PB0354 - Hybrid Transgenic Von Willebrand Factor Molecules Highlight Important Roles for the C-terminal Domains for Expression and Response to Shear Stress

Background: The C-terminal region of Von Willebrand Factor (VWF) spanning the D4-C6 domains remains uncharacterised. It is known that the VWF-C4 domain contains an RGD site that binds glycoprotein (GP)IIbIIIa, and the entire region forms a stem structure required for packaging into Weibel-Palade bodies. Furthermore, it has been suggested that the stem modulates VWF function under shear-stress. To investigate the evolutionary significance of these domains, we produced VWF molecules containing a C-terminal region from different evolutionary species.

Aims: To investigate the evolutionary significance of the VWF C-terminal regions.

Methods: Transgenic-full-length hybrid (hyb) VWF molecules were generated where the D4C1C2C3C4C5C6 region was removed and replaced with the same region from Hagfish, Catshark, Zebrafish, Frog, Mouse, Pig and Bat. Expression studies were carried out in HEK293T cells and VWF analysed using static and flow-based assays.

Results: Hagfish, catshark and zebrafish hyb-VWF failed to secrete and demonstrated intracellular retention, while frog, mouse, pig and bat hyb-VWF were secreted at reduced levels. Mouse, pig and bat hyb-VWF demonstrated normal multimers and collagen binding, while frog hyb-VWF multimers presented as a smear but had normal collagen binding. Mouse, pig and bat hyb-VWF also demonstrated normal GPIIbIIIa binding. Frog hyb-VWF lacking an RGD site did not bind GPIIbIIIa. Interestingly, despite having a functional human A1-domain, mouse, pig, bat and frog hyb-VWF all bound to gain-of-function-GPIbα with reduced affinity. Under shear-stress, frog hyb-VWF demonstrated less VWF-mediated platelet capture at 1500s-1 and 5000s-1, while mouse hyb-VWF enhanced platelet capture at 5000s-1. Finally frog hyb-VWF failed to form rolling VWF-platelet aggregates, however mouse hyb-VWF was more responsive to shear-stress, forming rolling aggregates at lower shear rates compared to full-length human VWF.

Conclusion(s): These data demonstrate that the C-terminal region of VWF plays an important role in modulating function, and different evolutionary species may have adapted C-terminal regions to respond to shear-stress differentially.