Carbohydrates and Activity of Natural and Recombinant Tissue Factor

Abstract The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF·factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (Vmax) of the complex increased in the order rTF1–243 (Escherichia coli) < rTF1–263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the Km of FX for rTF1–263-FVIIa remained unchanged after deglycosylation, whereas the kcat decreased slightly. A pronounced decrease, 4-fold, in kcat was observed for pTF·FVIIa upon deglycosylation, whereas the Km was minimally altered. The parameters of FX activation by both rTF1–263D-FVIIa and pTFD-FVIIa were identical and similar to those for rTF1–243-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF1–263 and pTF. The carbohydrates of rTF1–263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.