Fviia

Factors IXa, Xa, VIIa, XIa, and thrombin amidolytic activity were assayed by incubating purified enzymes with cystamine (0.2–200 mM, in a 2-fold serial dilution) and measuring the cleavage rate of chromogenic substrates (Pefachrome FIXa, FXa, FVIIa, FXa, and TG, respectively [Enzyme Research Laboratories, South Bend, IN, USA]). The K_i was determined by first calculating the apparent K_i (K_{i, app}) using the equation v(V₀)^-1 = 1-[I](K_{i, app}+[I])^-1. Since the inhibitor and substrate are in competition, the true K_i was then calculated using the equation K_i = K_{i, app} (K_m([S]+K_m)^-1).

Cell surface TF-fVIIa activity was measured by modification of previously described procedures [3 (link),24 (link),31 (link)]. Cells (5 × 10⁴) were seeded out into 48-well plates and transfected with the appropriate wild-type or mutant TF plasmids, as shown in Table 1. The cells were washed with phosphate-buffered saline (PBS) pH 7.4 and then pre-adapted to serum-free medium. The cells were incubated with PAR2-AP for up to 60 min and then washed and incubated with fVIIa (20 nM; Enzyme Research Labs, Swansea, UK) in HEPES-buffered saline (HBS) pH 7.4, containing 1% (w/v) bovine serum albumin (BSA) and 5 mM CaCl₂ (100 µL) for an additional 10 min. Finally, the samples were supplemented with fX (100 nM) together with fXa substrate (0.2 mM; Hyphen) diluted in the same buffer (100 µL). The samples were incubated for 60 min to develop the colour. Aliquots (150 µL) were then transferred to a 96-well plate containing 2% (v/v) acetic acid (50 µL) and the absorptions were measured immediately at 405 nm. The amount of fXa generated was determined using a standard curve prepared using fXa (Enzyme Research Labs). To inhibit cell surface TF activity, cells were pre-incubated with the inhibitory anti-TF antibody HTF1 (40 µg/mL; eBioscience/Thermo Scientific, Warrington, UK) prior to the addition of fVIIa [15 (link)].