LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B
Alexandre Kauskot, … , Cécile V. Denis, Dominique Baruch
Alexandre Kauskot, … , Cécile V. Denis, Dominique Baruch
Published October 6, 2016
Citation Information: JCI Insight. 2016;1(16):e88643. https://doi.org/10.1172/jci.insight.88643.
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Research Article Hematology

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B

Abstract

von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein’s multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.

Authors

Alexandre Kauskot, Sonia Poirault-Chassac, Frédéric Adam, Vincent Muczynski, Gabriel Aymé, Caterina Casari, Jean-Claude Bordet, Christelle Soukaseum, Chantal Rothschild, Valérie Proulle, Audrey Pietrzyk-Nivau, Eliane Berrou, Olivier D. Christophe, Jean-Philippe Rosa, Peter J. Lenting, Marijke Bryckaert, Cécile V. Denis, Dominique Baruch

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Figure 2

Platelet/proplatelet formation in vivo and in vitro is affected by the p.V1316M mutation.

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Platelet/proplatelet formation in vivo and in vitro is affected by the p...
(A) Whole-blood platelet counts of WT (solid line) or type 2B mutant vWF/p.V1316M (2B, dashed line) mice were obtained after immune-induced thrombocytopenia following intravenous injection of anti–mouse GPIbα antibodies. Platelet counts (left) and mean platelet volume (MPV) (right) were measured at indicated times (n = 9 for WT and 6 for 2B). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. **P < 0.01. (B) Quantification of shape change of megakaryocytes (MKs) in bone marrow explant experiments measured in 3 separate experiments (75–125 MKs were analyzed/experiment). (C) Mature MKs were incubated over a fibrinogen matrix for 5 hours. Representative images of MKs (left, scale bars: 50 μm) and representative images of α-tubulin staining in MKs (right, scale bars: 25 μm). (D) Quantification of the percentage of MKs forming proplatelets (left), the number of proplatelets/MK (middle), and the size of platelet-like structures (right) measured in 3 separate experiments. Statistical significance was determined by Student’s t test. *P < 0.05, **P < 0.01 (20–45 MKs were analyzed/experiment). (E) Representative images of WT and 2B MKs forming proplatelets during perfusion over a vWF matrix at a shear stress of 18 dyn/cm2. Black arrows indicate proplatelets attached to the cell, and white arrows indicate platelets and proplatelets released from the MKs. Scale bars: 20 μm. Graph of the quantification of the number of platelets and proplatelets released in the flow chamber during perfusion of mature MKs at 10, 20, 30, and 40 minutes over a vWF matrix. The number of platelets and proplatelets released from MKs and attached in the flow chamber was measured in 3 separate experiments (right). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. **P < 0.01.