Functional and Structural Properties of the 2nd Gain-of-Function Variant in the C4 Domain of von Willebrand Factor

von Willebrand factor (VWF) plays a key role in hemostasis by mediating platelet adhesion and aggregation at the site of vascular injury and by protecting factor VIII from rapid clearance in the circulation. Its deficiency causes a bleeding phenotype (von Willebrand disease, VWD) of variable severity, depending on the molecular defect of the multidomain and multifunctional protein induced by mutations. In addition to the numerous loss-of-function mutations, a number of gain-of-function (GOF) mutations have been identified in the VWF gene. The latter are located in the A1 domain and cause bleeding events, due to spontaneous binding and crosslinking of platelets in the circulation. The results are thrombocytopenia but also proteolytic degradation of the high-molecular-weight—functionally highly active—VWF multimers by the VWF-specific protease ADAMTS-13.

But VWF can also be regarded as prothrombotic factor, particularly if high VWF concentrations are present in the blood stream. We have recently described the prothrombotic properties of the common VWF variant p.Phe2561Tyr (Schneppenheim et al, 2019, Blood). It plays a significant role in premature and repeated events of myocardial infarction (MI) in a cohort of the Ludwigshafen Risk and Cardiovascular Health Study. This GOF variant is located in the VWF C4 domain, harboring the RGD binding sequence for platelet glycoprotein (GP) IIb/IIIa. Our functional studies have indicated an increase in force sensitivity of the VWF-GPIIb/IIIa interaction by the presence of p.Tyr2561 that shifts the onset of aggregation to lower shear rates compared to Phe2561-VWF.

We have now investigated the rare VWF variant p.Pro2555Arg, which is located in the first subdomain of C4, adjacent to the platelet GPIIb/IIIa binding site. We recombinantly expressed this variant and performed static and shear-based functional studies to characterize its properties. We found that GPIIb/IIIa binding is not altered under static conditions but if investigated under shear flow, p.Pro2555Arg also possesses GOF characteristics. In cone and plate aggregometry, p.Pro2555Arg exhibits the same increase in platelet aggregate size and surface coverage as p.Phe2561Tyr. But microfluidic assays revealed that, interestingly, p.Pro2555Arg seems to induce the prothrombotic GOF via a different mechanism than p.Phe2561Tyr. The GOF properties of these two variants, p.Phe2561Tyr and p.Pro2555Arg, further underline the prothrombotic character of VWF as a potential target for antithrombotic therapy.

Publication History

Article published online:
13 November 2020