von Willebrand Factor D4 and C-terminal Domain Deletions Alter VWF Synthesis and Function

Background: While the role of many of the Von Willebrand Factor (VWF) domains has been characterised, less is known about the D4 and C-terminal domains. Structural analysis revealed the D4 and 6 VWF C-domains assemble into a dimeric bouquet and stem structure, and it has been hypothesised that a more “open” stem renders the molecule more haemostatically active. Moreover, recently identified gain of function mutations in the VWF C-domains demonstrate that these domains play a crucial role in modulating VWF function, specifically under conditions of shear stress.

Aims: To investigate the importance of the VWF C-terminal domains in relation to expression and function.

Methods: Full-length VWF molecules lacking individual D4 and C-domains (ΔD4, ΔC1, ΔC2, ΔC2C3loop, ΔC3+ΔC2C3loop, ΔC3, ΔC4, ΔC5, ΔC6) or the entire D4-C6 region (ΔD4C6) were generated using infusion cloning and expressed in HEK293T cells. Functional analysis was performed using static and shear-based assays.

Results: While ΔD4 did not alter VWF expression, deletion of individual C-domains significantly decreased expression and abolished secretion for ΔC1, ΔC2C3loop and ΔC3. Conversely, ΔD4C6 expressed ~3-fold more than wild-type VWF (wtVWF) but formed significantly fewer pseudo-Weibel-Palade bodies in HEK293 cells. All deletion variants demonstrated normal multimers and static collagen binding. Static GPIbα binding revealed increased binding for ΔD4 and ΔC6 compared to wtVWF, while ΔC2, ΔC3+ΔC2C3Loop and ΔC4 demonstrated decreased binding. As expected, ΔC4 did not bind to GPIIbIIIa; however, ΔC2 and ΔC3+ΔC2C3loop demonstrated reduced and abolished GPIIbIIIa binding, respectively. Finally, flow-based assays demonstrated that all the deletion mutants had reduced platelet function under high shear stress conditions, with the ΔD4C6 variant failing to form any rolling VWF-platelet aggregates under high shear.

Conclusion(s): Our data demonstrates that the VWF C-terminal domains play an important role in protein synthesis and function, particularly under high shear stress and have undefined roles in mediating binding to GPIbα.