Original Article

Analysis of the Roles of 14-3-3 in the Platelet Glycoprotein Ib-IX–mediated Activation of Integrin _IIbß₃ Using a Reconstituted Mammalian Cell Expression Model

Correspondence to: Xiaoping Du, Department of Pharmacology, the University of Illinois, College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612. Tel:(312) 355-0237 Fax:(312) 996-1225 E-mail:xdu{at}uic.edu.

We have reconstituted the platelet glycoprotein (GP) Ib-IX–mediated activation of the integrin _IIbß₃ in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing _IIbß₃ adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin _IIbß₃ and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against _IIbß₃. vWF binding to GPIb-IX also activates soluble fibrinogen binding to _IIbß₃ indicating that GPIb-IX mediates a cellular signal leading to _IIbß₃ activation. Deletion of the 14-3-3–binding site in GPIb inhibited GPIb-IX–mediated fibrinogen binding to _IIbß₃ 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 GPIb induced an endogenous integrin-dependent cell spreading on vWF without requiring _IIbß₃, but inhibited vWF-induced fibrinogen binding to _IIbß₃. Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX–mediated activation of _IIbß₃ requires 14-3-3 interaction with GPIb.

Key Words: platelet, glycoprotein Ib-IX, integrin, 14-3-3, von Willebrand factor

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