Reversible Association of 14-3-3 with Platelet Glycoprotein Ibα Regulates the Ability of Cells to Adhere, Roll and Spread On VWF

Abstract 1061

Platelet adhesion to sites of vascular injury is required for the arrest of bleeding. Initial platelet adhesion is mediated by binding of von Willebrand factor (VWF) to the platelet glycoprotein (GP) Ib-IX-V complex, leading to the activation of integrin α _IIbβ₃ and other molecules that mediate firm adhesion, spreading and thrombus formation. The GPIb-IX-V complex comprises 4 polypeptides: GPIbα, GPIbβ, GPIX and GPV, in a 2:4:2:1 stoichiometry. Only the first three polypeptides are required for full VWF binding function. GPIbα is a 610 amino acid polypeptide that binds every known complex ligand within its N-terminal 300 amino acids. The cytoplasmic domain comprises 96 amino acids and contains binding sites for filamin, PI 3-kinase and the scaffolding protein 14-3-3. The association of 14-3-3 with the GPIbα cytoplasmic domain regulates the affinity for VWF. Typically, 14-3-3 requires phosphoserine- or phosphothreonine-containing motifs to bind target proteins. One such motif is in the GPIbα cytoplasmic domain surrounding Ser609, which is phosphorylated and known to bind 14-3-3. Mutation of Ser609 to Ala abrogates 14-3-3 association, which has been proposed to reduce the ability of GPIbα to bind VWF. Platelet aggregation results in the dissociation of 14-3-3 from a subpopulation of GPIbα. Ser609 also becomes dephosphorylated upon platelet spreading. To dissect further the functional roles of 14-3-3 association with GPIbα, we expressed in Chinese hamster ovary (CHO) cells GPIb-IX complexes (GPIbα, GPIbβ, and GPIX) containing either wild type GPIbα, or GPIbα mutants S609A or S609E. In other proteins, mutation of Ser to Glu at the 14-3-3 binding site mimics phosphoserine, recapitulates 14-3-3 binding and often prevents 14-3-3 dissociation. We first assessed the ability of the WT and mutant GPIbα to associate with 14-3-3. As expected, we detected little 14-3-3 binding to GPIbα S609A. GPIbα S609E bound 14-3-3 to the same extent as did WT GPIbα, indicating that the Glu substitution was able to mimic Ser phosphorylation at residue 609. We then assessed the ability of the CHO cells to attach to and roll on VWF under flow over a wide range of shear rates. At 3.26 and 10 dyne/cm² the α 609A and α 609E cells rolled twice as fast as the WT cells. Both CHO cells and platelets display a characteristic velocity nadir as shear rates increase. The α 609A and α 609E cell showed defective shear-enhanced adhesion; their slowest velocity was ∼3-fold faster than the WT cells. Because GPIbα is dephosphorylated upon platelet spreading, we also assessed the effect of the mutations on cell spreading on VWF. All three cell lines adhered similarly to VWF but a higher percentage of α 609A cells spread (67% vs 58% for WT and α 609E). Of the spread cells, the α S609E cells spread less well; their spread area was 15% less than the WT and α S609A cells. The morphology of the adherent, spread cells was dramatically different among the different cell lines. WT cells displayed a few filopodial extensions along with punctate phalloidin staining indicative of focal adhesions. In some cases the cells displayed stress fibers. The α S609A cells extended more and longer filopodia than the WT cells but had fewer focal adhesions and more stress fibers. The CHO α S609E cells extended thin filopodia that tended to be polarized at two sides of the cell body, and had fewer focal adhesions and no stress fibers. We also examined the effect of the mutations on localization of the GPIb-IX complex to lipid raft membrane microdomains, which is required for platelet adhesion to VWF. Raft GPIbα was reduced by 40% in the S609A cells but increased 1.6-fold in the S609E cells.

In summary, lack of 14-3-3 association decreased raft localization of the complex, reduced shear-induced cell adhesion, but increased cell spreading. Stable 14-3-3 association increased raft localization, but decreased shear-induced cell adhesion and decreased the ability of cells to fully spread. Together, our results demonstrate that regulated 14-3-3 association mediated by the phosphorylation status of S609 is required for coordinated adhesion, and cell spreading. Together, our results demonstrate that the functions of the GPIb-IX complex are regulated by the ability of GPIbα Ser609 to both bind and release 14-3-3 and suggest that it is not 14-3-3 binding per se that regulates GPIbα function.