Figure 6
Figure 6. TF mutant lacking Cys186-Cys209 disulfide bond has reduced affinity for factor VIIa but retains coagulant function. (A) HUVECs were transduced with adenovirus encoding wild-type TF (2 MOI/cell) or TFC186S, TFC209S, or TFC186S/C209S (50 MOI/cell) to express similar levels of TF antigen and were cultured for 48 hours before they were used. Cells were detached from the dish, and TF activity of intact cells was measured in a clotting assay. (B-I) HUVECs were transduced with adenovirus encoding wild-type TF (2 MOI/cell) or TFC186S/C209S (50 MOI/cell), and TF protein expression levels at the cell surface were evaluated by cell-surface biotinylation (B), in situ ELISA (C), and the binding of radiolabeled TF mAb or FVIIa (D) as described in the legend to Figure 5. (E) Specific binding of 125I-FVIIa to TF. Various concentrations of 125I-FVIIa were incubated with HUVEC monolayers expressing wild-type TF (○) or TFC186S/C209S (●) in the presence or absence of anti-TF IgG for 2 hours at 4°C. The amount of FVIIa bound to TF was determined as described in “Determination of 125I-FVIIa and 125I-TF monoclonal antibody binding to cells.” (F) TF coagulant function. HUVECs expressing wild-type TF (○), TFC186S/C209S (●), or uninfected HUVECs (◇) were incubated with various concentrations of FVIIa (0.0-200nM) for 5 minutes, then FX (1μM) was added to the cells, and the rate of FX activation was determined by measuring the amount of FXa generated in a chromogenic assay. (G) TF activity of HUVECs expressing wild-type TF and TFC186S/C209S in the presence of saturating concentration of FVIIa. HUVECs were incubated with FVIIa (100nM) and FX (1μM), and the rate of FXa generation was measured in a chromogenic assay. These assays were performed in parallel to the experiments in which TF antigen levels were determined. (H) Rate of FXa generation by cells expressing wild-type TF or TFC186S/C209S at various concentrations of FX and a saturating concentration of FVIIa (100nM). Wild-type TF, ○; TFC186S/C209S, ● (n = 3; mean ± SEM). (I) Specific activity derived, based on FVIIa or TF mAb bound to the cell surface.

TF mutant lacking Cys186-Cys209 disulfide bond has reduced affinity for factor VIIa but retains coagulant function. (A) HUVECs were transduced with adenovirus encoding wild-type TF (2 MOI/cell) or TFC186S, TFC209S, or TFC186S/C209S (50 MOI/cell) to express similar levels of TF antigen and were cultured for 48 hours before they were used. Cells were detached from the dish, and TF activity of intact cells was measured in a clotting assay. (B-I) HUVECs were transduced with adenovirus encoding wild-type TF (2 MOI/cell) or TFC186S/C209S (50 MOI/cell), and TF protein expression levels at the cell surface were evaluated by cell-surface biotinylation (B), in situ ELISA (C), and the binding of radiolabeled TF mAb or FVIIa (D) as described in the legend to Figure 5. (E) Specific binding of 125I-FVIIa to TF. Various concentrations of 125I-FVIIa were incubated with HUVEC monolayers expressing wild-type TF (○) or TFC186S/C209S (●) in the presence or absence of anti-TF IgG for 2 hours at 4°C. The amount of FVIIa bound to TF was determined as described in “Determination of 125I-FVIIa and 125I-TF monoclonal antibody binding to cells.” (F) TF coagulant function. HUVECs expressing wild-type TF (○), TFC186S/C209S (●), or uninfected HUVECs (◇) were incubated with various concentrations of FVIIa (0.0-200nM) for 5 minutes, then FX (1μM) was added to the cells, and the rate of FX activation was determined by measuring the amount of FXa generated in a chromogenic assay. (G) TF activity of HUVECs expressing wild-type TF and TFC186S/C209S in the presence of saturating concentration of FVIIa. HUVECs were incubated with FVIIa (100nM) and FX (1μM), and the rate of FXa generation was measured in a chromogenic assay. These assays were performed in parallel to the experiments in which TF antigen levels were determined. (H) Rate of FXa generation by cells expressing wild-type TF or TFC186S/C209S at various concentrations of FX and a saturating concentration of FVIIa (100nM). Wild-type TF, ○; TFC186S/C209S, ● (n = 3; mean ± SEM). (I) Specific activity derived, based on FVIIa or TF mAb bound to the cell surface.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal