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Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract

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Abstract

In the recent past, a low molecular mass serine protease, the Hag-protease that caused pro-coagulant activity and as well as local toxicity was isolated and characterized from the Hippasa agelenoides spider venom gland extract (Devaraja et al., Toxicon 52:130–138, 2008). In the current study, the pro-coagulant property has been investigated further and the results are presented. The Hag-protease reduced the re-calcification time of citrated human plasma. It reduced the activated partial thromboplastin time (APTT), and prothrombin time (PT) suggesting its participation in common pathway of blood coagulation. Interestingly, it coagulated the citrated human plasma in the absence of CaCl2 but, it was lacking thrombin-like activity as it did not clot the purified fibrinogen. Strikingly, the enzyme coagulated the factor X deficient congenital human plasma, suggesting the factor Xa-like activity. However, the cumulative augmented activity was observed in presence of CaCl2 and phospholipids. Further, the Hag-protease preferentially hydrolyzed the Aα chain and then the Bβ-chain, but not the γ-chain. As a result, truncated fibrinogen generated was lacking in the polymerization property. It hydrolyzed all the subunits of partially cross-liked fibrin clot (α-polymer, α-chain, β-chain, and γ–γ dimers). Further, at low concentrations, the Hag-protease stimulated the aggregation of human platelets in platelet rich plasma, but at high concentrations caused spontaneous clumping. In contrast, it inhibited the collagen induced aggregation of washed human platelets. In summary, the present study for the first time reporting the factor Xa-like activity of a serine protease especially from the spider venom that exhibited opposing effects on hemostasis, the pro-coagulant activity and the anti-coagulant activity including fibrin(ogen)olytic and platelet aggregation inhibition activities.

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Abbreviations

APTT:

Activated partial thromboplastin time

PT:

Prothrombin time

PMSF:

Phenyl methyl sulphonyl fluoride

EDTA:

Ethylene diamine tetra acetic acid

IAA:

Iodoacetic acid

PC:

Phosphatidyl choline

PS:

Phosphatidyl serine

PPP:

Platelet poor plasma

PRP:

Platelet rich plasma

References

  1. Young AR, Pincus SJ (2001) Comparison of enzymatic activity from three species of necrotizing arachnids in Australia: Loxosceles rufescens, Badumna insignis and Lampona cylindrata. Toxicon 39:391–400. doi:10.1016/S0041-0101(00)00145-8

    Article  CAS  PubMed  Google Scholar 

  2. Cunha RB, Barbaro KC, Muramatsu D, Portaro FCV, Fontes W, de Souza MV (2003) Purification and characterization of Loxnecrogin a dermonecrotic toxin from Loxosceles gaucho Brown spider venom. J Protein Chem 22:135–146. doi:10.1023/A:1023470829671

    Article  CAS  PubMed  Google Scholar 

  3. da Silveira RB, Pigozzo RB, Chaim OM, Appel MH, Dreyfuss JL, Toma L, Mangili OC, Gremski W, Dietrich CD, Nadu HB, Viega SS (2006) Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (Brown spider) venom gland. Biochimie 88:1241–1253. doi:10.1016/j.biochi.2006.02.008

    Article  PubMed  Google Scholar 

  4. Zanetti VC, da Silveira RB, Dreyfuss JL, Haoach J, Mangili OC, Veiga SS, Gremski W (2002) Morphological and biochemical evidence of blood vessel damage and fibrinogenolysis triggered by brown spider venom. Blood Coagul Fibrinolysis 13:135–148. doi:10.1097/00001721-200203000-00009

    Article  CAS  PubMed  Google Scholar 

  5. Veiga SS, Feitosa L, Santos VLP, de Souza GA, Ribeiro AS, Mangili OC, Porcionatto MA, Nada HB, Dietrich CP, Brentani RR, Gremski W (2000) Effect of Loxosceles intermedia (Brown spider) venom on basement membrane structures. Histochem J 32:397–408. doi:10.1023/A:1004031019827

    Article  CAS  PubMed  Google Scholar 

  6. Jackson CM (1984) Factor X. Prog Hemost Thromb 7:55–109

    CAS  PubMed  Google Scholar 

  7. Manjunatha Kini R (2005) Serine proteases affecting blood coagulation and fibrinolysis from snake venoms. Pathophysiol Haemost Thromb 34:200–204. doi:10.1159/000092424

    Article  PubMed  Google Scholar 

  8. Jackson CM, Nemerson Y (1980) Blood coagulation. Annu Rev Biochem 49:765–811. doi:10.1146/annurev.bi.49.070180.004001

    Article  CAS  PubMed  Google Scholar 

  9. Davie EW, Fujikawa K, Kisiel W (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30:10363–10370. doi:10.1021/bi00107a001

    Article  CAS  PubMed  Google Scholar 

  10. Devaraja S, Nagaraju S, Mahadeshwara sawmy YH, Girish KS, Kemparaju K (2008) A low molecular weight serine protease: purification and characterization from Hippasa agelenoides (Funnel web) spider venom gland extract. Toxicon 52:130–138. doi:10.1016/j.toxicon.2008.04.168

    Article  CAS  PubMed  Google Scholar 

  11. Satake M, Murata Y, Suzuki T (1963) Studies on snake venoms XIII. Chromatographic separation and properties of three proteinases from Agkistrodon halys blomhoffii venom. J Biochem 53:483–497

