von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3
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
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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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 1

Platelet aggregation, secretion, and αIIbβ3 integrin activation in mvWF/p.V1316M.

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Platelet aggregation, secretion, and αIIbβ3 integrin activation in mvWF/...
(A) Aggregation and integrin αIIbβ3 activation of washed platelets were initiated by adding thrombin (0.1 U/ml), PAR4-AP (150 μM), and collagen (1.2 μg/ml). Aggregation was expressed as the percentage change in light transmission, with the value for the blank (buffer without platelet) set at 100%. Traces are representative of 3 independent experiments. Integrin αIIbβ3 activation was assessed by flow cytometry. Platelets were incubated with a phycoerythrin-labeled rat anti-mouse integrin αIIbβ3 mAb (JON/A) specific for the activated conformation of the mouse integrin. Flow cytometry was performed without stirring. The level of activated integrin is indicated by the MFI ± SEM from at least 3 experiments. (B) Dense and α granule secretion were assessed by measuring ATP release and P-selectin exposure, respectively. ATP was quantified after platelet aggregation induced by various agonists (thrombin, PAR4-AP, collagen) and expressed as the amount of ATP released (pmoles). P-selectin exposure was evaluated by flow cytometry using FITC-labeled rat anti-mouse P-selectin mAb. The level of P-selectin exposed at the surface is expressed as MFI ± SEM from at least 3 experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (paired Student’s t test).