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.
Subject(s)
Actin Depolymerizing Factors/genetics , Lim Kinases/genetics , Thrombocytopenia/physiopathology , von Willebrand Disease, Type 2/physiopathology , von Willebrand Factor/genetics , Animals , Gene Knock-In Techniques , Humans , Male , Mice , Mutation , Signal Transduction , rho GTP-Binding Proteins , rhoA GTP-Binding Protein , von Willebrand Disease, Type 2/enzymologyABSTRACT
The haemostatic potential of von Willebrand factor, a glycoprotein expressed by endothelial cells as ultra-large polymers (UL-vWF)(1), increases with its length, which in turn is regulated proteolytically by ADAMTS13, a zinc-metalloprotease selectively cleaving vWF at the Tyr1605-Met1606 bond. We have recently shown that in vitro oxidation of Met1606, under conditions mimicking those found in diseases characterized by high oxidative stress, severely impairs proteolysis by ADAMTS13, with a resulting pro-thrombotic effect caused by the accumulation of UL-vWF species. Conversely, Val1607Asp mutation, found in vWF from patients with type 2A von Willebrand disease, accelerates proteolysis of vWF, with a final hemorrhagic effect. Considering the physio-pathological importance of ADAMTS13-vWF interaction and the absence of experimental structural data, here we produced by homology modeling techniques a three-dimensional model of ADAMTS13 metalloprotease domain (M13). Thereafter, the vWF(1604-1607) peptide, containing the cleavable Tyr1605-Met1606 bond, was manually docked into the protease active site and the resulting model complex provided us key information for interpreting on structural grounds the variable effects that chemical modifications/mutations in vWF have on proteolysis by ADAMTS13.
