A rapid, sensitive, and specific assay to measure TF activity based on chromogenic determination of thrombin generation.

Most tissue factor (TF) activity assays are based on measurement of factor X (FX) activation by TF in the presence of factor VII (FVII)/FVIIa. This requires long incubation, which may result in TF-independent activity of FX and inaccurate measurement of TF activity. To develop a sensitive and specific TF activity assay, which does not register a non-specific TF activity, using commercial coagulation factors. Tissue factor activity was measured based on the ability of TF to accelerate the activation of FX by FVIIa in the presence of factor V (FV)/Va, prothrombin, and phospholipids. Following 4 min incubation at 37°C, TF activity was quantified in test samples of different nature by thrombin generation using a chromogenic substrate. The TF activity assay proved high sensitivity (low fM range) and specificity, assessed by neutralization of TF activity by anti-TF antibody and the use of FVIIai. TF activity was detected in extracellular vesicles (EVs) derived from HAP1-TF+cells, while no activity was measured in EVs from HAP1-TF/KO cells. The assay was applicable for measurement of TF activity on the surface of live endothelial cells and monocytes activated in vitro, and cell lysates. Infusion of low dose lipopolysaccharide (2 ng/kg bodyweight endotoxin) caused a transient 8-fold increase (peaked at 4 h) in TF activity in EVs isolated from plasma of healthy volunteers. Our assay provides a fast, sensitive, and specific measurement of TF activity. It reliably quantifies TF activity on cell surface, cell lysate, and isolated EVs. The assay can be used for laboratory and clinical research.