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 14-3-3zeta and filamin A. Both 14-3-3zeta and filamin A associate with the cytoplasmic domain of GPIbalpha, 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 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX. METHODS AND RESULTS: A truncation mutant of GPIbalpha (Delta565) deleting the C-terminal 14-3-3zeta binding sites retained 14-3-3zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbalpha totally abolished 14-3-3zeta binding, indicating that the residues 551 to 564 of GPIbalpha are important in the interaction between 14-3-3zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbalpha eliminated the association of 14-3-3zeta with Delta565. 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 GPIbalpha 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 14-3-3zeta to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3zeta 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.
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 14-3-3zeta and filamin A. Both 14-3-3zeta and filamin A associate with the cytoplasmic domain of GPIbalpha, 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 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX. METHODS AND RESULTS: A truncation mutant of GPIbalpha (Delta565) deleting the C-terminal 14-3-3zeta binding sites retained 14-3-3zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbalpha totally abolished 14-3-3zeta binding, indicating that the residues 551 to 564 of GPIbalpha are important in the interaction between 14-3-3zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbalpha eliminated the association of 14-3-3zeta with Delta565. 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 GPIbalpha 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 14-3-3zeta to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3zeta 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.
Authors: Eelo Gitz; Cornelis A Koekman; Dave J van den Heuvel; Hans Deckmyn; Jan W Akkerman; Hans C Gerritsen; Rolf T Urbanus Journal: Haematologica Date: 2012-06-24 Impact factor: 9.941
Authors: Eelo Gitz; Charlotte D Koopman; Alèkos Giannas; Cornelis A Koekman; Dave J van den Heuvel; Hans Deckmyn; Jan-Willem N Akkerman; Hans C Gerritsen; Rolf T Urbanus Journal: Haematologica Date: 2013-06-10 Impact factor: 9.941
Authors: Lorenz Rognoni; Johannes Stigler; Benjamin Pelz; Jari Ylänne; Matthias Rief Journal: Proc Natl Acad Sci U S A Date: 2012-11-13 Impact factor: 11.205
Authors: Myriam Chimen; Helen M McGettrick; Bonita Apta; Sahithi J Kuravi; Clara M Yates; Amy Kennedy; Arjun Odedra; Mohammed Alassiri; Matthew Harrison; Ashley Martin; Francesca Barone; Saba Nayar; Jessica R Hitchcock; Adam F Cunningham; Karim Raza; Andrew Filer; David A Copland; Andrew D Dick; Joseph Robinson; Neena Kalia; Lucy S K Walker; Christopher D Buckley; Gerard B Nash; Parth Narendran; G Ed Rainger Journal: Nat Med Date: 2015-04-20 Impact factor: 53.440