Archives of Toxicology

, Volume 83, Issue 3, pp 271–279 | Cite as

Colombistatin: a disintegrin isolated from the venom of the South American snake (Bothrops colombiensis) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion

  • Elda E. Sánchez
  • Alexis Rodríguez-Acosta
  • Rene Palomar
  • Sara E. Lucena
  • Sajid Bashir
  • Julio G. Soto
  • John C. Pérez
Organ Toxicity and Mechanisms


Snake venoms are complex mixtures of proteins, which affect the vital biologic systems of prey, as well as humans. Envenomation leads to immobilization by paralysis, cardiac, and circulatory failure. These same venom proteins that cause havoc in the physiologic system could be used as therapeutic agents. Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are non-enzymatic proteins that inhibit cell–cell interactions, cell–matrix interactions, and signal transduction. These proteins may have potential in the treatment of strokes, heart attacks, cancers, osteoporosis, and diabetes. The present study describes the isolation and characterization of a disintegrin (colombistatin) found in the venom of the Venezuelan snake mapanare (Bothrops colombiensis). Colombistatin was purified by a two-step high-performance liquid chromatography procedure, which included reverse phase C18 and size exclusion protein Pak 60. Colombistatin inhibited ADP-induced platelet aggregation, human urinary (T24) and skin melanoma (SK-Mel-28) cancer cell adhesion to fibronectin, and cell migration. Colombistatin contained 72 amino acids with a mass of 7.778 kDa as determined by mass spectrometry. Colombistatin could be used as a therapeutic tool in the treatment of melanoma cancers and also thrombotic diseases.


Disintegrin, Venom, Mapanare Bothrops colombiensis T24 cells SK-Mel-28 Platelet aggregation Cell migration 


