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CC BY-NC-ND 4.0 · Thromb Haemost 2022; 122(02): 226-239
DOI: 10.1055/a-1344-4405
Cellular Haemostasis and Platelets

Gain-of-Function Variant p.Pro2555Arg of von Willebrand Factor Increases Aggregate Size through Altering Stem Dynamics

Volker Huck‡   *
1   Department of Dermatology and Venereology, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
2   Experimental Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
,
Po-Chia Chen‡   *
3   Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
,
Emma-Ruoqi Xu‡   *
4   European Molecular Biology Laboratory, Hamburg Unit, Hamburg, Germany
,
Alexander Tischer
5   Division of Hematology, Mayo Clinic, Rochester, Minnesota, United States
,
Ulrike Klemm
6   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Camilo Aponte-Santamaría
7   Max Planck Tandem Group in Computational Biophysics, University of los Andes, Bogotá, Colombia
8   Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany
,
Christian Mess
1   Department of Dermatology and Venereology, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Tobias Obser
1   Department of Dermatology and Venereology, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Fabian Kutzki
9   Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
10   Institute of Physical Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany
,
Gesa König
6   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Cécile V. Denis
11   Laboratory of Hemostasis, Inflammation and Thrombosis, Institut National de la Santé et de la Recherche Médicale UMR_1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Frauke Gräter
8   Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany
9   Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
,
Matthias Wilmanns
4   European Molecular Biology Laboratory, Hamburg Unit, Hamburg, Germany
12   University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Matthew Auton
5   Division of Hematology, Mayo Clinic, Rochester, Minnesota, United States
,
Stefan W. Schneider
1   Department of Dermatology and Venereology, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Reinhard Schneppenheim
6   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
,
Janosch Hennig   **
3   Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
,
Maria A. Brehm   **
1   Department of Dermatology and Venereology, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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Modulation of von Willebrand Factor Function by Domain C4 MutationsThromb Haemost 2022; 122(02): 176-176
DOI: 10.1055/a-1659-0729

Abstract

The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target.

Keywords

von Willebrand factor - hypercoagulability - GPIIb/IIIa

Authors' Contributions

V.H. performed the microfluidic experiments, designed the microfluidic settings with S.W.S., and analyzed the dataset with C.M. and S.W.S. P.C. and J.H. designed, performed, analyzed and interpreted NMR experiments. J.H. designed, supervised, and coordinated part of this study. P.C. and C.A.-S. designed and performed the MD simulations, P.C., C.A.-S., F.K., and F.G. analyzed the MD data. E.-R.X. expressed and purified C4 and D4N-CK constructs, performed and analyzed SAXS data. M.W. contributed to analysis of the data and coordination of the study. C.V.D. designed, performed, and analyzed the GPIIb/IIIa binding experiments. A.T. and M.A. purified proteins, performed and analyzed the CD experiments. U.K. performed and analyzed CPA experiments; G.K. and T.O. cloned and expressed VWF variants and constructs; G.K. performed cell-binding experiments, R.S. conceived, initiated, and coordinated this study; M.A.B. designed and coordinated this study and designed, supervised, and analyzed cell-binding and CPA experiments. All authors contributed in writing and editing of the manuscript.


* These authors contributed equally to this study.


** Co-senior authors.


Supplementary Material



Publication History

Received: 12 August 2020

Accepted: 28 December 2020

Accepted Manuscript online:
31 December 2020

Article published online:
14 April 2021

© 2020. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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