Abstract

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet function. The extracellular domain of GpIbα binds to von Willebrand factor (VWF) and rapidly effects shear-dependent platelet rolling, tether formation, adherence and aggregation onto freshly exposed subendothelium. Following VWF binding, GpIbα transduces proaggregatory signals through its cytoplasmic domain, and these signals may be modulated by the activity of GpIbβ and GpV. The expression of GpIbα, GpIbβ and GpIX in megakaryocytes also regulates terminal maturation and platelet release (its absence results in Bernard-Soulier syndrome) and there is human genetic and in vitro evidence that the cytoplasmic domain of GpIbα regulates megakaryocyte growth and surface expression of the complex. The cytoplasmic domain of GpIbα possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton, and there is evidence that filamin A binding to GpIbα directs the surface expression of GpIb-IX in CHO cells. To investigate the mechanism of this effect, we examined GpIbα biosynthesis in CHO cells stably co-expressing wild-type or mutant GpIbα with GpIbβ, GpIX and filamin A. We observed that surface GpIbα expression as measured by flow cytometry is enhanced ~ 1 log-order in CHO cells co-expressing human filamin A. In comparison with CHO-GpIbαβIX cell lysates, lysates from CHO-GpIbαβIX-filamin A cells showed greater amounts of immature (~ 65 kDa), incompletely glycosylated (~ 80 kDa and ~ 90 kDa) and fully mature GpIbα (~ 120 kDa), but lesser amounts of the ~ 15 kDa C-terminal peptide released when the extracellular domain of GpIbα (glycocalycin) is cleaved by surface proteases. To determine if the effect of filamin A is due to its binding to GpIbα, we examined GpIbα biosynthesis using several mutants of GpIbα co-expressed with GpIbβ, GpIX and filamin A. When filamin A binding is eliminated by truncation of GpIbα at C-terminal residue 557 or by a deletion in GpIbα between amino acids 542–570, the decreased synthesis of mature GpIbα is accompanied by nearly complete elimination of all immunodetectable immature GpIbα and by increased immunodetectable C-terminal peptide. To corroborate these data and control for cell selection and non-specific secondary structural changes, we examined the expression of three cell lines expressing two additional mutants of GpIbα in CHO cells stably transfected with GpIbβ, GpIX and filamin A. The expression of GpIbα with a C-terminal deletion between residues 560 and 570 (which inhibits filamin A binding), but not GpIbα with alanine substitutions at C-terminal residues 557 through 559 (which converts the sequence RGS to AAA but doesn’t inhibit filamin binding), results in the near-elimination of immature GpIbα. These results suggest that GpIbα binding to filamin enhances the surface expression of GpIb-IX by directing nascent protein trafficking away from a degradative pathway and towards the golgi. This leads to increased glycosylation, which further stabilizes the complex by attenuating the susceptibility of the extracellular domain to proteolytic cleavage.

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