title: A novel fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (Barnett´s pitviper) snake venom with anti-platelet properties.
abstract: BACKGROUND
  Viperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential.
  
  
  METHODS
  Bar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function.
  
  
  RESULTS
  Bar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC50=1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation.
  
  
  CONCLUSIONS
  A non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function.
  
  
  CONCLUSIONS
  This study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.
authors.affiliation: Research and Development Center, Ezequiel Dias Foundation, 30510-010, Belo Horizonte, MG, Brazil; Faculty of Biological Sciences, Nacional University of San Marcos, Lima-Peru. Electronic address: eladiooswaldo@gmail.com.
authors.forename: Eladio Flores
authors.initials: EF
authors.lastname: Sanchez
authors.affiliation: Research and Development Center, Ezequiel Dias Foundation, 30510-010, Belo Horizonte, MG, Brazil.
authors.forename: Michael
authors.initials: M
authors.lastname: Richardson
authors.affiliation: Department of Cell Biology, Federal University of Parana, Brazil.
authors.forename: Luiza Helena
authors.initials: LH
authors.lastname: Gremski
authors.affiliation: Department of Cell Biology, Federal University of Parana, Brazil.
authors.forename: Silvio Sanches
authors.initials: SS
authors.lastname: Veiga
authors.affiliation: Faculty of Biological Sciences, Nacional University of San Marcos, Lima-Peru.
authors.forename: Armando
authors.initials: A
authors.lastname: Yarleque
authors.affiliation: Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Niland
authors.affiliation: Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany.
authors.forename: Augusto Martins
authors.initials: AM
authors.lastname: Lima
authors.affiliation: Research and Development Center, Ezequiel Dias Foundation, 30510-010, Belo Horizonte, MG, Brazil; Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany.
authors.forename: Maria Inácia
authors.initials: MI
authors.lastname: Estevao-Costa
authors.affiliation: Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany.
authors.forename: Johannes Andreas
authors.initials: JA
authors.lastname: Eble
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000201:enzymology*; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology*; D038982:Integrin alpha2beta1 / Q000378:metabolism; D045726:Metalloproteases / Q000302:isolation & purification / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
chemical_list: D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D038982:Integrin alpha2beta1; D010975:Platelet Aggregation Inhibitors; D045726:Metalloproteases
keywords: Antithrombotic; Direct acting fibrinolytic enzyme; Integrins; Metalloproteinase; Snake venoms; von willebrand factor