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 7

Human type 2B mutant vWF/p.V1316M (2B) megakaryocytes (MKs) exhibit defective actin structure and proplatelet formation, which is rescued by LIM kinase (LIMK) inhibition.

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Human type 2B mutant vWF/p.V1316M (2B) megakaryocytes (MKs) exhibit defe...
(A) Control and patient 2B MKs after thrombopoietin-induced differentiation in culture were incubated over a fibrinogen matrix for 3 hours and stained for F-actin (green) and the Ser3-phosphorylated cofilin (p-cofilin, red). Scale bars: 10 μm. White squares indicate the magnification zone. (B) Western blot of p-cofilin and total cofilin in control (C) and in patient MKs (P). (C) Proplatelet formation in liquid culture in the presence or the absence of LIMKi at day 11, in control and in patient MKs. LIMKi (10 μM) was added at day 10; DMSO (0.2%) was used as vehicle. Scale bars: 50 μm. (D) Control and patient 2B MKs after thrombopoietin-induced differentiation in culture were spread over a polylysine-coated coverslip for 3 hours and stained for F-actin. Scale bars: 20 μm. (E) Proplatelet structure after F-actin staining and graph of the size of platelet-like structures in the 2B patient in the presence or absence of LIMKi. Scale bars: 5 μm. MKs in the presence of DMSO (n = 15) and MKs in the presence of LIMKi (n = 10) were analyzed for the size of platelet-like structures. Statistical significance was determined by Student’s t test. ***P < 0.001.