Measurement of TF activity in plasma and isolated EVs. In plasma-based activity assays, exogenous FVIIa and FX are added to plasma that already contains FVII and FX. FVII or FVIIa and FX are added to isolated EVs to determine TF activity. Plasma contains TFPI that can inhibit the TF-FVIIa complex and reduce the amount of FXa generated. Isolated EVs have a small amount of TFPI that is carried over from plasma during the isolation of the EVs. FXa cleavage of a chromogenic peptide substrate leads to the release of a chromophore. Professional illustration by Patrick Lane, ScEYEnce Studios.