Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B

In this study, biochemical and pharmacological characterization of Brevithrombolase, a fibrinolytic serine protease purified from Brevibacillus brevis strain FF02B has been reported. An assessment of its thrombolytic potency has also been made. The molecular mass of this monomeric protease was determined as 55 kDa, and 56043 Da, respectively, by SDS-PAGE and MALDI-TOF-MS. In the analytical studies, the N-terminal sequence of Brevithrombolase was found to be blocked; however, the peptide mass fingerprinting and amino acid composition analyses demonstrated the similarity of Brevithrombolase with endopeptidases in possessing serine in their catalytic triad. This finding was confirmed by the observation that the serine protease inhibitors decrease the catalytic (fibrinolytic) activity of Brevithrombolase. The secondary structure of Brevithrombolase was composed of 30.6% alpha helix and 69.4% random coil. Brevithrombolase showed the Km and Vmax values towards the chromogenic substrate for plasmin at 0.39 mM and 14.3 μmol/min, respectively. Brevithrombolase demonstrated optimum fibrinolytic activity at pH 7.4 and 37 °C, and showed marginal hydrolytic activity towards globulin, casein and fibrinogen. The anticoagulant potency of Brevithrombolase was comparable to the low molecular mass heparin/antithrombin-III and warfarin. Among the three enzymes-Brevithrombolase, plasmin and streptokinase-the fibrinolytic activity and in vitro thrombolytic potency of Brevithrombolase was found to be superior. The RP-HPLC and SDS-PAGE analyses suggested a similar pattern of fibrin degradation by Brevithrombolase and plasmin, indicating that former enzyme is a plasmin-like fibrinolytic serine protease. Brevithrombolase did not show in vitro cytotoxicity on HT29 and HeLa cells or hemolytic activity. At a dose of 10 mg/kg, Brevithrombolase did not exhibit lethality or toxicity on Wistar strain albino rats. Brevithrombolase did not inhibit factor Xa, and its mechanism of anticoagulant action was associated with the enzymatic cleavage of thrombin. The combined properties of Brevithrombolase indicate its therapeutic potential in peptide-based cardiovascular drug development.