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The platelet glycoprotein GPIbβ intracellular domain participates in von Willebrand factor induced‐filopodia formation independently of the Ser 166 phosphorylation site
Author(s) -
DAVID T.,
STRASSEL C.,
ECKLY A.,
CAZENAVE J.P.,
GACHET C.,
LANZA F.
Publication year - 2010
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2009.03590.x
Subject(s) - filopodia , microbiology and biotechnology , intracellular , von willebrand factor , glycoprotein ib , phosphorylation , filamin , chemistry , platelet membrane glycoprotein , cytoskeleton , biology , platelet , glycoprotein , actin , biochemistry , immunology , cell
See also Gardiner EE. A GPIb‐IX‐V complex signaling environment. This issue, pp 1075–6. Summary. Background : Circulating platelets are initially recruited at the site of vessel injury by von Willebrand factor (VWF) immobilized on collagen fibers. This process, mediated by the GPIb–V–IX complex, is accompanied by specific intracellular signaling leading to reorganization of the platelet actin cytoskeleton and extension of filopodia. Objectives / methods : To evaluate the GPIbα and GPIbβ intracellular domains contribution to this signaling, we generated Chinese hamster ovary (CHO) cells expressing a GPIb–IX complex with mutant forms of the two subunits and we measured their ability to extend filopodia upon adhesion on a VWF matrix. Results : Complete intracellular deletion or elimination of the filamin or the 14‐3‐3ζ binding sites in GPIbα did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu 150 –Arg 160 ) or central (Ala 159 –Pro 170 ) intracellular segment of GPIbβ resulted in a 21% and 23% reduction in the number of cells extending filopodia, respectively. This occurred without decreasing adhesion efficiency or GPIb–IX association with filamin A or 14‐3‐3ζ. Alanine scanning mutagenesis of the Leu 150 –Pro 170 segment identified Arg 164 , Leu 165 , Leu 167 , Thr 168 and Pro 170 as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser 166 PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIbβ subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock‐in mice expressing a GPIbβ intracellular deletion mutant. Conclusions : Altogether, our results strongly support participation of GPIbβ and its intracellular region in GPIb‐dependent platelet activation and shape change triggered by a VWF matrix.