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.
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
Submitter: Margaret Rand | email@example.com
Authors: Shannon C. Jackson, Stephanie Cloutier, and Man-Chiu Poon.
The Hospital for Sick Children
Published December 13, 2013
In a recent study in the JCI (1), Casari et al. report a comprehensive analysis of effects of von Willebrand factor (VWF) with the V1316M gain-of-function mutation (VWF/V1316M) on platelet function, finding that this mutation inhibits integrin αIIbβ3 activation. They conclude that platelet dysfunction may be a mechanism leading to the bleeding phenotype in type 2B von Willebrand disease (VWD2B), in which high molecular weight (HMW) multimers of circulating mutated VWF spontaneously interact with glycoprotein (GP)Ibα (1,2). The authors point out 2 earlier publications on platelet dysfunction in VWD2B, but did not mention the most thoroughly-investigated family with the V1316M mutation, that was indeed initially described to have a platelet disorder.
50 years ago, a French-Canadian kindred was reported with severe mucocutaneous bleeding, and thrombocytopenia with large, clumped platelets, inherited autosomal dominantly (3). Frojmovic and colleagues termed the disorder the Montreal Platelet Syndrome (MPS) (4), demonstrating that MPS platelets aggregate spontaneously in plasma, and that this was enhanced by stirring. A defect in thrombin-induced aggregation was observed, after correcting for spontaneous aggregation (5). MPS platelets were found to be deficient in cytosolic calpain (6).
In more recent investigations of the MPS kindred, affected family members were shown to have a laboratory phenotype consistent with VWD2B, and to be heterozygous for a VWF 3946 G>A (V1316M) mutation at exon 28 (7). Binding of HMW multimers of VWF/V1316M to platelets in vivo explains Milton et al.’s observations that control platelets, either washed or in platelet-rich plasma, did not aggregate spontaneously when added to MPS plasma, while washed MPS platelets resuspended into control plasma or calcium-free buffer aggregated spontaneously (5). In this regard, it is remarkable that Casari et al.’s (1) experiments with washed platelets from their VWD2B/V1316M patient, and with control washed murine or human platelets pretreated with recombinant murine or human VWF/V1316M, respectively, were not confounded by spontaneous aggregation – or more correctly, agglutination. This same group did previously report that recombinant murine VWF/V1316M induced spontaneous interactions with washed murine platelets (8).
Thus, platelet dysfunction certainly does appear to occur in VWD2B/V1316M, as was first recognized in early studies on MPS (4-6). The detailed analysis by Casari et al. (1) of signaling defects brought about by VWF/V1316M binding on platelet activation has shed light into the mechanisms by which this occurs. The question remains whether the signaling defect also explains the dysmegakaryocytopoiesis observed in VWD2B (9), but not discussed in (1).
Margaret L. Rand,1 Shannon C. Jackson,2 Stephanie Cloutier,3 and Man-Chiu Poon4
1Division of Haematology/Oncology, Department of Paediatrics and Research Institute, The Hospital for Sick Children, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada. 2 Division of Hematology, Department of Medicine, St. Paul’s Hospital, University of British Columbia, Vancouver, Canada. 3 Division of Hematology, Department of Medicine, Université Laval and CHA-Hôpital de l’Enfant-Jesus, Quebec City, Canada. 4 Division of Hematology and Hematologic Malignancies, Department of Medicine, University of Calgary and Calgary Health Region, Calgary, Canada.
Conflict of interest statement: The authors have no conflicts of interest to declare.
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