Download PDF
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
› Author Affiliations
› Further Information
Also available at
   Permissions and Reprints

Comment on:

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/)

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Wagner DD. Cell biology of von Willebrand factor. Annu Rev Cell Biol 1990; 6: 217-246
    CrossrefPubMedGoogle Scholar
  • 2 Weiss HJ, Sussman II, Hoyer LW. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest 1977; 60 (02) 390-404
    CrossrefPubMedGoogle Scholar
  • 3 Sadler JE, Budde U, Eikenboom JC. et al; Working Party on von Willebrand Disease Classification. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006; 4 (10) 2103-2114
    CrossrefPubMedGoogle Scholar
  • 4 Schneppenheim R, Budde U. von Willebrand factor: the complex molecular genetics of a multidomain and multifunctional protein. J Thromb Haemost 2011; 9 (Suppl. 01) 209-215
    CrossrefPubMedGoogle Scholar
  • 5 Ruggeri ZM, Zimmerman TS. von Willebrand factor and von Willebrand disease. Blood 1987; 70 (04) 895-904
    CrossrefPubMedGoogle Scholar
  • 6 Wieberdink RG, van Schie MC, Koudstaal PJ. et al. High von Willebrand factor levels increase the risk of stroke: the Rotterdam study. Stroke 2010; 41 (10) 2151-2156
    CrossrefPubMedGoogle Scholar
  • 7 Levi M, Scully M, Singer M. The role of ADAMTS-13 in the coagulopathy of sepsis. J Thromb Haemost 2018; 16 (04) 646-651
    CrossrefPubMedGoogle Scholar
  • 8 Zachariah U, Nair SC, Goel A. et al. Targeting raised von Willebrand factor levels and macrophage activation in severe COVID-19: consider low volume plasma exchange and low dose steroid. Thromb Res 2020; 192: 2
    CrossrefPubMedGoogle Scholar
  • 9 Winkelmann BR, März W, Boehm BO. et al; LURIC Study Group (LUdwigshafen RIsk and Cardiovascular Health). Rationale and design of the LURIC study--a resource for functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease. Pharmacogenomics 2001; 2 (01, Suppl 1): S1-S73
    CrossrefPubMedGoogle Scholar
  • 10 Schneppenheim R, Hellermann N, Brehm MA. et al. The von Willebrand factor Tyr2561 allele is a gain-of-function variant and a risk factor for early myocardial infarction. Blood 2019; 133 (04) 356-365
    CrossrefPubMedGoogle Scholar
  • 11 Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA, Ginsberg MH. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Natl Acad Sci U S A 1985; 82 (23) 8057-8061
    CrossrefPubMedGoogle Scholar
  • 12 Xu ER, von Bülow S, Chen PC. et al. Structure and dynamics of the platelet integrin-binding C4 domain of von Willebrand factor. Blood 2019; 133 (04) 366-376
    CrossrefPubMedGoogle Scholar
  • 13 Müller JP, Mielke S, Löf A. et al. Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction. Proc Natl Acad Sci U S A 2016; 113 (05) 1208-1213
    CrossrefPubMedGoogle Scholar
  • 14 Legendre P, Navarrete AM, Rayes J. et al. Mutations in the A3 domain of von Willebrand factor inducing combined qualitative and quantitative defects in the protein. Blood 2013; 121 (11) 2135-2143
    CrossrefPubMedGoogle Scholar
  • 15 Legendre P, Salsmann A, Rayes J, Trassard O, Kieffer N, Baruch D. CHO cells expressing the high affinity alpha(IIb)beta3 T562N integrin demonstrate enhanced adhesion under shear. J Thromb Haemost 2006; 4 (01) 236-246
    CrossrefPubMedGoogle Scholar
  • 16 Caron C, Hilbert L, Vanhoorelbeke K, Deckmyn H, Goudemand J, Mazurier C. Measurement of von Willebrand factor binding to a recombinant fragment of glycoprotein Ibalpha in an enzyme-linked immunosorbent assay-based method: performances in patients with type 2B von Willebrand disease. Br J Haematol 2006; 133 (06) 655-663
    CrossrefPubMedGoogle Scholar
  • 17 Ruggeri ZM, Orje JN, Habermann R, Federici AB, Reininger AJ. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood 2006; 108 (06) 1903-1910
    CrossrefPubMedGoogle Scholar
  • 18 Chen H, Fallah MA, Huck V. et al. Blood-clotting-inspired reversible polymer-colloid composite assembly in flow. Nat Commun 2013; 4: 1333
    CrossrefPubMedGoogle Scholar
  • 19 Brehm MA, Huck V, Aponte-Santamaría C. et al. von Willebrand disease type 2A phenotypes IIC, IID and IIE: a day in the life of shear-stressed mutant von Willebrand factor. Thromb Haemost 2014; 112 (01) 96-108
    Article in Thieme ConnectPubMedGoogle Scholar
  • 20 Meyer dos Santos S, Klinkhardt U, Schneppenheim R, Harder S. Using ImageJ for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions. Platelets 2010; 21 (01) 60-66
    CrossrefPubMedGoogle Scholar
  • 21 Franke D, Petoukhov MV, Konarev PV. et al. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J Appl Cryst 2017; 50 (Pt 4): 1212-1225
    CrossrefPubMedGoogle Scholar
  • 22 Konarev PV, Volkov VV, Sokolova AV. et al. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Cryst 2003; 36: 1277-1282
    CrossrefPubMedGoogle Scholar
