title: A fibrinolytic protease AfeE from Streptomyces sp. CC5, with potent thrombolytic activity in a mouse model.
abstract: Fibrinolytic proteases have potential applications in cardiovascular disease therapy. A novel fibrinolytic protease, AfeE, with strong thrombolytic activity was purified from Streptomyces sp. CC5. AfeE displayed maximum activity at 40°C in the pH range of 7.0-12.0. It was strongly inhibited by serine protease inhibitor phenylmethanesulfonylfluoride, soybean trypsin inhibitor, tosyl-l-lysine chloromethyl ketone and tosyl-l-phenylalanine chloromethyl ketone. The activity of the enzyme was partially inhibited by Cu(2+), Co(2+) and Zn(2+). AfeE exhibited higher substrate specificity for fibrin than fibrinogen, which has rarely been reported in fibrinolytic enzymes. AfeE also showed high thrombolytic activity in a carrageenan-induced mouse tail thrombosis model. AfeE prolonged prothrombin time, activated partial thromboplastin time, and thrombin time in rat blood. A bleeding time assay revealed that AfeE did not prolong bleeding time in mice at a dose of 1mg/kg. No acute cytotoxicity was observed for AfeE at 320μg/well in human umbilical vein endothelial cells. The afeE gene was cloned from the genome of Streptomyces sp. CC5. Full-length AFE-CC5E contained 434 amino acids and was processed into a mature form consisting 284 amino acids by posttranslational modification, as revealed by high-resolution mass spectrometry analysis. These results indicate that AfeE is a prospective candidate for antithrombotic drug development.
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Liu
authors.affiliation: College of Life Sciences and Technology, China Pharmaceutical University, Nanjing 210009, People's Republic of China.
authors.forename: Guangyan
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Biying
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Dong
authors.affiliation: College of Life Sciences and Technology, China Pharmaceutical University, Nanjing 210009, People's Republic of China.
authors.forename: Xingli
authors.initials: X
authors.lastname: Su
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People's Republic of China. Electronic address: czl@njau.edu.cn.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
authors.affiliation: College of Life Sciences and Technology, China Pharmaceutical University, Nanjing 210009, People's Republic of China. Electronic address: yikong668@163.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Kong
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D004195:Disease Models, Animal; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008297:Male; D008670:Metals / Q000737:chemistry; D051379:Mice; D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D013927:Thrombosis / Q000097:blood / Q000188:drug therapy
chemical_list: D005343:Fibrinolytic Agents; D007477:Ions; D008670:Metals; D010450:Endopeptidases
keywords: Fibrinolytic protease; Streptomyces sp. CC5; Thrombolytic activity