Fig. 2: Inhibition of thrombin by DTIs.
From: Efficacy and safety of next-generation tick transcriptome-derived direct thrombin inhibitors
a Amidolytic activity of thrombin (0.8 nM) on chromogenic substrate (S2238, 100 µM) in the presence of various concentrations of inhibitors were monitored as an increase in absorbance over time. A representative progression curve of thrombin inhibition by DAA34688.1 repeat 1 is depicted. All experiments were repeated independently as indicated below with similar results. The linear progression curves are characteristic of fast-binding inhibitors. b Residual thrombin amidolytic activity in the presence of various concentrations of various peptides were fitted to a kinetic equation describing tight-binding inhibitors to estimate apparent Ki (\({K}_{{{{{{\mathrm{i}}}}}}}^{{\prime} }\)), n = 3. c Ultravariegin showed a linear increase of \({K}_{{{{{{\mathrm{i}}}}}}}^{{\prime} }\) with increasing concentrations of substrate [100 µM (n = 7); 150 µM (n = 6); 200 µM (n = 6); 300 µM (n = 5); 400 µM (n = 6)], indicating competitive inhibition. The Ki were calculated to be 4.0 ± 0.5 pM. d Despite cleavage by thrombin, the cleaved peptide C-terminus to scissile bond for ultravariegin (UV011, coloured salmon) retained strong inhibition against thrombin amidolytic activity with IC50 = 1.66 ± 0.76 nM (n = 3). In contrast, the cleaved peptide C-terminus to scissile bond for bivalirudin (BV001, coloured olive) does not inhibit thrombin but instead paradoxically activates thrombin amidolytic activity by around 20% at high concentrations (1–100 µM, n = 4). e Inhibition of the amidolytic activity of various serine proteases by ultravariegin (n = 3), note the difference in peptide concentrations tested against thrombin compared to other serine proteases. All data are mean ± standard deviation (SD), n is number of independent experiments.
