Mouse fviia

Samples (50 μl each) were incubated with the 1H1 anti-TF antibody at 100 μg/ml (Genentech) for murine samples, HTF-1 anti-TF antibody at 8 μg/ml (BD Bioscience) for human samples or respective IgG controls for 15 min at room temperature. Next, 50 μl of HBSA containing 10 nM mouse FVIIa (Enzyme Research Laboratories, South Bend, IN), 300 nM human FX (Enzyme Research Laboratories) and 10 mM CaCl₂ were added to the sample and incubated for 2 hours at 37 °C in a 96-well plate. FXa generation was stopped by the addition of 25 μl of 25 mM EDTA buffer. Finally, 25 μl of the chromogenic substrate Pefachrome FXa 8595 (4 mM, Pentapharm, Switzerland) was added and the mixture incubated at 37 °C for 15 min. Absorbance at 405 nm was measured using a Spectramax microplate reader (Molecular Devices, San Jose, CA). The procoagulant activity (PCA) of the sample was calculated by reference to a standard curve generated using recombinant human relipidated TF (0– 55 pg/ml, Siemens, Munich, Germany). The TF-dependent PCA generation (pg/ml) was determined by subtracting the amount of PCA generated in the presence of blocking antibodies from the amount of total PCA generated in the presence of the IgG controls.