The platelet glycoprotein GPIbβ intracellular domain participates in von Willebrand factor induced-filopodia formation independently of the Ser 166 phosphorylation site

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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

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Edité par CCSD ; Wiley -

International audience. 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 GPIbalpha and GPIbbeta 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 14-3-3zeta binding sites in GPIbalpha did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu(150)-Arg(160)) or central (Ala(159)-Pro(170)) intracellular segment of GPIbbeta 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 14-3-3zeta. 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 GPIbbeta subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock-in mice expressing a GPIbbeta intracellular deletion mutant.Conclusions: Altogether, our results strongly support participation of GPIbbeta and its intracellular region in GPIb-dependent platelet activation and shape change triggered by a VWF matrix.

Langue: en

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