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Thromb Haemost 2012; 108(03): 570-578
DOI: 10.1160/TH12-04-0235
New Technologies, Diagnostic Tools and Drugs
Schattauer GmbH

Functional characterisation of Vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi

Daniella Gorete Lourenço Oliveira
1   Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil
,
Miryam Paola Alvarez-Flores
1   Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil
2   Center of Applied Toxinology (CAT/CEPID), Butantan Institute, São Paulo, São Paulo, Brazil
,
Adriana Rios Lopes
1   Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil
,
Ana Marisa Chudzinski-Tavassi
1   Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil
2   Center of Applied Toxinology (CAT/CEPID), Butantan Institute, São Paulo, São Paulo, Brazil
› Author Affiliations Financial support: This study was supported in part by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 07/54257–1 and FAPESP 98/14307–9) and by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/INCT-TOX) and CAT/CEPID-FAPESP.
Further Information

Publication History

Received: 13 April 2012

Accepted after minor revision: 17 May 2012

Publication Date:
25 November 2017 (online)

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Summary

The strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Bloodsucking animals have in their salivary glands mixtures of anticoagulants, which could be used for designing novel antithrombotic compounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi. Vizottin was purified by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (Ki 2 nM) like FXa inhibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the prothrombinase complex and Gla-domainless FXa. Moreover, vizottin did not interfere with FX activation induced by RVV-X, a known enzyme that requires the Gla-domain of FX for activation. Competition experiments in the presence of FXa and GGACK-FXa (active site blocked) demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. This novel inhibitor appears to exert its inhibitory effects through direct binding to the active site of FXa in a time-dependent manner, but not involving a tight-binding model. In this context, vizottin is a promising model for designing novel anticoagulants for the treatment of thrombotic diseases.

Keywords

Leech salivary gland - Haementeria - factor Xa inhibitor - factor Xa active site - extrinsic tenase complex
 

