Correlation between TF antigen and TF activity levels at the cell surface in endothelial cells expressing wild-type TF or TF lacking Cys186-Cys209 disulfide bond. HUVECs cultured in 6- or 24-well plates were transduced with adenovirus encoding wild-type TF, TF_C186S, TF_C209S, TF_C186S/C209S, or a control virus (β-Gal; 25 MOI/cell) and cultured for 48 hours. (A) To measure TF antigen at the cell surface, the monolayers were labeled with cell-impermeant NHS-SS-biotin (0.5 mg/mL) for 30 minutes at 4°C. Total cell extracts were then subjected to immunoprecipitation with neutravidin-agarose and immunoblotted with anti-TF polyclonal antibodies. (B) Monolayers were incubated with ¹²⁵I-labeled TF mAb or FVIIa (100nM) for 2 hours at 4°C, and the amount of TF mAb and FVIIa associated with the cell surface was determined. To determine TF-specific FVIIa binding, parallel experiments were conducted in which cells were preincubated with polyclonal anti-TF IgG (100 μg/mL) for 30 minutes before the addition of ¹²⁵I-FVIIa, and the radioactivity associated with cells in the presence of anti-TF was subtracted from the values obtained in the absence of anti-TF (total binding). (C) Higher concentrations of FVIIa were required to saturate TF Cys186 and Cys209 mutants compared with wild-type TF. Monolayers of HUVECs expressing wild-type TF or TF mutant were incubated with various concentrations of FVIIa (0.05-100nM) for 5 minutes at room temperature before substrate FX (1μM) was added to the cells. The amount of FXa generated was measured in a chromogenic assay. The symbols are as follows: HUVECs expressing wild-type TF, ●; TF_C186S, ▲; TF_C209S, ■; TF_C186S/C209S, □; control virus (β-gal), ○ (n = 4, mean ± SEM). (D) HUVEC monolayers were incubated with FVIIa (100nM) for 5 minutes at room temperature, and then substrate FX (1μM) was added to the cells. The amount of FXa generated was measured in a chromogenic assay (n = 3-4; mean ± SEM). (E) TF-specific activity at the surface was determined as the amount of FXa formed (pM)/fmol of TF, as measured by TF mAb binding studies (■) or fmol FVIIa bound (▩), as determined by TF-specific binding of ¹²⁵I-FVIIa. The concentrations of FVIIa and FX used were 100nM and 1μM, respectively. *Because of low expression of TF mutant, TF_C186S, it was difficult to measure FVIIa binding to these cells accurately; therefore, TF-specific activity in relation to FVIIa bound to cells was not calculated.