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Circulation Research

Year: 2009  |  Volume: 105  |  Issue: 12  |  Page No.: 1177 - 1185

Identification of a Novel 14-3-3{zeta} Binding Site Within the Cytoplasmic Domain of Platelet Glycoprotein Ib{alpha} That Plays a Key Role in Regulating the von Willebrand Factor Binding Function of Glycoprotein Ib-IX

Y Yuan, W Zhang, R Yan, Y Liao, L Zhao, C Ruan, X Du and K. Dai

Abstract

Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3 and filamin A. Both 14-3-3 and filamin A associate with the cytoplasmic domain of GPIb, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX.

Objective: To explore the mechanism underlying the roles of 14-3-3 and filamin A in regulating the VWF binding function of GPIb-IX.

Methods and Results: A truncation mutant of GPIb (565) deleting the C-terminal 14-3-3 binding sites retained 14-3-3 binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb totally abolished 14-3-3 binding, indicating that the residues 551 to 564 of GPIb are important in the interaction between 14-3-3 and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIb suggesting phosphorylation of a population of GPIb molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb eliminated the association of 14-3-3 with 565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIb with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions.

Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3 to central filamin A binding site of GPIb, and the dimeric 14-3-3 binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

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