Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activat ion of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model , and show that 14-3-3 is important in GPIb-IX signaling. CHO cells express ing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin (alpha(IIb)beta(3) and GPIb-IX adhere and spread on VWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3) vWF bi nding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)bet a(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(II b)beta(3) activation. Deletion of the 14-3-3-binding site in GPIb alpha inh ibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell sp reading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX sig naling. Expression of a dominant negative 14-3-3 mutant inhibited cell spre ading on vWF, suggesting an important role for 14-3-3. Deleting both the 14 -3-3 and filamin-binding sites of GPIb alpha induced an endogenous integrin -dependent cell spreading on VWF without requiring alpha(IIb)beta(3), but i nhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while d ifferent activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requi res 14-3-3 interaction with GPIb alpha.