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

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 zeta and filamin A. Both 14-3-3 zeta and filamin A associate with the cytoplasmic domain of GPIb alpha, 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 zeta and filamin A in regulating the VWF binding function of GPIb-IX. Methods and Results: A truncation mutant of GPIb alpha (Delta 565) deleting the C-terminal 14-3-3 zeta binding sites retained 14-3-3 zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb alpha totally abolished 14-3-3 zeta binding, indicating that the residues 551 to 564 of GPIb alpha are important in the interaction between 14-3-3 zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIb alpha suggesting phosphorylation of a population of GPIb alpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb alpha eliminated the association of 14-3-3 zeta with Delta 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 alpha 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 zeta to central filamin A binding site of GPIb alpha, and the dimeric 14-3-3 zeta 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. (Circ Res. 2009; 105: 1177-1185.)