Carbohydrates and Activity of Natural and Recombinant Tissue Factor

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 (V_max) of the complex increased in the order rTF_1–243 (Escherichia coli) < rTF_1–263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the K_m of FX for rTF_1–263-FVIIa remained unchanged after deglycosylation, whereas the k_cat decreased slightly. A pronounced decrease, 4-fold, in k_cat was observed for pTF·FVIIa upon deglycosylation, whereas the K_m was minimally altered. The parameters of FX activation by both rTF_1–263D-FVIIa and pTF_D-FVIIa were identical and similar to those for rTF_1–243-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF_1–263 and pTF. The carbohydrates of rTF_1–263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.