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Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands*

  • Dongying Ma
    Footnotes
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Yipeng Wang
    Footnotes
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Hailong Yang
    Footnotes
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Jing Wu
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Shu An
    Affiliations
    School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230026, China, and

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Li Gao
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Xueqing Xu
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
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  • Ren Lai
    Correspondence
    To whom correspondence should be addressed: Kunming Inst. of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. Tel.:86-871-5196202; Fax:86-871-5199086;
    Affiliations
    Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China,

    Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China,
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  • Author Footnotes
    * This work was supported by the Chinese National Natural Science Foundation (30830021), Chinese Academy of Sciences (KSCXZ-YW-R-088, KSCX2-YW-G-024), and the Ministry of Science (2008AA02Z133, 2007AA100602).
    The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
    ¶ These authors contributed equally to this paper.
Open AccessPublished:June 16, 2009DOI:https://doi.org/10.1074/mcp.M900186-MCP200
      Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their saliva has been thought to contain wide range of physiologically active molecules. In traditional Eastern medicine, horseflies are used as anti-thrombosis material for hundreds of years. By proteomics coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which exert mainly on anti-thrombosis functions, were identified and characterized from 60,000 pairs of salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I) ten fibrin(ogen)olytic enzymes, which hydrolyze specially alpha chain of fibrin(ogen) and are the first family of fibrin(ogen)olytic enzymes purified and characterized from arthropods; (II) another fibrin(ogen)olytic enzyme, which hydrolyzes both alpha and beta chain of fibrin(ogen); (III) ten Arg-Gly-Asp-motif containing proteins acting as platelet aggregation inhibitors; (IV) five thrombin inhibitor peptides; (V) three vasodilator peptides; (VI) one apyrase acting as platelet aggregation inhibitor; (VII) one peroxidase with both platelet aggregation inhibitory and vasodilator activities. The first three families are belonging to antigen five proteins, which show obvious similarity with insect allergens. They are the first members of the antigen 5 family found in salivary glands of blood sucking arthropods to have anti-thromobosis function. The current results imply a possible evolution from allergens of blood-sucking insects to anti-thrombosis agents. The extreme diversity of horsefly anti-thrombosis components also reveals the anti-thrombosis molecular mechanisms of the traditional Eastern medicine insect material.
      Antihemostatic compounds of blood-sucking arthropods have been distinguished into several groups such as inhibitors of coagulation factors (Factors VII, V, thrombin, and Xa) and platelet functions, fibrin(ogen)olytic enzymes, and vasoactive peptides (
      • Ribeiro J.M.
      Blood-feeding arthropods: live syringes or invertebrate pharmacologists?.
      ,
      • Charlab R.
      • Valenzuela J.G.
      • Rowton E.D.
      • Ribeiro J.M.
      Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.
      ,
      • Takác P.
      • Nunn M.A.
      • Mészáros J.
      • Pechánová O.
      • Vrbjar N.
      • Vlasáková P.
      • Kozánek M.
      • Kazimírová M.
      • Hart G.
      • Nuttall P.A.
      • Labuda M.
      Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.
      ,
      • Ribeiro J.M.C.
      Characterization of a vasodilator from the salivary glands of the yellow fever mosquito Aedes aegypti.
      ,
      • Arcá B.
      • Lombardo F.
      • de Lara
      • Capurro M.
      • della Torre A.
      • Dimopoulos G.
      • James A.A.
      • Coluzzi M.
      Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae.
      ,
      • Arocha-Piñango C.L.
      • Marchi R.
      • Carvajal Z.
      • Guerrero B.
      Invertebrate compounds acting on the hemostatic mechanism.
      ,
      • Markwardt F.
      Coagulation inhibitors from animals feeding on blood.
      ,
      • Kazimírová M.
      • Sulanová M.
      • Kozánek M.
      • Takác P.
      • Labuda M.
      • Nuttall P.A.
      Identification of anticoagulant activities in salivary gland extracts of four horsefly species (Diptera, tabanidae).
      ,
      • Wikel S.K.
      The immunology of host-ectoparasitic arthropod relationships.
      ,
      • Ribeiro J.M.C.
      Ixodes dammini: salivary anti-complement activity.
      ). No fibrin(ogen)olytic enzyme from insects was characterized although a tick fibrin(ogen)olytic metalloprotease has been reported previously (
      • Francischetti I.M.
      • Mather T.N.
      • Ribeiro J.M.
      Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis.
