Glycosylation of tissue factor is not essential for its transport or functions

Glycosylation plays an important role in protein function. The importance of glycosylation for tissue factor (TF) function is unclear.

The aim of the present study is to investigate the importance of TF glycosylation in transport to the cell surface and its coagulant and signaling functions.

Endothelial cells and peripheral blood mononuclear cells (PBMC) were treated with tunicamycin to inhibit N-linked glycosylation. Site-specific mutagenesis of one or more potential N-linked glycosylation sites in TF was used to generate TF mutants lacking glycans. TF expression at the cell surface was determined in binding assays using (125)I-FVIIa or (125)I-TF mAb and confocal microscopy. TF coagulant activity was measured by factor (F) Xa generation assay, and TF signaling function was assessed by measuring cleavage of protease activated receptor 2 (PAR2) and activation of p44/42 MAPK.

Tunicamycin treatment reduced TF activity at the endothelial cell surface; however, this reduction was found to be the result of decreased TF protein production in tunicamycin-treated cells. Tunicamycin treatment had no significant effect on TF activity or antigen levels in PBMC. No significant differences were observed in TF protein expression and procoagulant activity among cells transfected to express either wild-type TF or TF mutants. A fully non-glycosylated TF is shown to bind FVIIa and interact with FX with the same efficiency as that of wild-type TF. Non-glycosylated TF is also capable of supporting FVIIa cleavage of PAR2 and PAR2-dependent p44/42 MAPK activation.

Glycosylation is not essential for TF transport and coagulant or signaling functions.