  1. American Cancer Society (2008) Cancer Facts and Figs 2008. American Cancer Society, AtlantaGoogle Scholar
  2. Bajwa SS, Markland FS, Russell FE (1980) Fibrinolytic enzyme(s) in western diamondback rattlesnake (Crotalus atrox) venom. Toxicon 18:185–290CrossRefGoogle Scholar
  3. Brando C, Marcinkiewicz C, Goldman B, McLane MA, Niewiarowski S (2000) EC3, a heterodimeric disintegrin from Echis carinatus, inhibits human and murine alpha 4 integrin and attenuates lymphocyte infiltration of Langerhans islets in pancrease and salivary glands in nonobese diabetic mice. Biochem Biophys Res Commun 267:413–417PubMedCrossRefGoogle Scholar
  4. Chang CP, Chang JC, Chang HH, Tsai WJ, Lo SJ (2001) Positional importance of Pro53 adjacent to the Arg49-Gly50-Asp51 of rhodostomin in binding to integrin alphaIIbbeta. Biochem J 357:57–64PubMedCrossRefGoogle Scholar
  5. Coplen DE, Brown EJ, McGarr J, Ratliff TL (1991) Characterization of fibronectin attachment by human transitional cell carcinoma line, T-24. J Urol 145:1312–1315PubMedGoogle Scholar
  6. Dennis MS, Henzel WJ, Pitti RM, Lipari MT, Napier MA, Deisher TA, Bunting S, Lazarus RA (1989) Platelet glycoprotien IIb-IIIa protein antagonist from snake venoms: evidence for a family of platelet-aggregation inhibitors. Proc Natl Acad Sci USA 87:2471–2475CrossRefGoogle Scholar
  7. Fisher JE, Caulfield MP, Sato M, Quartuccio HA, Gould RJ, Garsky VM, Rodan GA, Rosenblatt M (1993) Inhibition of osteoclastic bone resorption in vivo by echistatin, an “arginyl-glycyl-aspartyl” (RGD)-containing protein. Endrocrinology 132:1411–1413CrossRefGoogle Scholar
  8. Galán JA, Sánchez EE, Bashir S, Pérez JC (2005) Characterization and identification of disintegrins in Crotalus horridus venom by liquid chromatography and tandem matrix-assisted laser desorption ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) mass spectrometry. Can J Chem 83:1124–1131CrossRefGoogle Scholar
  9. Galán JA, Sánchez EE, Rodríguez-Acosta A, Soto JG, Bashir S, McLane MA, Paquette-Straub C, Pérez JC (2008) Inhibition of lung tumor colonization and cell migration with the disintegrin crotatroxin 2 isolated from the venom of Crotalus atrox. Toxicon 51:1186–1189PubMedCrossRefGoogle Scholar
  10. Garcia M, Jemal A, Ward EM, Center MM, Hao Y, Siegel RL, Thun MJ (2007) Global Cancer Facts and Figs 2007. American Cancer Society, AtlantaGoogle Scholar
  11. Glenn JL, Straight RC (1978) Mojave rattlesnake Crotalus scutulatus scutulatus venom: variation in toxicity with geographical origin. Toxicon 16:81–84PubMedCrossRefGoogle Scholar
  12. Glenn JL, Straight RC (1982) The rattlesnakes and their venom yield and lethal toxicity. In: Tu AT (ed) Rattlesnake venoms: their actions and treatment. Marcel Dekker, Inc., New York, pp 99Google Scholar
  13. Gould RJ, Polokoff MA, Friedman PA, Huang T-F, Holt JC, Cook JJ, Niewiarowski S (1990) Minireview. Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc Soc Expl Biol Med 195:168–171Google Scholar
  14. Huang T-F, Sheu J-R, Teng C-M (1991) Mechanism of action of a potent antiplatelet peptide, triflavin from Trimeresurus flavoviridis snake venom. Thromb Haemost 66:489–493PubMedGoogle Scholar
  15. Jin H, Varner JA (2004) Integrins: roles in cancer development and as treatment targets. Br J Cancer 90:561–565PubMedCrossRefGoogle Scholar
  16. Kini RM, Evans HJ (1992) Structural domains in venom proteins: evidence that metalloproteinases and nonenzymatic platelet aggregation inhibitors (disintegrins) from snake venoms are derived by proteolysis from a common precursor. Toxicon 30:265–293PubMedCrossRefGoogle Scholar
  17. Knight LC, Romano JE, Cosenza SC, Iqbal NM, Marcinkiewics C (2007) Differences in binding of 99mTc-disintegrins to integrin αvβ3 on tumor and vascular cells. Nucl Med Biol 34:371–381PubMedCrossRefGoogle Scholar
  18. Kuroda K, Brown EJ, Telle WB, Russell DG, Ratliff TL (1993) Characterization of the internalization of Bacillus Calmette-Guerin by human bladder tumor cells. J Clin Invest 91:69–76PubMedCrossRefGoogle Scholar
  19. Marcinkiewicz C, Weinreb PH, Calvete JJ, Kisiel DG, Mousa SA, Tuszynski GP, Lobb RR (2003) Obstustatin: a potent selective inhibitor of alpha 1 beta 1 integrin in vitro and angiogenesis in vivo. Cancer Res 63:2020–2023PubMedGoogle Scholar
  20. Markland FS, Zhou Q (2002) Snake venom disintegrins: an effective inhibitor of breast cancer growth and dissemination. In: Tu AT, Gaffield W (eds) Natural and synthetic toxins, biological implications. American Chemical Society, Washington DC, pp 262–282Google Scholar
  21. McLane MA, Sanchez EE, Wong A, Paquette-Straub C, Perez JC (2004) Disintegrins. Curr Drug Targets Cardiovasc Haematol Disord 4:327–355PubMedCrossRefGoogle Scholar
  22. McLane MA, Joerger T, Mahmoud A (2008) Disintegrins in health and disease. Front Biosci 13:6617–6637PubMedCrossRefGoogle Scholar
  23. Mercer B, Markland F, Minkin C (1998) Contortrostatin, a homodimeric snake venom disintegrin, is a potent inhibitor of osteoclast attachment. J Bone Miner Res 13:409–414PubMedCrossRefGoogle Scholar
  24. Minton SA, Minton MR (1969) Venomous reptiles. Scribner, New YorkGoogle Scholar
  25. Moursi AM, Globus RK, Damsky CH (1997) Interaction between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro. J Cell Sci 110:2187–2196PubMedGoogle Scholar
  26. Nakamura I, Tanaka H, Rodan GA, Duong LT (1998) Echistatin inhibits the migration of murine prefusion osteoclasts and the formation of multinucleated osteoclast-like cells. Endocrinology 139:5182–5193PubMedCrossRefGoogle Scholar
