N p tosyl gly pro arg p nitroanilide acetate salt

Different concentrations of each recombinant protein were incubated with three different serine proteases in Tris buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% BSA; pH 7.4): factor Xa (FXa) (5 × 10⁻⁵ U), thrombin (2.5 × 10⁻³ U), or trypsin (9 U) (Sigma-Aldrich®) at 37°C for 15 min in 0.5 ml microtubes. Then, the tube contents were placed in 96-well plates and the enzymatic reactions were triggered in the presence of the specific substrates CH₃OCO-D-CHA-GlyArg-pNA-AcOH (Sigma-Aldrich®) for FXa, Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma-Aldrich®) for trypsin, and N-(p-tosyl)-Gly-Pro-Arg p-nitroanilide acetate salt (Sigma-Aldrich®) for thrombin at 4 mM concentration and 100 µl final volume. Immediately after the addition of the substrate, the absorbance variation was assessed at 405 nm every 10 s with 2 s agitation between reads in a microplate reader (VersaMax™, Molecular Devices) for 30 min. The maximum reaction velocity (Vmax) values were obtained for reactions lacking inhibitors and were used as a reference for reactions in the presence of different concentrations of inhibitors.

Thrombin (EC 3.4.21.5) from bovine plasma, fibrinogen from bovine plasma, N-(p-Tosyl)-Gly-Pro-Arg-p-nitroanilide acetate salt, ethylene glycol tetraacetic acid (EGTA), hemoglobin from bovine blood, bovine serum albumin (BSA), venom from the snake Oxyuranus scutellatus, and heparin-agarose were purchased from Sigma Chemical Co. Cephalit Kit for APTT, and Kit for PT were bought from bioMẻrieux. All other chemicals were of analytical grade.