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.