Analysis of the Roles of 14-3-3 in the Platelet Glycoprotein Ib-IX–Mediated Activation of Integrin αIIbβ3 Using a Reconstituted Mammalian Cell Expression Model

Abstract

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.

A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase

“…GPIb-IX is a unique receptor that, unlike thromboxane A2 or collagen receptors, does not appear to require secretion of granules to induce activation of integrin. GPIb-IX-mediated integrin activation can be reconstituted in the cultured Chinese hamster ovary cells that do not have platelet-specific granules (19,20,33,44), although it is significantly amplified by ADP secre- FIG. 6.…”

“…The glycoprotein VI/Fc receptor ␥ chain pathway mediates collagen-induced platelet activation (28 -30). The GPIb-IX-dependent activation pathway mediates von Willebrand factor-induced platelet activation (11,(31)(32)(33) and is also important in low dose thrombininduced platelet activation (34 -37). We reported recently that PKG plays a stimulatory role in GPIb-IX-dependent integrin activation (19,20).…”

Platelet biogenesis and functions require correct protein O-glycosylation

“…Platelet spreading on immobilized VWF or fibrinogen after stimulation with botrocetin and bovine thrombin was performed as previously described (45) using fluorescein-labeled phalloidin (Invitrogen) (46). Additional information on flow cytometry and platelet spreading is presented in SI Materials and Methods.…”

Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

“…Under high shear rate flow conditions, initial platelet adhesion is mediated by the binding of the platelet von Willebrand Factor (VWF) 1 receptor, the glycoprotein (GP) Ib-IX-V complex, to subendothelial-bound VWF (1,2). The GPIb-IX-VWF interaction also initiates an intracellular signaling cascade leading to platelet shape change, spreading, secretion, and aggregation (3)(4)(5)(6)(7).…”