      ). Horseflies are hematophagous insects. Horseflies feed from hemorrhagic pools after lacerating their host's skin while injecting saliva (
      • Dickerson G.
      • Lavoipierre M.M.
      Studies on the methods of feeding of blood-sucking arthropods. III. The methods by which Haematopota pluvialis (Diptera, Tabanidae) obtains its blood-meal from the mammalian host.
      ). Female horseflies require substantial amounts of blood (up to 0.5 ml) for egg production. They can ingest up to 200 mg of blood within only 1–3 min, suggesting that they must contain very potent antihemeostatic ability (
      • Takác P.
      • Nunn M.A.
      • Mészáros J.
      • Pechánová O.
      • Vrbjar N.
      • Vlasáková P.
      • Kozánek M.
      • Kazimírová M.
      • Hart G.
      • Nuttall P.A.
      • Labuda M.
      Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.
      ,
      • Hollander A.L.
      • Wright R.E.
      Impact of tabanids on cattle: blood meal size and preferred feeding sites.
      ). Similar to other hematophagous arthropods, such as mosquitoes (
      • Arcá B.
      • Lombardo F.
      • de Lara
      • Capurro M.
      • della Torre A.
      • Dimopoulos G.
      • James A.A.
      • Coluzzi M.
      Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae.
      ), flies (
      • Charlab R.
      • Valenzuela J.G.
      • Rowton E.D.
      • Ribeiro J.M.
      Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.
      ,
      • Takác P.
      • Nunn M.A.
      • Mészáros J.
      • Pechánová O.
      • Vrbjar N.
      • Vlasáková P.
      • Kozánek M.
      • Kazimírová M.
      • Hart G.
      • Nuttall P.A.
      • Labuda M.
      Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.
      ), and ticks (
      • Urioste S.
      • Hall L.R.
      • Telford 3rd, S.R.
      • Titus R.G.
      Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism.
      ,
      • Kopecký J.
      • Kuthejlová M.
      Suppressive effect of Ixodes ricinus salivary gland extract on mechanisms of natural immunity in vitro.
      ,
      • Ferreira B.R.
      • Silva J.S.
      Successive tick infestations selectively promote a T-helper 2 cytokine profile in mice.
      ,
      • Kopecky J.
      • Kuthejlová M.
      • Pechová J.
      Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-γ by the upregulation of interleukin-10.
      ,
      • Koník P.
      • Slavíková V.
      • Salát J.
      • Reznícková J.
      • Dvoroznáková E.
      • Kopecký J.
      Anti-tumour necrosis factor-alpha activity in Ixodes ricinus saliva.
      ), horsefly saliva contains a wide range of physiologically active molecules that are crucial for attachment to the host or for the transmission of pathogens, and that interact with host processes, including coagulation and fibrinolysis, immunity and inflammation. As an important hematophagous arthropod, there have been comparatively few studies on antihemostaic substances in horseflies. In our previous report, two platelet inhibitors containing RGD
      The abbreviations used are:
      RGD
      Arg-Gly-Asp
      SGE
      salivary gland extract
      PBS
      phosphate-buffered solution
      RP-HPLC
      reverse phase high-performance liquid chromatography
      MALDI-TOF MS
      matrix-assisted laser desorption ionization time-of-flight mass spectrometer
      FPLC
      fast protein liquid chromatography
      ADP
      adenosine 3′,5′-diphosphate.
      1The abbreviations used are:RGD
      Arg-Gly-Asp
      SGE
      salivary gland extract
      PBS
      phosphate-buffered solution
      RP-HPLC
      reverse phase high-performance liquid chromatography
      MALDI-TOF MS
      matrix-assisted laser desorption ionization time-of-flight mass spectrometer
      FPLC
      fast protein liquid chromatography
      ADP
      adenosine 3′,5′-diphosphate.
      sequence, a thrombin inhibitor peptide and vasoactive peptide have been found in the salivary glands of the horsefly of Tabanus yao (
      • Xu X.
      • Yang H.
      • Ma D.
      • Wu J.
      • Wang Y.
      • Song Y.
      • Wang X.
      • Lu Y.
      • Yang J.
      • Lai R.
      Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands.
      ). A fibrinogenolytic factor with a molecular mass of 36 kDa has been purified from the salivary glands of T. yao; however its amino acid sequence is unknown (
      • Xu X.
      • Yang H.
      • Ma D.
      • Wu J.
      • Wang Y.
      • Song Y.
      • Wang X.
      • Lu Y.
      • Yang J.
      • Lai R.
      Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands.
      ).
      In China and some other Eastern countries, horseflies have been used as anti-thrombosis materials for hundreds of years (
      • Yang X.
      • Hu K.
      • Yan G.
      • Zhang Y.
      • Pei Y.
      Fibronogenolytic componments in Tabanid'an ingredient in traditional Chinese medicine and their properties (Chinese).
      ), but the functional components of anti-thrombosis have not been investigated from the insects. In order to identify and characterize interesting salivary compounds for understanding the molecular mechanisms of the anti-thrombosis and help in identifying novel anti-thrombosis compounds, we used proteomics and transcriptomes analysis coupling with pharmacological testing to investigate anti-thrombosis molecules in the salivary glands of the horsefly, T. yao Macquart.

