Analysis of TF procoagulant activity and TF antigen expression in CHO cells transfected with wild-type TF or TF mutants. (A) Monolayers of CHO cells transfected transiently with equal amounts of wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S plasmid DNA were incubated with FVIIa (10nM) and FX (175nM), and FXa generation was measured in a chromogenic assay (n = 3; mean ± SEM). (B) CHO cells transfected transiently with equal amounts of wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S plasmid DNA were incubated with ¹²⁵I-FVIIa (10nM) for 2 hours at 4°C in the presence or absence of anti-TF IgG (100 μg/mL), and the amount of ¹²⁵I-FVIIa associated with cell-surface TF was determined (n = 3; mean ± SEM). (C) Same as in panel A, except CHO cells were stably transfected with wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S (n = 6; mean ± SEM). (D) CHO cells stably expressing wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S were incubated with saturating concentrations of ¹²⁵I-FVIIa (± anti-TF IgG) or ¹²⁵I-TF mAb (10H10 or 5G9; 100nM) for 2 hours at 4°C, and the amount of radioactivity associated with the cell surface was determined. ¹²⁵I-FVIIa binding shown in the figure was TF specific (n = 3; mean ± SEM). (E) Immunoblot analysis of wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S. Cell lysates of cells stably expressing wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions followed by immunoblot analysis with polyclonal anti-TF antibodies.