4.8. Procoagulant Activity of TF

Cell TF activity was studied by measuring TF capacity to generate factor Xa. Cells were lysed with lysis buffer containing Tris-HCl pH 7.5, NaCl, and 0.1% Triton X-100. Three cycles of freezing/defreezing were performed to lyse the cells while preserving TF activity. Protein concentration was measured with the Pierce™ Bicinchoninic Acid Protein Assay (ThermoFisher Scientific, Villebon-sur-Yvette, France). Samples were diluted ½ in HEPES buffer then incubated with a blocking anti-TF antibody (clone VD8, SEKISUI Diagnostics, Pfungstadt, Germany) or an irrelevant IgG1 mAb to determine the TF contribution in factor Xa generation.

Cell lysates were incubated during 2 h at 37 °C in buffer containing factor X, factor VII and calcium to allow factor Xa generation. Reaction was stopped with EDTA. A fluorescent substrate of factor Xa was added and fluorescence values (excitation 390nm/emission 460nm) were measured during 15 min at 37 °C using a fluoroskan Ascent (ThermoFisher Scientific, Villebon-sur-Yvette, France). Data were normalized with protein level, and results were expressed in fM of FXa produced per minute per mg protein.