Absence of the TF allosteric disulfide results in abolished coagulant function. (A) BHK cells were stably transfected to express TF^WT or TF^Cys209Ala. TF cell surface exposure was determined by labeling with 1mM NHS-biotin in HBS, and subsequently precipitation of biotinylated TF. To obtain total TF protein, immunoprecipitation with the TF mAb 9C3 was performed with n-octyl β-D-glucopyranoside (OG) cell lysates. Western Blotting was performed using Goat anti-TF antibody. The arrow indicates TF dimers. (B) Reactivity of a polyclonal goat anti-TF and 5G9 (both 10 μg/mL) with BHK cell surface levels of TF^WT or TF^Cys209Ala. (C) TF procoagulant activity on nontransfected BHK cells, BHK-TF^WT and BHK-TF^Cys209Ala was measured kinetically after addition of the indicated concentrations of VIIa and 100nM FX. (D) BHK cells expressing TF^WT, TF^Cys209Ser and TF^{Cys186/209Ser}. Cell surface expression was determined after NHS-biotin labeling and total TF expression was determined in total lysate. Note the absence of a dimer fraction in case of the TF^{Cys186/209Ser} mutant. The arrow indicates TF dimers. (E) TF procoagulant activity on nontransfected BHK cells, BHK-TF^WT, BHK-TF^Cys209Ser and BHK-TF^{Cys186/209Ser} was determined as described before. (F) BHK-TF^WT and BHK-TF^Cys209Ser were incubated with 10mM N-ethylmaleimide for 1 hour. Presence of dimer factions was determined on Western blot and procoagulant activity was performed as described before, using 1nM VIIa.