Structural analysis of VWF gain-of-function variants by atomic force microscopy

Introduction The multimeric glycoprotein von Willebrand factor (VWF) is a highly adhesive protein, found in the bloodstream [1]. At sides of vascular injury, VWF is subjected to elevated hydrodynamic forces, leading to the activation of VWF’s A1 domain, initiating primary hemostasis via binding to platelet GPIbα. Later on, the thrombus is further stabilized by crosslinking of platelet receptor GPIIb/IIIa with VWF – via its C4 domain – and fibrin [2].

Recently, VWF gain-of-function (GOF) variants were identified exhibiting an increased prothrombotic potential [3]. For example, Huck et al. characterized variant p.Pro2555Arg, located in the C4 domain, which exhibits increased VWF-platelet-complex size at the same critical shear rate as wildtype (wt)VWF. The GOF effect was hypothesized to be induced by a decreased reformation rate of the closed stem conformation within VWF dimers [4].

Method We performed conformational studies using atomic force microscopy (AFM) imaging of different recombinant VWF dimer and multimer constructs. The proteins were immobilized in a near physiological buffer on a poly-L-lysine functionalized mica surface and dried by a gentle stream of nitrogen.

AFM imaging in air was performed to analyze the distribution of VWF stem conformations employing a NanoWizard®​ ULTRA Speed 2 (JPK, Bruker) and MFP3D (Asylum Research) in tapping mode with silicon nitride probes. The resulting height profiles were evaluated by measuring the stem length and the distance between the CK domain and the abrupt increase in height of the N-terminal domains to normalize the length of the closed stem. Based on the stem conformation distribution and the statistical analysis, three groups of conformations were determined. The initial tertial of the normalized stem length distribution was defined as fully-opened. Closed stem segments with a minimum length of 80% relative to the normalized full stem length were considered to be fully-closed. The length distribution in between was classified as flexible intermittent.

Results AFM imaging revealed for wtVWF that 14% of the stems were present in a fully-opened conformation while 52% exhibited a flexible intermittent conformation and 33% were fully-closed. In contrast, p.Pro2555Arg dimers showed 36% fully-opened, 33% flexible intermittent and 31% fully-closed stems. The full-length multimeric variants exhibited a similar tendency: within the multimers, wtVWF and p.Pro2555Arg exhibited 20% and 36% fully-opened stems, respectively.

Conclusion In this study, we used AFM imaging to visualize and analyze the structural variation of stem conformations in wtVWF and p.Pro2555Arg. The results of this analysis support the hypothesis that p.Pro2555Arg stems are more prone to an open confirmation. This effect was observed in isolated dimers as well as in dimers within multimers. If this effect can also be observed in additional, newly identified, GOF variants is subject of an ongoing study.

Conflict of Interest

The authors have no conflicts of interest to declare.

• References

• 1 Sadler J.E.. Biochemistry and genetics of von Willebrand factor. In: Annual review of biochemistry . 1998. 67. S 395-424
• 2 Wagner D.D.. Cell biology of von Willebrand factor. In: Annual review of cell biology. 1990. 6. S 217-246
• 3 Schneppenheim R.. et al. The von Willebrand factor Tyr2561 allele is a gain-of-function variant and a risk factor for early myocardial infarction. In: Blood. 2019. 133. 04 S. 356-365
• 4 Volker H. et al. Gain-of-Function Variant p.Pro2555Arg of von Willebrand Factor Increases Aggregate Size through Altering Stem Dynamics. In: Thrombosis and haemostasis. 2022. 122. 02 S. 226-239

Publication History

Article published online:
26 February 2024