Published online before print October 29, 2009, doi: 10.1161/CIRCRESAHA.109.204669
Submitted on July 8, 2009
Revised on October 14, 2009
Accepted on October 20, 2009
Identification of a Novel 14-3-3
Binding Site Within the Cytoplasmic Domain of Platelet Glycoprotein Ib
That Plays a Key Role in Regulating the von Willebrand Factor Binding Function of Glycoprotein Ib-IX
Yanhong Yuan ;
From the School of Biological Science and Medical Engineering (Y.Y., W.Z., R.Y., Y.L., L.Z., K.D.), Beijing University of Aeronautics and Astronautics, Beijing, China; Jiangsu Institute of Hematology (C.R.), Key Laboratory of Thrombosis and Hemostasis, First Affiliated Hospital of Soochow University, Ministry of Health, Suzhou, China; and Department of Pharmacology (X.D.), University of Illinois, College of Medicine, Chicago.
* To whom correspondence should be addressed. E-mail: kdai{at}buaa.edu.cn.
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 14-3-33
and filamin A. Both 14-3-33 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 14-3-33 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 14-3-33 binding sites retained 14-3-33 binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb totally abolished 14-3-33 binding, indicating that the residues 551 to 564 of GPIb are important in the interaction between 14-3-33 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 14-3-33 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 14-3-33 to central filamin A binding site of GPIb, and the dimeric 14-3-33 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.
Key words: platelets • GPIb
•
14-3-3 •
von Willebrand factor •
thrombosis