von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3
Caterina Casari, … , Cécile V. Denis, Marijke Bryckaert
Caterina Casari, … , Cécile V. Denis, Marijke Bryckaert
Published November 25, 2013
Citation Information: J Clin Invest. 2013;123(12):5071-5081. https://doi.org/10.1172/JCI69458.
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Research Article

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3

Abstract

von Willebrand disease type 2B (vWD-type 2B) is characterized by gain-of-function mutations in von Willebrand factor (vWF) that enhance its binding to the glycoprotein Ib-IX-V complex on platelets. Patients with vWD-type 2B have a bleeding tendency that is linked to loss of vWF multimers and/or thrombocytopenia. In this study, we uncovered evidence that platelet dysfunction is a third possible mechanism for bleeding tendency. We found that platelet aggregation, secretion, and spreading were diminished due to inhibition of integrin αIIbβ3 in platelets from mice expressing a vWD-type 2B–associated vWF (vWF/p.V1316M), platelets from a patient with the same mutation, and control platelets pretreated with recombinant vWF/p.V1316M. Impaired platelet function coincided with reduced thrombus growth. Further, αIIbβ3 activation and activation of the small GTPase Rap1 were impaired by vWF/p.V1316M following exposure to platelet agonists (thrombin, ADP, or convulxin). Conversely, thrombin- or ADP-induced Ca2+ store release, which is required for αIIbβ3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLCγ2 activation following collagen receptor signaling, further implying that vWF/p.V1316M acts directly on or downstream of Ca2+ release. These data indicate that the vWD-type 2B mutation p.V1316M is associated with severe thrombocytopathy, which likely contributes to the bleeding tendency in vWD-type 2B.

Authors

Caterina Casari, Eliane Berrou, Marilyne Lebret, Frédéric Adam, Alexandre Kauskot, Régis Bobe, Céline Desconclois, Edith Fressinaud, Olivier D. Christophe, Peter J. Lenting, Jean-Philippe Rosa, Cécile V. Denis, Marijke Bryckaert

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Figure 9

Cvx-induced activation of human control platelets pretreated with recombinant hvWF/p.V1316M.

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Cvx-induced activation of human control platelets pretreated with recomb...
Washed human control platelets pretreated for 10 minutes with recombinant hvWF/p.V1316M or WT were stimulated with Cvx in the (A) absence or (B–E) presence of apyrase (2 U/ml) or (F) in the presence of antagonists of ADP receptors (AR-C69931MX, 10 μM; MRS 2179, 10 μM). (A and B) Aggregation and secretion of washed platelets were initiated by adding concentrations of thrombin (0.02–0.5 U/ml). Dense granule secretion was assessed by measuring the amount of ATP release. (C) Integrin αIIbβ3 activation induced by Cvx (1–10 nM) was assessed by flow cytometry using the integrin αIIbβ3 mAb PAC1 specific for the activated conformation of the human integrin. The level of activated integrin is indicated by MFI. (D) Washed platelets in suspension were activated by 4 nM Cvx for 30 and 60 seconds in the absence of stirring, and tyrosine Syk-P and PLCγ2-P was assessed by immunoblotting with anti–Syk-P and anti–PLCγ2-P, respectively. (E) Ca2+ signaling was monitored using Oregon Green 488 BAPTA1-AM. Curves represent the area under the curve of Ca2+ store release and Ca2+ influx. (F) Rap1 activity was measured by pull-down assay after 30 seconds of stimulation with Cvx (4 nM) in the absence of stirring and identified by immunoblotting with anti-Rap1 antibody. The presence of comparable amounts of Rap1 in aliquots of the all platelet samples is shown in a parallel gel; lanes of the Rap1-GTP blot were derived from the same gel but were noncontiguous, as indicated by the black line. Data from 1 experiment carried out in triplicate are presented as mean ± SEM. *P < 0. 05, ***P < 0.001 (unpaired Student’s t test). Results are representative of 3 independent experiments.