Analysis of the Roles of 14-3-3 in the Platelet Glycoprotein Ib-IX–Mediated Activation of Integrin α(IIb)β(3) Using a Reconstituted Mammalian Cell Expression Model

We have reconstituted the platelet glycoprotein (GP) Ib-IX–mediated activation of the integrin α(IIb)β(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing α(IIb)β(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin α(IIb)β(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against α(IIb)β(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to α(IIb)β(3) indicating that GPIb-IX mediates a cellular signal leading to α(IIb)β(3) activation. Deletion of the 14-3-3–binding site in GPIbα inhibited GPIb-IX–mediated fibrinogen binding to α(IIb)β(3) 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 α(IIb)β(3), but inhibited vWF-induced fibrinogen binding to α(IIb)β(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX–mediated activation of α(IIb)β(3) requires 14-3-3 interaction with GPIbα.