    Google Scholar 

  12. Quick AJ (1935) The prothrombin in hemophilia and obstructive jaundice. J Biol Chem 107:73–85

    Google Scholar 

  13. Denson KWE (1969) Coagulation and anticoagulant actions of snake venoms. Toxicon 7:5–11. doi:10.1016/0041-0101(69)90154-8

    Article  CAS  PubMed  Google Scholar 

  14. Ouyang C, Teng CM (1976) Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom. Biochim Biophys Acta 420:298–308

    CAS  PubMed  Google Scholar 

  15. Chakrabarty D, Kausiki D, Antony G, Debasish B (1999) Hemorrhagic protein of Russell’s viper venom with fibrinolytic and esterolytic activities. Toxicon 38:1475–1490. doi:10.1016/S0041-0101(99)00243-3

    Article  Google Scholar 

  16. Ardlie NG, Han P (1974) Enzymatic basis for platelet aggregation and release: the significance of the “platelet atmosphere” and the relationship between platelet function and blood coagulation. J Haematol 26:331–356. doi:10.1111/j.1365-2141.1974.tb00477.x

    Article  CAS  Google Scholar 

  17. Born GVR (1962) Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194:27–29. doi:10.1038/194927b0

    Article  Google Scholar 

  18. Rajesh R, Nataraju A, Gowda CDR, Frey BM, Frey FJ, Vishvanath BS (2006) Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot. Biochimie 88:1313–1322. doi:10.1016/j.biochi.2006.06.007

    Article  CAS  PubMed  Google Scholar 

  19. Reis CV, Portaro FC, Andrade SA, Fritzen M, Fernandes BL, Sampaio CA, Camargo AC, Chudzinski-Tavassi AM (2001) A prothrombin activator serine protease from the Lonoma oblique caterpillar venom (Lopap) biochemical characterization. Thromb Res 102:427–436. doi:10.1016/S0049-3848(01)00265-1

    Article  CAS  PubMed  Google Scholar 

  20. Jing Z, Rong PJH, Ying L, Dong FL, Rong QH (2007) Eisenia fetida protease-III-1 functions in both fibrinolysis and fibrogenesis. J Biomed Biotechnol 10:1155–1165

    Google Scholar 

  21. Kini RM, Morita T, Rosing J (2001) Classification and nomenclature of prothrombin activators isolated from snake venoms. Thromb Haemost 86:710–711

    CAS  Google Scholar 

  22. Morita T, Iwanaga S (1978) purification and properties of prothrombin activator from the venom of Echis carinatus. J Biochem Tokya 83:559–570

    CAS  Google Scholar 

  23. Rao VS, Kini RM, Pseutarin C (2002) a prothrombin activator from Pseudonaja textilis venom: its structural and functional similarity to mammalian coagulation factor Xa–Va complex. Thromb Haemost 88(4):611–619

    CAS  PubMed  Google Scholar 

  24. Persson EP, Hogg J, Stenflo J (1993) Effects of Ca2+ binding on the protease module of factor Xa and its interaction with factor Va. Evidence for two Gla-independent Ca(2+) binding sites in factor Xa. J Biol Chem 268:22531–22539

    CAS  PubMed  Google Scholar 

  25. Nagaraju S, Mahadeshwaraswamy YH, Girish KS, Kemparaju K (2006) Venom from spiders of the genus Hippasa: biochemical and pharmacological studies. Comp Biochem Physiol 144:1–9

    CAS  Google Scholar 

  26. da Silveira RB, dos Santos Filho JF, Mangili OC, Veiga SS, Gremski W, Nader HB, von Dietrich CP (2002) Identification of proteases in the extract of venom glands from brown spiders. Toxicon 40:815–822. doi:10.1016/S0041-0101(02)00078-8

    Google Scholar 

  27. da Silveira RB, Wille AC, Chaim OM, Appel MH, Silva DT, Franco CR, Toma L, Mangili OC, Gremski W, Dietrich CP, Nader HB, Veiga SS (2007) Identification, cloning, expression and functional characterization of and astacin-like metallporotese toxin from Loxosceles intermedia (Brown spider) venom. Biochem J 406:355–363. doi:10.1042/BJ20070363

    Article  PubMed  Google Scholar 

  28. Veiga SS, Feitosa L, Santos VLP, de Souza GA, Ribeiro AS, Mangili OC, Porcionatto MA, Nada HB, Dietrich CP, Brentani RR, Gremski W (2000) Effect of Loxosceles intermedia (Brown spider) venom on basement membrane structures. J Histochem 32:397–408. doi:10.1023/A:1004031019827

    Article  CAS  Google Scholar 

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Acknowledgments

The authors thank Prof. B.S. Vishwanath, DOS in Biochemistry, University of Mysore for his support. The authors also thank Dr. S. Nagaraju, Department of Orthopedic Research, University of Virginia, USA, for his valuable suggestions during the study. S. Devaraja thanks the University grant commission (RGNF), New Delhi, India, for financial assistance.

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Authors and Affiliations

  1. Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India

    S. Devaraja, K. S. Girish, V. R. Devaraj & K. Kemparaju

  2. Department of Biochemistry, Bangalore University, Central College Campus, Bangalore, 560 001, India

    V. R. Devaraj

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  1. S. Devaraja
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  2. K. S. Girish
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  3. V. R. Devaraj
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  4. K. Kemparaju
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Correspondence to K. Kemparaju.

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Devaraja, S., Girish, K.S., Devaraj, V.R. et al. Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract. J Thromb Thrombolysis 29, 119–126 (2010). https://doi.org/10.1007/s11239-009-0341-3

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  • DOI: https://doi.org/10.1007/s11239-009-0341-3

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