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

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• 作者：Ashis K. Mukherjee^a ; ¹ ; ^{akm@tezu.ernet.in} ; Sudhir K. Rai^a ; ² ; Rupamoni Thakur^a ; Pronobesh Chattopadhyay^b ; Santosh K. Kar^c
• 关键词：Bacillus protease ; Thrombolytic activity ; Fibrinolytic enzyme ; Anticoagulant protease ; Plasmin-like enzyme ; Peptide mass fingerprinting
• 刊名：Biochimie
• 出版年：2012
• 期刊代码：24_03009084
• 类别：bio
• 出版时间：June, 2012
• 卷：94
• 期：6
• 页码：1300-1308
• 文件大小：470 K

摘要

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聽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 K_m and V_max values of Bafibrinase towards fibrin were determined as 0.24聽渭M and 2.8聽渭mol/min, respectively. It showed a K_m value of 0.139聽mM towards a chromogenic substrate for plasmin (d-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37聽掳C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an 伪,尾-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2聽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聽vitro thrombus. The in聽vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.