Antithrombin III, also known as Serpin C1, is a member of the Serpin superfamily of the serine protease inhibitors. It is the principal plasma Serpin of blood clotting proteases and inhibits thrombin as well as several factors such as Xa. Similar to Serpins A5 and D1, its thrombin inhibitory activity is enhanced by heparin. Hereditary and acquired Serpin C1 deficiency is the cause of an increased thrombotic tendency in many cases. For example, acquired Serpin C1 deficiency is a common condition in sepsis, after major trauma or surgery. The activity of recombinant human antithrombin III was measured by its ability to inhibit thrombin cleavage of a fluorogenic peptide substrate Boc-VPR-AMC in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. Thrombin was diluted to 0.5 U with heparin at 50 μg/ml in the assay buffer and 10 ul different concentrations of recombinant human antithrombin III (MW: 50 KD) was incubated with 10 ul diluted thrombin at 37 ℃ for 30?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 200?μM substrate. Include a substrate blank containing 50?μL of assay buffer and 50?μL of 200?μM substrate. Then read at excitiation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant human antithrombin III significantly decreased thrombin activity. The inhibition IC50 was <8 nM.