  • 23 Svergun DI. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst 1992; 25: 495-503
    CrossrefPubMedGoogle Scholar
  • 24 Carr HY, Purcell EM. Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 1954; 94 (03) 630-638
    CrossrefPubMedGoogle Scholar
  • 25 Meiboom S, Gill D. Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 1958; 29 (08) 688-691
    CrossrefPubMedGoogle Scholar
  • 26 Tollinger M, Skrynnikov NR, Mulder FA, Forman-Kay JD, Kay LE. Slow dynamics in folded and unfolded states of an SH3 domain. J Am Chem Soc 2001; 123 (46) 11341-11352
    CrossrefPubMedGoogle Scholar
  • 27 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 1995; 6 (03) 277-293
    CrossrefPubMedGoogle Scholar
  • 28 Lee W, Tonelli M, Markley JL. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 2015; 31 (08) 1325-1327
    CrossrefPubMedGoogle Scholar
  • 29 Ahlner A, Carlsson M, Jonsson BH, Lundström P. PINT: a software for integration of peak volumes and extraction of relaxation rates. J Biomol NMR 2013; 56 (03) 191-202
    CrossrefPubMedGoogle Scholar
  • 30 Niklasson M, Otten R, Ahlner A. et al. Comprehensive analysis of NMR data using advanced line shape fitting. J Biomol NMR 2017; 69 (02) 93-99
    CrossrefPubMedGoogle Scholar
  • 31 Walker O, Varadan R, Fushman D. Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from (15)N relaxation data using computer program ROTDIF. J Magn Reson 2004; 168 (02) 336-345
    CrossrefPubMedGoogle Scholar
  • 32 Abraham AJ, Murtola T, Schulz R. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 2015; 1–2 (September): 19-25
    CrossrefPubMedGoogle Scholar
  • 33 König G, Obser T, Marggraf O. et al. Alteration in GPIIb/IIIa binding of VWD-associated von willebrand factor variants with C-terminal missense mutations. Thromb Haemost 2019; 119 (07) 1102-1111
    Article in Thieme ConnectPubMedGoogle Scholar
  • 34 Wu YP, Vink T, Schiphorst M. et al. Platelet thrombus formation on collagen at high shear rates is mediated by von Willebrand factor-glycoprotein Ib interaction and inhibited by von Willebrand factor-glycoprotein IIb/IIIa interaction. Arterioscler Thromb Vasc Biol 2000; 20 (06) 1661-1667
    CrossrefPubMedGoogle Scholar
  • 35 Aponte-Santamaría C, Huck V, Posch S. et al. Force-sensitive autoinhibition of the von Willebrand factor is mediated by interdomain interactions. Biophys J 2015; 108 (09) 2312-2321
    CrossrefPubMedGoogle Scholar
  • 36 Huck V, Schneider MF, Gorzelanny C, Schneider SW. The various states of von Willebrand factor and their function in physiology and pathophysiology. Thromb Haemost 2014; 111 (04) 598-609
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Constantinescu-Bercu A, Grassi L, Frontini M, Salles-Crawley II, Woollard K, Crawley JT. Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2. eLife 2020; 9: 9
    CrossrefPubMedGoogle Scholar
  • 38 de Witt SM, Swieringa F, Cavill R. et al. Identification of platelet function defects by multi-parameter assessment of thrombus formation. Nat Commun 2014; 5: 4257
    CrossrefPubMedGoogle Scholar
  • 39 Deng W, Xu Y, Chen W. et al. Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor. Nat Commun 2016; 7: 12863
    CrossrefPubMedGoogle Scholar
  • 40 Micsonai A, Wien F, Kernya L. et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A 2015; 112 (24) E3095-E3103
    CrossrefPubMedGoogle Scholar
  • 41 Fu X, Chen J, Gallagher R, Zheng Y, Chung DW, López JA. Shear stress-induced unfolding of VWF accelerates oxidation of key methionine residues in the A1A2A3 region. Blood 2011; 118 (19) 5283-5291
    CrossrefPubMedGoogle Scholar
  • 42 Wieland DC, Garamus VM, Zander T. et al. Studying solutions at high shear rates: a dedicated microfluidics setup. J Synchrotron Radiat 2016; 23 (02) 480-486
    CrossrefPubMedGoogle Scholar
  • 43 Wieland DCF, Zander T, Garamus VM. et al. Complex solutions under shear and pressure: a rheometer setup for X-ray scattering experiments. J Synchrotron Radiat 2017; 24 (Pt 3): 646-652
    CrossrefPubMedGoogle Scholar
  • 44 Ruggeri ZM. Type IIB von Willebrand disease: a paradox explains how von Willebrand factor works. J Thromb Haemost 2004; 2 (01) 2-6
    CrossrefPubMedGoogle Scholar
  • 45 Kulkarni S, Dopheide SM, Yap CL. et al. A revised model of platelet aggregation. J Clin Invest 2000; 105 (06) 783-791
    CrossrefPubMedGoogle Scholar
  • 46 Ruggeri ZM. Mechanisms of shear-induced platelet adhesion and aggregation. Thromb Haemost 1993; 70 (01) 119-123
    Article in Thieme ConnectPubMedGoogle Scholar
  • 47 Ikeda Y, Handa M, Kawano K. et al. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest 1991; 87 (04) 1234-1240
    CrossrefPubMedGoogle Scholar
  • 48 Panigada M, Bottino N, Tagliabue P. et al. Hypercoagulability of COVID-19 patients in intensive care unit: a report of thromboelastography findings and other parameters of hemostasis. J Thromb Haemost 2020; 18 (07) 1738-1742
    CrossrefPubMedGoogle Scholar