| A platelet receptor for von Willebrand factor (vWF), the glycoprotein Ib-IX (GPIb-IX), plays an important role in platelet activation and thrombus formation under flow conditions in arteries and capillaries. This thesis investigates the mechanisms involved in regulating the ligand binding function of GPIb-IX. First, it was found that the receptor function of GPIb-IX is regulated intracellulary via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIbα markedly enhanced ristocetin-induced vWF binding and allowed GPIb-IX expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D, that deploymerizes actin, also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. Secondly, the ligand binding function of GPIb-IX was also found to be negatively regulated through phosphorylation of GPIbβ at Ser166 by cAMP-dependent protein kinase (PKA). Expression of Ser166 phosphorylation-deficient mutant of GPIbβ with wild type GPIbα and GPIX enhanced ristocetin-induced vWF binding and cell adhesion to immobilized vWF. The PKA inhibitor, PKI, reduced phosphorylation of GPIbβ at Ser166 and enhanced vWF binding to wild type GPIb-IX. In contrast, a PKA stimulator, forskolin, reduced vWF binding to GPIb-IX and vWF-induced platelet agglutination. Thus, PKA-mediated phosphorylation of GPIbβ at Ser166 negatively regulates vWF binding to GPIb-IX. Finally, it is suggested that 14-3-3ζ is involved in PKA- and membrane skeleton-dependent regulation of vWF binding to GPIb-IX. GPIbα mutants of GPIb-IX that do not bind or weakly bind 14-3-3ζ reversed the enhancing effects of GPIbβ Ser166 dephosphorylation on vWF binding function of GPIb-IX. Furthermore, phosphorylation-deficient S166A mutant of GPIb-IX showed a decreased association with the membrane skeleton, which is reversed by the removal of the 14-3-3 binding site. Thus, these data provides support for the hypothesis that 14-3-3 is involved in regulating vWF-binding to GPIb-IX. |
