Prothrombotic Variants of von Willebrand Factor (VWF)

Abstract

The von Willebrand factor (VWF) circulates as a multimeric, multidomain glycoprotein in the bloodstream and initiates hemostasis at sites of vascular injuries. Through shear mediated mechanical activation, the structurally exposed binding site for the platelet receptor GPIbα in the A1 domain of VWF enables reversible platelet adhesion. This interaction exhibits a shear-dependent, biphasic ‘catch-slip’ binding behavior, which leads to signal transduction and intracellular calcium mobilization at increased shear forces. The subsequent intermediate activation of the previously inactive GPIIb/IIIa receptor enables binding to agonists, such as the VWF C4 domain and fibrin, and cross linking of platelets. However, full activation of GPIIb/IIIa, and thus stable irreversible aggregation, only occurs after platelet activation via extracellular calcium influx in presence of prolonged increased shear rates. Recently, two single nucleotide polymorphisms (SNPs) p.Phe2561Tyr and p.Pro2555Arg in the C4 domain of VWF were identified in vitro as gain-of-function (GOF) variants. While variant p.Phe2561Tyr shows a higher sensitivity to shear forces, the variant p.Pro2555Arg forms larger platelet aggregates. The prevalence of variant p.Phe2561Tyr was associated with a significantly increased risk of myocardial infarction in young women. In this study, a SNP was identified in another cohort, in a patient with myocardial infarction. Together with 8 other SNPs in the C4 and C6 domain of VWF this SNP was classified as a GOF variant in the course of an in vitro characterization of the recombinant protein using light transmission aggregometry and microfluidic experiments. Additionally, 9 loss-of-function (LOF) variants in C2 and C4 domain were identified which, when recombinantly expressed in HEK293F cells, either showed a secretion or multimerization defect, or exhibited reduced activity in shear-dependent assays. The identified GOF variants were phenotypically similar to variants p.Pro2555Arg or p.Phe2561Tyr. While aggregate size in microfluidic experiments showed a strong dependence on the GPIIb/IIIa interaction, the increased shear sensitivity was demonstrated to be GPIIb/IIIa-independent. In vivo studies in mice underlined a prothrombotic effect of variant p.Pro2555Arg and revealed a tendency to unstable thrombi formation and thus a possible increased risk of thromboembolism for variant p.Phe2561Tyr. Taking into account all the data collected in the present study and in agreement with previously published results, a new mechanism can be postulated that could be responsible for the GOF of shear-sensitive variants: Variant p.Phe2561Tyr and similar variants appear to impair the stability of the dimeric stem region, resulting in the formation of more elongated multimers. This reduces the shear force required for platelet adhesion via GPIbα, which accelerates the formation of the GPIbα-VWF-A1 ‘catch-bond’, allowing intermediate activation of GPIIb/IIIa to be achieved at lower shear rates. Due to the pre-activated GPIIb/IIIa receptor, the platelet aggregates are cross-linked via GPIIb/IIIa VWF C4 interactions at lower shear rates. However, since the required tensile force for complete activation is not present, platelet activation might be incomplete and the formed aggregates may be unstable, allowing it to be torn away. This could therefore increase the risk of thromboembolism, which is in agreement with the increased risk of myocardial infarction described for p.Phe2561Tyr. On the other hand, variants such as p.Pro2555Arg appear to promote vascular occlusion due to increased GPIIb/IIIa-mediated cross-linking of platelets. The present study has thus contributed significantly to generating new, clinically relevant insights into the GOF mechanism that could account for the prothrombotic effect of VWF GOF variants.