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Thromb Haemost 1999; 82(05): 1469-1473
DOI: 10.1055/s-0037-1614857
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A New Factor Xa Inhibitor (Lefaxin) from the Haementeria depressa Leech

Fernanda Faria
1   From Laboratorio de Fisiopatologia, Instituto Butantan; São-Paulo, Brasil
,
Eva Maria A. Kelen
1   From Laboratorio de Fisiopatologia, Instituto Butantan; São-Paulo, Brasil
,
Claudio A. M. Sampaio
2   Departamento de Bioquímica e Biologia Molecular, Universidade Federal de São Paulo (UNIFESP-EPM) São Paulo, Brasil
,
Cassian Bon
3   Unité des Venins, Institut Pasteur, Paris, France
,
Nathalie Duval   *
3   Unité des Venins, Institut Pasteur, Paris, France
,
Ana Marisa Chudzinski-Tavassi
1   From Laboratorio de Fisiopatologia, Instituto Butantan; São-Paulo, Brasil
› Author Affiliations
Further Information

Publication History

Received 15 December 1998

Accepted after resubmission 07 June 1999

Publication Date:
09 December 2017 (online)

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Summary

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent KI of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the tick Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.

Keywords

Leeches - FXa inhibitor - Haementeria depressa

* Present address: Dr. N. Duval, Unité de Neurovirologie et Régénération du Système Nerveux, Institut Pasteur, Paris, France


 

