Active Tissue Factor Pathway Inhibitor 2 (TFPI2)

Tissue Factor Pathway Inhibitor 2 (TFPI2) takes part in the regulation of plasmin-mediated matrix remodeling. Inhibits trypsin, plasmin, factor VIIa/tissue factor and weakly factor Xa. TFPI2 does not have any influence on thrombin. TFPI2 also can inhibit MMP activity, which can hydrolyze gelatin under certain conditions. Thus, the activity of TFPI2 can be measured by inhibit MMP-2 hydrolyze gelatin. Gelatin zymography is mainly used for the detection of the gelatinases, 2μg/mL was denatured by SDS loading buffer, electrophoresed through sodium dodecylsulphate–polyacrylamide gel (SDS–PAGE; 8%gels) containing gelatin (1mg/mL) with nonreducing conditions. After renaturation, incubate with various concentrations of recombinant human TFPI2, then staining with coomassie brilliant blue G250, active MMP-2 would hydrolyze gelatin nearby, which was indicated by the white binds on the gel; if the activity of MMP-2 inhibit by TFPI2, there was none white binds on the gel. The result was shown in figure 1.
As the figure1 shown, MMP-2 can be inhibited by recombinant human TFPI2 at least 5μg/mL.

The activity of recombinant human TFPI2 was also 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 human TFPI2 (MW: 25.89 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 2 and it was obvious that recombinant human TFPI2 significantly decreased trypsin activity. The inhibition IC50 was <60 nM.