      CONCLUSION

      The current work identified eleven fibrinogenolytic enzymes, eight RGD-containing anti-platelet aggregation disintegrins, four thrombin inhibitors, two vasodilator peptides, one peroxidase and apyrase from the salivary glands of the horsefly, T. yao Macquart (GenBank accession numbers FJ469601-21, FJ477724-6) (Table I). In our previous work, two RGD-containing anti-platelet aggregation disintegrins, one thrombin inhibitor, and vasodilator peptide have been identified from the saliva of T. yao Macquart (
      • Xu X.
      • Yang H.
      • Ma D.
      • Wu J.
      • Wang Y.
      • Song Y.
      • Wang X.
      • Lu Y.
      • Yang J.
      • Lai R.
      Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands.
      ). Therefore, 31 pharmacological molecules with vasodilatory, anti-clotting, and anti-platelet aggregation activities that are capable of inhibiting thrombus formation were identified from the salivary components of this horsefly. The extreme diversity of anti-thrombosis components in horsefly saliva makes the salivary glands a potential reservoir to explore novel pharmaceutical compounds to treat thrombus or platelet aggregation-associated disorders.
      Table IAnti-thrombosis proteins or peptides from salivary glands of horsefly, T. yao
      Proteins/PeptidesMolecular massFunctions
      kDa
      Tablysins 2–624–26Hydrolyze α chain of fibrinogen
      Tablysin 726Hydrolyzes both α and β chains of fibrinogen
      Predicted tablysins 7–1123.5–26
      Tabinhibitins 1–722.5–25Inhibit aggregation of platelet
      Predicted tabinhibitins 8–1023.5–26
      Tabkunins 1–56Inhibit coagulation
      Vasotab TYs 1–36Inhibit vasoconstriction
      Peroxindase TY65Inhibits aggregation of platelet/inhibit vasoconstriction
      Apyrase TY65Inhibit aggregation of platelet

      Acknowledgments

      We are grateful to Professor Jose Ribeiro for the valuable comments and kind help in the manuscript preparation.

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