title: Functional characterization of a slow and tight-binding inhibitor of plasmin isolated from Russell's viper venom.
abstract: BACKGROUND
  Snake venoms are rich in Kunitz-type protease inhibitors that may have therapeutic applications. However, apart from trypsin or chymotrypsin inhibition, the functions of most of these inhibitors have not been elucidated. A detailed functional characterization of these inhibitors may lead to valuable drug candidates.
  
  
  METHODS
  A Kunitz-type protease inhibitor, named DrKIn-II, was tested for its ability to inhibit plasmin using various approaches such as far western blotting, kinetic analyses, fibrin plate assay and euglobulin clot lysis assay. In addition, the antifibrinolytic activity of DrKIn-II was demonstrated in vivo.
  
  
  RESULTS
  DrKIn-II potently decreased the amidolytic activity of plasmin in a dose-dependent manner, with a global inhibition constant of 0.2nM. Inhibition kinetics demonstrated that the initial binding of DrKIn-II causes the enzyme to isomerize, leading to the formation of a much tighter enzyme-inhibitor complex. DrKIn-II also demonstrated antifibrinolytic activity in fibrin plate assay and significantly prolonged the lysis of the euglobulin clot. Screening of DrKIn-II against a panel of serine proteases indicated that plasmin is the preferential target of DrKIn-II. Furthermore, DrKIn-II treatment prevented the increase of FDP in coagulation-stimulated mice and significantly reduced the bleeding time in a murine tail bleeding model.
  
  
  CONCLUSIONS
  DrKIn-II is a potent, slow and tight-binding plasmin inhibitor that demonstrates antifibrinolytic activity both in vitro and in vivo.
  
  
  CONCLUSIONS
  This is the first in-depth functional characterization of a plasmin inhibitor from a viperid snake. The potent antifibrinolytic activity of DrKIn-II makes it a potential candidate for the development of novel antifibrinolytic agents.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan. Electronic address: adamcheng@gate.sinica.edu.tw.
authors.forename: An-Chun
authors.initials: AC
authors.lastname: Cheng
authors.forename: Inn-Ho
authors.initials: IH
authors.lastname: Tsai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000933:Antifibrinolytic Agents / Q000494:pharmacology*; D007611:Aprotinin / Q000737:chemistry; D001777:Blood Coagulation / Q000187:drug effects*; D026481:Blotting, Far-Western; D004546:Elapid Venoms / Q000737:chemistry; D005337:Fibrin / Q000378:metabolism; D063285:Fibrin Clot Lysis Time; D005341:Fibrinolysin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology*; D011517:Prothrombin Time; D017840:Daboia; D017386:Sequence Homology, Amino Acid; D014757:Viper Venoms / Q000494:pharmacology*
chemical_list: D000933:Antifibrinolytic Agents; D004546:Elapid Venoms; D011480:Protease Inhibitors; D014757:Viper Venoms; C413544:textilinin; D005337:Fibrin; D007611:Aprotinin; D005341:Fibrinolysin
keywords: APTT; Antifibrinolytic agent; BPTI; Daboia russelii Kunitz Inhibitor-II; DrKIn-II; FDP; HPLC; Kunitz-type protease inhibitor; PT; Plasmin; RVV; Russell's viper venom; Tight-binding inhibition; Time-dependent inhibition; activated partial thromboplastin time; bovine pancreatic trypsin inhibitor; fibrin/fibrinogen degradation product; high performance liquid chromatography; prothrombin time; tPA; tissue plasminogen activator; uPA; urokinase plasminogen activator