The type 2B p.R1306W natural mutation of von Willebrand factor dramatically enhances the multimer sensitivity to shear stress

Authors

  • G. L. Scaglione,

    1. Istituto di Medicina Interna e Geriatria, Servizio Malattie Emorragiche e Trombotiche, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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    • These authors contributed equally to this study.
  • S. Lancellotti,

    1. Istituto di Medicina Interna e Geriatria, Servizio Malattie Emorragiche e Trombotiche, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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    • These authors contributed equally to this study.
  • M. Papi,

    1. Istituto di Medicina Interna e Geriatria, Servizio Malattie Emorragiche e Trombotiche, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
    2. Istituto di Fisica, Centro di Microscopia, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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    • These authors contributed equally to this study.
  • M. De Spirito,

    1. Istituto di Fisica, Centro di Microscopia, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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  • A. Maiorana,

    1. Istituto di Fisica, Centro di Microscopia, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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  • L. Baronciani,

    1. U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano and Luigi Villa Foundation, Milan, Italy
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  • M. T. Pagliari,

    1. U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano and Luigi Villa Foundation, Milan, Italy
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  • A. Arcovito,

    1. Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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  • E. Di Stasio,

    1. Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
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  • F. Peyvandi,

    1. U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano and Luigi Villa Foundation, Milan, Italy
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  • R. De Cristofaro

    Corresponding author
    1. Istituto di Medicina Interna e Geriatria, Servizio Malattie Emorragiche e Trombotiche, Facoltà di Medicina e Chirurgia, Università Cattolica S. Cuore, Roma, Italy
    • Correspondence: Raimondo De Cristofaro, Istituto di Medicina Interna e Geriatria, Servizio Malattie Emorragiche e Trombotiche, Facoltà di Medicina e Chirurgia ‘A. Gemelli’, Università Cattolica S. Cuore, Largo F. Vito, 1, 00168 Roma, Italy.

      Tel.: +39 630156329; fax: +39 630155915.

      E-mail: rdecristofaro@rm.unicatt.it

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  • Manuscript handled by: P. H. Reistma
  • Final decision: P. H. Reitsma, 23 June 2013

Summary

Background

Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self-association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα.

Objectives

To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor.

Methods

The interaction of GPIbα with wild-type (WT) and p.R1306W VWF multimers and A1–A2–A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress-induced self-association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0–60 dyn cm−2 range.

Results

WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL−1 already bound to the receptor. By contrast, the WT and p.R1306W-A1–A2–A3 constructs showed comparable affinities for GPIbα (Kd ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of < 14 dyn cm−2, the p.R1306W multimers rapidly changed conformation, entering a regime of self-aggregation, which, in contrast, was induced for WT VWF by shear forces of > 30 dyn cm−2. Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein.

Conclusions

The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites.

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