Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation.

The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.