We have reconstituted the platelet glycoprotein (GP) Ib-IX–mediated activation of the integrin αIIbβ3 in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing αIIbβ3 adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin αIIbβ3 and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against αIIbβ3. vWF binding to GPIb-IX also activates soluble fibrinogen binding to αIIbβ3 indicating that GPIb-IX mediates a cellular signal leading to αIIbβ3 activation. Deletion of the 14-3-3–binding site in GPIbα inhibited GPIb-IX–mediated fibrinogen binding to αIIbβ3 and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbα induced an endogenous integrin-dependent cell spreading on vWF without requiring αIIbβ3, but inhibited vWF-induced fibrinogen binding to αIIbβ3. Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX–mediated activation of αIIbβ3 requires 14-3-3 interaction with GPIbα.

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb␣ markedly enhances ristocetin-(or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes 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. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeletondependent inside-out signal.

The ␣ chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIb␣ is phosphorylated at Ser 609 ; this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIb␣ C-terminal sequence S 606 GHSL 610 only when Ser 609 is phosphorylated. Immunoabsorption with antipS609 removed almost all of the GPIb␣ from platelet lysates, indicating a high proportion of GPIb␣ phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3 binding. A synthetic phosphopeptide corresponding to the GPIb␣ C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3. Thus, phosphorylation at Ser 609 of GPIb␣ is important for 14-3-3 binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIb␣ staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIb␣ molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser 609 of GPIb␣ regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading.