Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency

• Published in: Biochimie 2012-06, Vol.94 (6), p.1300-1308
• Main Authors: Mukherjee, Ashis K., Rai, Sudhir K., Thakur, Rupamoni, Chattopadhyay, Pronobesh, Kar, Santosh K.
• Format: Article
• Language: English
• Subjects: Animals; Anticoagulant protease; Anticoagulants - pharmacology; Bacillus - enzymology; Bacillus protease; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacterial Proteins - pharmacology; Fibrin - metabolism; Fibrinolysis - drug effects; Fibrinolytic Agents - metabolism; Fibrinolytic Agents - pharmacology; Fibrinolytic enzyme; Humans; Mice; Peptide mass fingerprinting; Plasmin-like enzyme; Serine Endopeptidases - metabolism; Serine Proteases - isolation & purification; Serine Proteases - metabolism; Serine Proteases - pharmacology; Thrombolytic activity; Thrombolytic Therapy
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2012.02.027

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent Km and Vmax values of Bafibrinase towards fibrin were determined as 0.24 μM and 2.8 μmol/min, respectively. It showed a Km value of 0.139 mM towards a chromogenic substrate for plasmin (d-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,β-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases. ► A 32.3 kDa fibrinolytic serine protease was purified from Bacillus sp. AS-S20-I. ► Novelty of the purified protease was deduced from its peptide mass fingerprinting. ► Protease showed better thrombolytic and anticoagulant properties than human plasmin. ► Protease was found as non-toxic and non-hemorrhagic in in vitro and in animal model. ► Our study suggested application of this protease as a better thrombolytic drug.