Majumdar Sourav, Dutta Sumita, Das Tanusree, Chattopadhyay Pronobesh, Mukherjee Ashis K
Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences, Tezpur University, Tezpur 784028, Assam, India.
Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India.
Int J Biol Macromol. 2015 Aug;79:477-89. doi: 10.1016/j.ijbiomac.2015.04.075. Epub 2015 May 8.
Fibrin(ogen)olytic enzymes offer great promise for the treatment of thrombosis associated disorders. The present study describes the characterization of an extracellular fibrin(ogen)olytic serine protease (named Bacethrombase) purified from the Bacillus cereus strain FF01. The molecular mass of the Bacethrombase was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectroscopy analyses at 39.5 kDa and 38,450.51 Da, respectively. The peptide mass fingerprinting and analyses of the composition of the amino acids revealed the similarity of the Bacethrombase to the bacterial serine proteases. The secondary structure of the Bacethrombase was composed of 14% helix, 6.6% beta-sheet, and 79.4% random coil. Bacethrombase was found to contain 48% sialic acid and it preferentially degraded the Aα-chain of fibrinogen, as well as fibrin. The anticoagulant potency of the Bacethrombase was comparable with that of warfarin and heparin, and was corroborated by its fibrinogenolytic activity rather than the inhibition of thrombin, prothrombin or FXa. Bacethrombase demonstrated antiplatelet activity, and dose-dependently inhibited the ADP-induced platelet aggregation. Bacethrombase (10 mg/kg) did not show toxicity after i.v. administration in Wistar rats; however, it revealed an in vivo anticoagulant effect and significantly inhibited the carrageenan-induced in vivo thrombus formation in rats.
纤维蛋白（原）溶解酶在治疗血栓相关疾病方面具有巨大潜力。本研究描述了从蜡样芽孢杆菌菌株FF01中纯化得到的一种细胞外纤维蛋白（原）溶解丝氨酸蛋白酶（命名为芽孢溶纤酶）的特性。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和基质辅助激光解吸/电离飞行时间质谱分析，确定芽孢溶纤酶的分子量分别为39.5 kDa和38,450.51 Da。肽质量指纹图谱和氨基酸组成分析显示芽孢溶纤酶与细菌丝氨酸蛋白酶具有相似性。芽孢溶纤酶的二级结构由14%的螺旋、6.6%的β-折叠和79.4%的无规卷曲组成。发现芽孢溶纤酶含有48%的唾液酸，它优先降解纤维蛋白原的Aα链以及纤维蛋白。芽孢溶纤酶的抗凝效力与华法林和肝素相当，并且通过其纤维蛋白原溶解活性而非对凝血酶、凝血酶原或FXa的抑制作用得到证实。芽孢溶纤酶表现出抗血小板活性，并剂量依赖性地抑制ADP诱导的血小板聚集。芽孢溶纤酶（10 mg/kg）经静脉注射给予Wistar大鼠后未显示毒性；然而，它显示出体内抗凝作用，并显著抑制角叉菜胶诱导的大鼠体内血栓形成。
