Cys186-Cys209 disulfide bond formation is not essential for de-encryption of TF. HUVECs were transduced with the same number of adenovirus encoding wild-type TF or TFC186S/C209S (25 MOI/cell; A-B) or different MOI/cell (wild-type TF, 2 MOI/cell; TFC186S/C209S, 50 MOI/cell) to express similar levels of TF (C-D). After culturing the cells for 48 hours, the monolayers were treated with HgCl2 (100μM) for 3 minutes or ionomycin (10μM) for 5 minutes at 37°C, the cells were washed with buffer B, and the cell-surface TF activity was measured in FX activation assay (A,C). (B,D) Cell lysates were made by scraping the cells in buffer B followed by repeated (3) freeze-thaw cycles. The lysates were diluted 1:10 in buffer B before they were used in the assay. TF activity in intact monolayers and cell lysates was determined by adding FVIIa (100nM) and FX (1μM) and measuring the rate of FX activation in a chromogenic assay. The rate of FX activation measured with intact and untreated cells was taken as 100% (A-B). *Significant increase in TF activity in treated cells versus the untreated cells (n = 3-6; mean ± SD; P < .05, t test). †The fold increase did not significantly differ from the fold increase observed with wild-type TF (n = 3-6; mean ± SD; P > .1, t test).
