The effect of Batroxase on coagulation was evaluated using human plasma (200 μL) incubated with different concentrations of the metalloproteinase (0.1, 0.2, 0.4, 0.8, 1.6 and 2.0 μg/25 μL) at 37 °C. As a control, human plasma (200 μL) was added to 25 μL of CaCl2 BIRB 796 nmr at 0.25 mM, which induced clot formation within 3 min (Selistre et al., 1990). The minimum coagulant dose (MCD) was calculated as the minimum amount of protein that was able to induce plasma clotting in 60 seconds. The fibrinolytic activity was assessed

in Petri plates containing fibrin according to Leitão et al. (2000). Aliquots of 30 μL containing different concentrations of Batroxase (0.5, 1.0, 4.0, 6.0, 8.0, 10, 20 and 40 μg) were added to cavities on the fibrin gel and incubated at 37 °C for 24 h. The fibrinolytic activity was evaluated visually and quantified according to the halo diameter, which was compared to a positive control (plasmin 10 μg) and a negative control (PBS only). The ability Selleck Nutlin3a of Batroxase to digest fibrinogen was

evaluated using the method published by Edgar and Prentice (1973), with some modifications. A 25 μl aliquot of fibrinogen solution (2.0 mg/mL in 25 mM Tris–HCl pH 7.4) was incubated with several concentrations of Batroxase (0.25, 0.5, 1, 2, 6, 8 and 10 μg in 5 μL 25 mM Tris–HCl pH 7.4) at 37 °C for 90 min. The reaction was stopped with 20 μL of 50 mM Tris–HCl pH 6.8 containing 10% glycerol (v/v), 4% SDS (w/v), 0.05% bromophenol blue (v/v) and 4% β-mercaptoethanol (v/v), followed by heating at 100 °C for 5 min. After reduction and denaturation, the samples were assayed for fibrinogen hydrolysis by 13.5% SDS-PAGE. The fibrinogen digestion kinetics were evaluated by incubating a fixed concentration of Batroxase with fibrinogen for different time intervals (0, 5, 10, 15, 30, 60 and 120 min) at 37 °C. The fibrinogenolytic activity was also tested under different pH values (2.5; 3.0; 4.0; 5.0; 6.0; 7.0; 9.0

and 10.0) and temperature conditions (−80, Amylase −20, 5, 37, 50 and 100 °C). Protease inhibitors (EDTA, EGTA, PMSF) and β-mercaptoethanol were assayed for inhibition of fibrinogen hidrolysis by Batroxase. The GE Life Sciences molecular mass standards were used. Type IV collagen solution (4 μg/μL) was prepared in 10 mM Tris–HCl pH 7.4 containing 10 mM NaCl and incubated with different concentrations of Batroxase. The reaction was stopped by adding 20 μL of 50 mM Tris–HCl pH 6.8 containing 10% glycerol (v/v), 4% SDS (w/v), 0.05% bromophenol blue (v/v) and 4% β-mercaptoethanol (v/v), followed by heating at 100 °C for 5 min. The substrate digestion was analyzed by 7.5% SDS-PAGE. Fibronectin (4 μg/μL) in 10 mM Tris–HCl pH 7.4 and 10 mM NaCl was incubated with Batroxase at a molar ratio of 1:50 enzyme:substrate at 37 °C for 2, 6, 12 and 24 h. The hydrolysis was interrupted by adding 20 μL of 50 mM Tris–HCl pH 6.