Poster Board II-973
The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX.
A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ 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), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . 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.
These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3ζ 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.