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 9

In vivo administration of LIM kinase inhibitor (LIMKi) increases the platelet count and reduces the mean platelet volume in type 2B mutant vWF/p.V1316M (2B) mice but does not reverse the hemostatic defect.

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In vivo administration of LIM kinase inhibitor (LIMKi) increases the pla...
Whole-blood platelet counts in WT (A) and 2B mice (B) of DMSO-treated and LIMKi-treated mice before and after 3 injections of LIMKi (30 mg/kg) or DMSO (1.6 μl/g body weight) at various times. n = 3. Mean platelet volume (MPV) (C) of DMSO-treated 2B mice and LIMKi-treated 2B mice. n = 4–12 mice in each group. Statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. (D) Tail bleeding time in WT (n = 3) and 2B mice (n = 6 and 9) of DMSO-treated mice and LIMKi-treated mice after 3 injections of LIMKi (30 mg/kg) or DMSO. Integrin αIIbβ3 expression (n = 4) (E) and activation (n = 3) (F) were assessed by flow cytometry using a CD41 antibody and integrin αIIbβ3 mAb (JON/A) specific for the activated conformation of the mouse integrin, respectively. The level of activated integrin is indicated by the mean fluorescence intensity (MFI) ratio JON/A:CD41, calculated with the WT JON/A:CD41 ratio equal to 1.