  27. Nakamura I, Pilkington MF, Lakkakorpi PT, Lipfert L, Sims SM, Dixon SJ, Rodan GA, Duong LT (1999) Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone. J Cell Sci 112(pt 22):3985–3993PubMedGoogle Scholar
  28. Niewiarowski S, Huang T-F, Rucinski B, Cook JJ, Williams JA, Musial J, Edmunds LH Jr, Bush L, Shebuski R, Friedman PA (1989) Inhibition of platelet adhesion to surface of extracorporeal circuit by RGD containing peptides from viper venoms. Thromb Haemost 62:319Google Scholar
  29. Niewiarowski S, McLane MA, Kloczewiak M, Stewart GJ (1994) Disintegrins and other naturally occuring antagonists of platelet fibrinogen receptors. Semin Hematol 31:289–300PubMedGoogle Scholar
  30. Omori-Satoh T, Sadahiro S, Ohsaka A, Murata R (1972) Purification and characterization of an antihemorrhagic factor in the serum of Trimeresurus flavoviridis, a crotalid. Biochim Biophys Acta 285:414–426PubMedGoogle Scholar
  31. Ouyang C, Yeh H-I, Huang T-F (1983) A potent platelet aggregation inhibitor purified from Agkistrodon halys (Mamushi) snake venom. Toxicon 21:797–804PubMedCrossRefGoogle Scholar
  32. Pattabhiraman TR, Russell FE, Whigham H (1978) Some chemical and physiopharmacological properties of fractions from the venom of Crotalus viridis helleri and Crotalus scutulatus scutulatus. In: Rosenberg P (ed) Toxins: animal, plant and microbial. Pergamon Press, New York, pp 211–222Google Scholar
  33. Peltonen J, Larjava H, Jaakkola S, Grainick H, Akiyama SK, Yamada SS, Yamada KM, Uitto J (1989) Localization of integrin receptors for fibronectin, collagen, and laminin in human skin: variable expression in basal and squamous cell carcinomas. J Clin Invest 84:1916–1923PubMedCrossRefGoogle Scholar
  34. Poole K, Müller D (2005) Flexible, actin-based ridges colocalise with b1 integrin on the surface of melanoma cells. Br J Cancer 92:1499–1505PubMedCrossRefGoogle Scholar
  35. Rahman S, Lu X, Kakkar VV, Authi KS (1995) The integrin alphaIIb beta3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake venom RGD proteins are related to the amino acid environment flanking the sequence RGD. Biochem J 312:223–232PubMedGoogle Scholar
  36. Rodriguez-Acosta A, Mondolfi A, Orihuela R, Aguilar M (1995) Que hacer frente a un accidente ofidico?. Venediciones, CaracasGoogle Scholar
  37. Rouslahti E (1988) Fibronectin and its receptors. Annu Rev Biochem 57:375–413CrossRefGoogle Scholar
  38. Rucinski B, Niewiarowski S, Holt JC, Soszka T, Knudsen KA (1990) Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin. Biochim Biophys Acta 1054:257–262PubMedCrossRefGoogle Scholar
  39. Sánchez EE, Ramirez MS, Galán JA, Lopez G, Rodríguez-Acosta A, Pérez JC (2003) Cross reactivity of three antivenoms against North American snake venoms. Toxicon 41:315–320PubMedCrossRefGoogle Scholar
  40. Sánchez EE, Galán JA, Russell WK, Russell DH, Soto JG, Pérez JC (2006) Isolation and characterization of two disintegrins from the venom of Crotalus scutulatus scutulatus (Mohave rattlesnake). Toxicol Appl Pharmacol 212:59–68PubMedCrossRefGoogle Scholar
  41. Scarborough RM, Rose JW, Naughton MA, Phillips DR, Nannizzi L, Arfsten A, Campbell AM, Charo IF (1993) Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J Biol Chem 268:1058–1065PubMedGoogle Scholar
  42. Scott G, Liang H (1995) pp125FAK in human melanocytes and melanoma: expression and phosphorylation. Exp Cell Res 219:197–203PubMedCrossRefGoogle Scholar
  43. Sheu JR, Lin CH, Chung JL, Teng CM, Huang TF (1992) Triflavin, and arg-gly-asp containing antiplatelet peptide inhibits cell-substratum adhesion and melanoma cell-induced lung colonization. J Cancer Res 83:885–983Google Scholar
  44. Sohn YD, Hong SY, Cho KS, Choi WS, Song SW, Bae JS, Kim DS, Chung KH (2008) Acute and repeated dose toxicity studies of recombinant saxatilin, a disintegrin from the Korean snake (Gloydius saxatilis). Toxicon 51:406–417PubMedCrossRefGoogle Scholar
  45. Takada J, Huang C, Hemler ME (1987) Fibronectin receptor structures in the VLA family of heterodimers. Nature (London) 326:1459–1471CrossRefGoogle Scholar
  46. Tian J, Paquette-Straub C, Sage EH, Funk SE, Patel V, Galileo D, McLane MA (2007) Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin. Toxicon 49:899–908PubMedCrossRefGoogle Scholar
  47. Wierzbicka-Patynowski I, Niewiarowski S, Marcinkiewicz C, Calvete JJ, Marcinkiewicz MM, McLane MA (1999) Structural requirements of echistatin for the recognition of alpha v beta 3 and alpha 5 beta 1 integrins. J Biol Chem 274:37809–37814PubMedCrossRefGoogle Scholar
  48. Yamada D, Shin Y, Morita T (1999) Nucleotide sequence of a cDNA encoding a common precursor of disintegrin flavostatin and hemorrhagic factor HR2a from the venom of Trimeresurus flavoviridis. FEBS Lett 451:299–302PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Elda E. Sánchez
    • 1
    • 2
  • Alexis Rodríguez-Acosta
    • 2
  • Rene Palomar
    • 1
  • Sara E. Lucena
    • 1
  • Sajid Bashir
    • 3
  • Julio G. Soto
    • 4
  • John C. Pérez
    • 1
  1. 1.Natural Toxins Research Center, College of Arts and ScienceTexas A&M University-KingsvilleKingsvilleUSA
  2. 2.Instituto de Medicina TropicalUniversidad Central de VenezuelaCaracasVenezuela
  3. 3.Laboratory for Mass Spectrometry, Department of ChemistryTexas A&M University-KingsvilleKingsvilleUSA
  4. 4.Biological Sciences DepartmentSan José State UniversitySan JoséUSA

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