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.
View: Text | PDF
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

×

Figure 6

Inhibition of LIM kinase (LIMK) rescues proplatelet formation in type 2B mutant vWF/p.V1316M (2B) megakaryocytes (MKs).

Options: View larger image (or click on image) Download as PowerPoint
Inhibition of LIM kinase (LIMK) rescues proplatelet formation in type 2B...
(A) Western blot of phospho-cofilin (p-cofilin) in 2B MKs in the presence or absence of LIMK inhibitor (LIMKi). Mature MKs were incubated for 3 hours with 10 μM LIMKi or DMSO (1:500 vol/vol) and then lysed in SDS denaturing buffer. n = 3. (B) Representative images of 4 separate experiments of the actin structure (white arrows) in 2B MKs treated or not with LIMKi (10 μM). DMSO was used as control. Scale bars: 20 μm. (C) Mature MKs after thrombopoietin-induced differentiation in culture were incubated over a fibrinogen matrix for 5 hours in the presence or absence of LIMKi (10 μM). DMSO was used as control. Representative images of MKs forming proplatelets from 3 separate experiments. Scale bars: 50 μm. Quantification of the percentage of proplatelet-forming MKs in the presence or absence of LIMKi (right). The percentage of cells was measured in 3 separate experiments (50–80 MKs were analyzed/experiment). (D) Representative images (left) of 2B MKs in the presence or absence of LIMKi. Scale bars: 20 μm. Graph of the size of platelet-like structures (right) was measured in 3 separate experiments. Statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01.