title: Purification and characterization of a fibrinolytic enzyme from Petasites japonicus.
abstract: A new, direct-acting chymotrypsin-like fibrinolytic serine protease was purified from Petasites japonicus, a medicinal herb. The molecular mass of the discovered enzyme was estimated to be 40.0 kDa as determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, fibrin zymography, and gel filtration chromatography. The N-terminal sequence of the purified enzyme was determined to be GQEDHFLQVSLTSA. The proteolytic activity of the enzyme was found to be inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(amidinophenyl) methanesulfonyl fluoride. An assay of enzyme activity on fibrin plates revealed that it could hydrolyze the fibrin directly. The enzyme displayed a potent fibrin(ogen)olytic activity, hydrolyzing the Aα-, α-, and Bβ-subunits of the human fibrinogen. The enzyme prolonged activated partial thromboplastin time and had little effect on prothrombin time. It prevented carrageenan-induced thrombus formation in mouse tails and did not increase the bleeding time. Our findings indicate that the extracted enzyme we present here has the potential for clinical use as an agent for the treatment of thrombosis.
authors.forename: Dae-Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Jun-Hui
authors.initials: JH
authors.lastname: Choi
authors.forename: Se-Eun
authors.initials: SE
authors.lastname: Park
authors.forename: Seung
authors.initials: S
authors.lastname: Kim
authors.forename: Kumar
authors.initials: K
authors.lastname: Sapkota
authors.forename: Sung-Jun
authors.initials: SJ
authors.lastname: Kim
mesh_terms: D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D008297:Male; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D036901:Petasites / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification*; D013927:Thrombosis / Q000473:pathology
chemical_list: D000577:Amides; D000925:Anticoagulants; D000991:Antithrombins; D005340:Fibrinogen; D057057:Serine Proteases