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.


OBJECTIVE
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.
David
T
T
EFS-Alsace, Inserm, UMR-S949, Université Louis Pasteur, Strasbourg, France.
Strassel
C
C
Eckly
A
A
Cazenave
J-P
JP
Gachet
C
C
Lanza
F
F
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012694:Serine