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

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 GPIb alpha and GPIb beta 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 14-3-3 zeta binding sites in GPIb alpha did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu150-Arg160) or central (Ala159-Pro170) intracellular segment of GPIb beta 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 14-3-3 zeta. Alanine scanning mutagenesis of the Leu150-Pro170 segment identified Arg164, Leu165, Leu167, Thr168 and Pro170 as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser166 PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIb beta subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock-in mice expressing a GPIb beta intracellular deletion mutant. Conclusions: Altogether, our results strongly support participation of GPIb beta and its intracellular region in GPIb-dependent platelet activation and shape change triggered by a VWF matrix.