  • References

  • 1 Weitz JI, Linkins LA. Beyond heparin and warfarin: the new generation of anticoagulants. Expert Opin Investig Drugs 2007; 16: 271-282.
    CrossrefPubMedSearch in Google Scholar
  • 2 Samama MM, Gerotziafas GT. Newer anticoagulants in 2009. J Thromb Thrombolysis 2010; 29: 92-104.
    CrossrefPubMedSearch in Google Scholar
  • 3 Galanis T, Thomson L, Palladino M. et al. New oral anticoagulants. J Thromb Thrombolysis 2011; 31: 310-320.
    CrossrefPubMedSearch in Google Scholar
  • 4 Weitz JI. Factor Xa and thrombin as targets for new oral anticoagulants. Thromb Res 2011; 127 (Suppl. 02) S5-S12.
    PubMedSearch in Google Scholar
  • 5 Koh CY, Kini RM. Anticoagulants from hematophagous animals. Expert Rev Hematol 2008; 01: 135-139.
    CrossrefPubMedSearch in Google Scholar
  • 6 Ledizet M, Harrison LM, Koskia RA. et al. Discovery and pre-clinical development of antithrombotics from hematophagous invertebrates. Curr Med Chem Cardiovasc Hematol Agents 2005; 03: 1-10.
    CrossrefPubMedSearch in Google Scholar
  • 7 Koh CY, Kini RM. Molecular diversity of anticoagulants from haematophagous animals. Thromb Haemost 2009; 102: 437-453.
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Walsmann P. Isolation and characterization of hirudin from Hirudo medicinalis. Semin Thromb Hemost 1991; 17: 83-87.
    Thieme ConnectPubMedSearch in Google Scholar
  • 9 Waxman L, Smith DE, Arcuri KE. et al. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 1990; 248: 593-596.
    CrossrefPubMedSearch in Google Scholar
  • 10 Vlasuk GP. Structural and functional characterization of tick anticoagulant peptide (TAP): a potent and selective inhibitor of blood coagulation factor Xa. Thromb Haemost 1993; 70: 212-216.
    Thieme ConnectPubMedSearch in Google Scholar
  • 11 Lee AY, Vlasuk GP. Recombinant nematode anticoagulant protein c2 and other inhibitors targeting blood coagulation factor VIIa/tissue factor. J Intern Med 2003; 254: 313-321.
    CrossrefPubMedSearch in Google Scholar
  • 12 Jackson CM. Factor X. Prog Hemost Thromb 1984; 07: 55-109.
    PubMedSearch in Google Scholar
  • 13 Mann KG, Nesheim ME, Church WR. et al. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
    PubMedSearch in Google Scholar
  • 14 Morita T. Proteases which activate factor X. In Enzymes from snake venoms. Fort Collins, CO: Alaken Inc; 1998: 179-208.
    Search in Google Scholar
  • 15 Kalafatis M, Egan JO, van 'tVeer C. et al. The regulation of clotting factors. Crit Rev Eukaryot Gene Expr 1997; 07: 241-280.
    CrossrefPubMedSearch in Google Scholar
  • 16 Mann KG, Kalafatis M. Factor V: a combination of Dr Jekyll and Mr Hyde. Blood 2003; 101: 20-30.
    CrossrefPubMedSearch in Google Scholar
  • 17 Leadley Jr. RJ. Coagulation factor Xa inhibition: biological background and rationale. Curr Top Med Chem 2001; 01: 151-159.
    CrossrefPubMedSearch in Google Scholar
  • 18 Porshinsky BS, Saha S, Grossman MD. et al. Clinical uses of the medicinal leech: a practical review. J Postgrad Med 2011; 57: 65-71.
    CrossrefPubMedSearch in Google Scholar
  • 19 Nutt E, Gasic T, Rodkey J. et al. The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure. J Biol Chem 1988; 263: 10162-10167.
    PubMedSearch in Google Scholar
  • 20 Condra C, Nutt E, Petroski CJ. et al. Isolation and structural characterization of a potent inhibitor of coagulation factor Xa from the leech Haementeria ghilianii. Thromb Haemost 1989; 61: 437-441.
    Thieme ConnectPubMedSearch in Google Scholar
  • 21 Faria F, Kelen EM, Sampaio CA. et al. A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech. Thromb Haemost 1999; 82: 1469-1473.
    Thieme ConnectPubMedSearch in Google Scholar
  • 22 Chopin V, Salzet M, Baert J. et al. Therostasin, a novel clotting factor Xa inhibitor from the rhynchobdellid leech, Theromyzon tessulatum. J Biol Chem 2000; 275: 32701-32707.
    CrossrefPubMedSearch in Google Scholar
  • 23 Eriksson BI, Turpie AG, Lassen MR. et al. Prevention of venous thromboembolism with an oral factor Xa inhibitor, YM150, after total hip arthroplasty. A dose finding study (ONYX-2). J Thromb Haemost 2010; 08: 714-721.
    CrossrefPubMedSearch in Google Scholar
  • 24 Lee YK, Player MR. Developments in factor Xa inhibitors for the treatment of thromboembolic disorders. Med Res Rev 2011; 31: 202-283.
    CrossrefPubMedSearch in Google Scholar
  • 25 Barenholz Y, Gibbes D, Litman BJ. et al. A simple method for the preparation of homogeneous phospholipid vesicles. Biochemistry 1977; 16: 2806-2810.
    CrossrefPubMedSearch in Google Scholar
  • 26 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685.
    CrossrefPubMedSearch in Google Scholar
  • 27 Gao X, Shi L, Zhou Y. et al. Characterization of the anticoagulant protein Rhipilin-1 from the Rhipicephalus haemaphysaloides tick. J Insect Physiol 2011; 57: 339-343.
    CrossrefPubMedSearch in Google Scholar
  • 28 Francischetti IM, Valenzuela JG, Andersen JF. et al. Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex. Blood 2002; 99: 3602-3612.
    CrossrefPubMedSearch in Google Scholar
  • 29 John FM. The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions. Trends in Biochemical Sciences 1982; 07: 102-105.
    CrossrefPubMedSearch in Google Scholar
  • 30 Segel IH. editor Enzyme kinetics. New York: 1975
  • 31 Purich DL. Enzyme kinetics: catalysis & control : a reference of theory and best-practice methods. Elsevier; 2010
  • 32 Alvarez-Flores MP, Furlin D, Ramos OH. et al. Losac, the first hemolin that exhibits procogulant activity through selective factor X proteolytic activation. J Biol Chem 2011; 286: 6918-6928.
    CrossrefPubMedSearch in Google Scholar
  • 33 Takeya H, Nishida S, Miyata T. et al. Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains. J Biol Chem 1992; 267: 14109-14117.
    Search in Google Scholar
  • 34 Takeda S, Igarashi T, Mori H. Crystal structure of RVV-X: an example of evolutionary gain of specificity by ADAM proteinases. FEBS Lett 2007; 581: 5859-5864.
    CrossrefPubMedSearch in Google Scholar
  • 35 Weitz JI. Emerging anticoagulants for the treatment of venous thromboembolism. Thromb Haemost 2006; 96: 274-284.
    Thieme ConnectPubMedSearch in Google Scholar
  • 36 Batista IF, Ramos OH, Ventura JS. et al. A new Factor Xa inhibitor from Amblyomma cajennense with a unique domain composition. Arch Biochem Biophys 2010; 493: 151-156.
    CrossrefPubMedSearch in Google Scholar
  • 37 Stassens P, Bergum PW, Gansemans Y. et al. Anticoagulant repertoire of the hookworm Ancylostoma caninum. Proc Natl Acad Sci USA 1996; 93: 2149-2154.
    CrossrefPubMedSearch in Google Scholar
  • 38 Joo SS, Won TJ, Kim JS. et al. Inhibition of coagulation activation and inflammation by a novel Factor Xa inhibitor synthesized from the earthworm Eisenia andrei. Biol Pharm Bull 2009; 32: 253-258.
    CrossrefPubMedSearch in Google Scholar
  • 39 Stark KR, James AA. Isolation and characterization of the gene encoding a novel factor Xa-directed anticoagulant from the yellow fever mosquito, Aedes aegypti. J Biol Chem 1998; 273: 20802-20809.
    CrossrefPubMedSearch in Google Scholar
  • 40 Dunwiddie C, Thornberry NA, Bull HG. et al. Antistasin, a leech-derived inhibitor of factor Xa. Kinetic analysis of enzyme inhibition and identification of the reactive site. J Biol Chem 1989; 264: 16694-16699.
    PubMedSearch in Google Scholar
  • 41 Bauer KA. New anticoagulants: anti IIa vs anti Xa--is one better?. J Thromb Thrombolysis 2006; 21: 67-72.
    CrossrefPubMedSearch in Google Scholar
  • 42 Crawley JT, Lane DA. The haemostatic role of tissue factor pathway inhibitor. Arterioscler Thromb Vasc Biol 2008; 28: 233-242.
    PubMedSearch in Google Scholar
  • 43 Monteiro RQ, Rezaie AR, Bae JS. et al. Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite. Protein Sci 2008; 17: 146-153.
    PubMedSearch in Google Scholar
  • 44 Huang ZF, Wun TC, Broze Jr. GJ. Kinetics of factor Xa inhibition by tissue factor pathway inhibitor. J Biol Chem 1993; 268: 26950-26955.
    PubMedSearch in Google Scholar