The Association of 14-3-3ζ and Filamin A with the Central Cytoplasmic Domain of GPIbα Involves in Regulation of the Von Willebrand Factor Binding and Signaling Functions of Platelet Glycoprotein Ib-IX.

Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate platelet tethering and simultaneously trigger signaling cascades leading to platelet adhesion, spreading, and thrombus formation. Although the intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and involve in the regulation of VWF binding function, whereas, the mechanisms still remain unclear. We have recently identified a novel 14-3-3ζ binding site with partial phosphorylation of Ser559 in resting platelets located in the cytoplasmic filamin A binding domain of GPIbα. Disruption of the central cytoplasmic 14-3-3ζ binding site of GPIbα did not abolish the interaction between 14-3-3ζ and GPIb-IX, whereas obviously diminished VWF binding function. Here we show that disruption of 14-3-3ζ binding to GPIbα by GPIbα mutants or cell permeable peptides enhances the association of filamin A with GPIb-IX and the expression of GPIb-IX in the plasma membrane. In contrast, disruption of 14-3-3ζ binding to GPIbβ, which would enhance the opportunity for 14-3-3ζ monomer to bind to GPIbα, reduces filamin A-GPIb-IX interaction and GPIb-IX membrane expression. Compared with a GPIb-IX mutant truncating the whole cytoplasmic domain of GPIbα (Δ551), GPIb-IX mutant retaining only the central 14-3-3ζ binding site of the cytoplasmic domain of GPIbα (Δ565) showed an enhanced cell adhesion to VWF under flow and ristocetin-induced VWF binding. Furthermore, the two cell permeable peptides, MP-P and MPαC, which had been confirmed to block the central 14-3-3ζ binding site of GPIbα (MP-P) or diminished the association of 14-3-3ζ with GPIb-IX complex (MPαC), obviously inhibited ristocetin induced wild-type GPIb-IX cell aggregation. In addition, MP-P and MPαC inhibited botrocetin-induced platelet spreading and lamellipodia formation on VWF matrix, and GPIb-IX-VWF interaction induced co-immunoprecipitation of Src with GPIb-IX. Taken together, these data indicate that the binding of 14-3-3ζ and filamin A to the central cytoplasmic domain of GPIbα involves in regulation of surface expression, VWF binding and signaling functions of glycoprotein Ib-IX.

No relevant conflicts of interest to declare.