  • References

  • 1 Bagdy D, Barabas E, Graf L, Petersen TE, Magnusson S. Naturally occurring protease inhibitors. Methods Enzymol 1976; 45: 669-78.
    PubMedGoogle Scholar
  • 2 Maliconico SM, Katz JB, Budzynski AZ. Fibrinogen degradation by hementin, a fibrinogenolytic anticoagulant from the salivary glands of the leech H. ghilianii . J Lab Clin Med 1984; 104: 842-54.
    PubMedGoogle Scholar
  • 3 Kelen EMA, Rosenfeld G. Fibrinogenolytic substance (Hementerin) of Brazilian blood sucking leeches (Haementeria lutzi Pinto 1920). Haemostas 1975; 4: 51-64.
    PubMedGoogle Scholar
  • 4 Tuszynski GP, Gasic TB, Gasic GJ. Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation. J Biol Chem 1987; 262: 9718-23.
    PubMedGoogle Scholar
  • 5 Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 1990; 248: 593-6.
    CrossrefPubMedGoogle Scholar
  • 6 Apitz-Castro R, Bèguin S, Tablante A, Bartoli F, Holt JC, Hemker HC. Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus) . Thromb Haemost 1995; 73: 94-100.
    PubMedGoogle Scholar
  • 7 Markwardt F. Hirudin as an inhibitor of thrombin. In: Methods in Enzymology. Perlmann GE, Lorand L. eds. New York: Academic Press; 1970. 19 924-32.
    Google Scholar
  • 8 Seymour JL, Henzel WJ, Nevins B, Stults JT, Lazarus A. Decorsin, a potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdela decora . J Biol Chem 1990; 265: 10143-7.
    PubMedGoogle Scholar
  • 9 Marzur P, Henzel WJ, Seymour JL, Lazarus RA. Ornatins: potent glycoprotein IIb-IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella ornata . Eur J Biochem 1991; 202: 1073-82.
    CrossrefPubMedGoogle Scholar
  • 10 Rigbi M, Levy H, Iraqui F, Teitelbaum M, Orevi M, Alajoutsijarvi A, Galum R. The saliva of the medicinal leech Hirudo medicinalis. Biochemical characterization of the high molecular weight fraction. Comp Biochem Physiol [B] 1987; 87: 567-73.
    PubMedGoogle Scholar
  • 11 Sellner C, Mentele R, Eckerskorn C, Fritz H, Sommerhoff CP. Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis . Eur J Biochem 1994; 219: 937-43.
    CrossrefPubMedGoogle Scholar
  • 12 Lyle EM, Fujita T, Conner MW, Connolly TM, Vlasuk GP, Lynch JL. Effect of inhibitors of factor Xa or platelet adhesion, heparin, and aspirin on platelet deposition in an atherosclerotic rabbit model of angioplasty injury. J Pharmacol Toxicol Methods 1995; 33: 53-61.
    CrossrefPubMedGoogle Scholar
  • 13 Deckmyn H, Stassen JM, Vreys I, Van Houte E, Sawyer RT, Vermylen J. Calin from Hirudo medicinalis, an inhibitor platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters. Blood 1995; 85: 712-9.
    PubMedGoogle Scholar
  • 14 Dunwiddie C, Thromberry NA, Bull HG, Sardana M, Friedman PA, Jacobs JW, Simpson E. Antistasin, a leech-derived inhibitor of factor Xa. Kinetic analysis of enzyme inhibition and identification of the reactive site. J Biol Chem 1988; 264: 16694-9.
    PubMedGoogle Scholar
  • 15 Budzynski AZ, Olex SA, Sawyer RT. Composition of salivary glands extracts from the leech Haementeria ghilianii . Proc Soc Exp Biol Med 1981; 168: 259-65.
    CrossrefPubMedGoogle Scholar
  • 16 Chudzinski-Tavassi AM, Kelen EMA, Paula Rosa AP, Loyau S, Sampaio CAM, Bon C, Anglés-Cano E. Fibrino(geno)lytic properties of a purified hementerin, metalloproteinase from the leech Haementeria depressa . Thromb Haemost 1998; 80: 155-60.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Stassens P, Bergum PW, Gansemans Y, Jespers L, Laroche Y, Huang S, Maki S, Messens J, Lauwereys M, Cappello M, Hotez PJ, Vlasuk GP. Anti-coagulant repertoire of the hookworm Ancilostoma caninum . Proc Natl Acad Sci USA 1996; 93: 2149-54.
    CrossrefPubMedGoogle Scholar
  • 18 Champagne DE, Nussenzveig RH, Ribeiro JMC. Purification, partial characterization, and cloning of nitric oxide-carrying heme proteins (Nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus . J Biol Chem 1995; 270: 8691-5.
    CrossrefPubMedGoogle Scholar
  • 19 Ribeiro JMC, Schneider M, Guimaraes JA. Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus . Biochem J 1995; 308: 243-9.
    CrossrefPubMedGoogle Scholar
  • 20 Sun J, Yamaguchi M, Yuda M, Miura K, Takeya H, Hirai M, Matsuoka H, Ando K, Watanabe T, Suzuki K, Chinzei Y. Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin S), from the salivary glands of the blood sucking bug, Rhodnius prolixus . Thromb Haemost 1996; 75: 573-7.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Laskowski M, Kato I. Protein Inhibitors of Proteinases. Annu Rev Biochem 1980; 49: 593-626.
    CrossrefPubMedGoogle Scholar
  • 22 Green NM, Work E. Pancreatic trypsin inhibitor. Biochem J 1953; 54: 349-52.
    PubMedGoogle Scholar
  • 23 Nagahara T, Yokoyama Y, Inamura K, Katakura S, Komoriya S, Yamaguchi H, Hara T, Iwamoto M. Dibasic (amidinoaryl) propanoic acid derivatives as novel blood coagulation factor Xa inhibitors. J Med Chem 1994; 37: 1200-7.
    CrossrefPubMedGoogle Scholar
  • 24 Markwell MAK, Hass SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Bioche 1978; 87: 206-10.
    PubMedGoogle Scholar
  • 25 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedGoogle Scholar
  • 26 Morrissey JH. Silver stain for proteins in polyacrylamide gels, a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117: 311-4.
    CrossrefPubMedGoogle Scholar
  • 27 Weber K, Osborne M. The reliability of molecular weight determination by sodium dodecyl sulfate polyacrylamide gels electrophoresis. J Biol Chem 1968; 244: 4406-12.
    PubMedGoogle Scholar
  • 28 Morrisson JF. Kinectis of the reversible inhibition of enzyme-catalyzed reactions by tight binding inhibitors. Biochim Biophys Acta 1982; 185: 269-86.
    PubMedGoogle Scholar
  • 29 Nutt E, Gasic T, Rodkey J, Gasic GJ, Jacobs JW, Friedman PA, Simpson E. The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure. J Biol Chem 1988; 263: 10162-7.
    PubMedGoogle Scholar
  • 30 Neeper MP, Waxman L, Smith DC, Schulman CA, Sardana M, Ellis RW, Schaffer L, Siegl PK, Vlasuk GP. Characterization of recombinant tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa. J Biol Chem 1990; 265: 17796-52.
    PubMedGoogle Scholar
  • 31 Takagi T, Cox JA. Primary structure of myohemerythrin from the annelid Nereis diversicolor . FEBS Lett 1991; 285: 25-7.
    CrossrefPubMedGoogle Scholar