Analysis of single nucleotide polymorphisms in the C4 domain of vWF reveals the novel gain-of-function variant p.Ser2564Arg

Introduction Von Willebrand Factor (vWF) is a 2813 amino acid large multidomain protein that assembles into multimers. It is exclusively expressed by endothelial cells, megakaryocytes and platelets and is secreted into the peripheral circulation. There it can sense vascular damage and recruit platelets, thereby initiating primary hemostasis. While, on the one hand, dysfunction and loss of vWF cause bleeding symptoms, on the other hand, hyperactivity and elevated plasma levels of vWF can promote thromboembolic events. Recently, two single nucleotide polymorphism (SNP) have been identified that specifically enhance the coagulative potency of vWF by increasing its function. These gain-of-function (GOF) variants are both located in the C4 domain of vWF and are characterized by either higher shear sensitivity, as shown for p.Phe2561Tyr or increased aggregate formation as observed for p.Pro2555Arg.

Method Using the published NMR structure of the vWF-C4 domain as well as the SNP database of Ensembl several SNPs from humans were selected and investigated for their impact on vWF function. The vWF with the SNP of interest was obtained by site-directed mutagenesis of a vWF plasmid, transfection and homozygous expression of vWF in HEK293F cells. These variants were analyzed for common parameters such as secretion, multimer pattern, binding to common interaction partner collagen, GPIbα and GPIIb/IIIa by ELISA, light transmission aggregometry, and platelet capture under flow using a microfluidic system.

Results 11 of 14 analyzed vWF variants showed normal expression and a normal multimer pattern when recombinantly expressed as full-length protein in HEK293F cells. Two variants were not secreted probably due to defects in intracellular trafficking. Among all expressed variants, six showed a mildly increased binding to collagen and platelet receptors GPIbα. Among them, the variant p.Ser2564Arg displayed an increased platelet agglutination in light transmission aggregometry (LTA) and led to accelerated platelet capture under flow in a microfluidic system. These data indicate that the variant p.Ser2564Arg is more sensitive for mechanical forces as it forms platelet aggregation already at lower shear rates.

Conclusion Prothrombotic variants of vWF have only been known for three years and display different phenotypes in vitro. Less is known about the exact mechanism of how these variants alter the vWF structure and function and why they vary among each other. Our study characterizes 14 different variants in the C4 domain of vWF that occur in humans, providing more data that help to identify a pattern and to understand the underlying mechanism for prothrombotic variants in the C4 domain. In addition, our screening revealed the novel GOF SNP p.Ser2564Arg which specifically increases the shear-dependent vWF function in vitro.

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Artikel online veröffentlicht:
20. Februar 2023