TFPI, also known as lipoprotein-associated coagulation inhibitor (LACI) and extrinsic pathway inhibitor (EPI), is a physiological inhibitor of extrinsic pathway of coagulation and has biological functions of anticoagulation and anti-inflammation. It is a secreted protein with a N-terminal acidic region, three Kunitz (K)?domains separated with by two linker regions, and a C-terminal basic region. The activity of recombinant rat TFPI was measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. Trypsin was diluted to 50 ug/ml in the assay buffer and 20 ul different concentrations of recombinant rat TFPI (MW: 61.9 KD) was incubated with 20 ul diluted trypsin at 37 ℃ for 15?minutes. Loading 50?μL of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50?μL of 20?μM substrate. Include a substrate blank containing 50?μL of assay buffer and 50?μL of 20?μM substrate. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant rat TFPI significantly decreased trypsin activity. The inhibition IC50 was <10 nM.