被台灣眼鏡蛇咬傷之後除了會影響神經性的麻痺之外，另一個主要的症狀會使傷口癒合速度變慢且造成局部性組織的壞死。我們利用SPFF陽離子交換樹脂的方式從台灣眼鏡蛇蛇毒中純化到一蛇毒金屬蛋白水解酵素 - atragin。在纖維母細胞傷口癒合實驗中我們發現到40nM atragin 與 5μM CTX A5均有約30%的抑制癒合的能力，且與細胞毒性無關。另ㄧ個有趣的現象則是，同時加入40nM atragin及5μM CTX A5會產生協同性的放大抑制傷口癒合的能力達65%，但似乎與產生了細胞毒性有關。Apoatragin為失去活性之atragin，此時apoatragin便不具有抑制傷口癒合的能力。同時檢驗atragin與apoatragin，發現兩者間二級結構差異性不大且apoatragin與整合素αvβ3結合能力仍不受影響，推測atragin內部的金屬蛋白水解酵素區在傷口癒合上的重要性。在細胞貼附實驗中，大量表現β3整合素之中國倉鼠卵巢細胞相對於大量表現α5與野生種的中國倉鼠卵巢細胞而言，與atragin的貼附能力較強，且對於atragin的毒性更為敏感。最後，我們試者先將atragin與纖維母細胞作用後再與數種細胞外間質蛋白進行細胞貼附實驗，發現同為整合素αvβ3受質之纖維粘連蛋白、玻連蛋白及纖維蛋白原與纖維母細胞的貼附能力變差，而與膠原蛋白則較不明顯。最後總結，我們發現atragin可能是透過了選擇性地分解細胞外間質蛋白及細胞外表面整合素αvβ3因此產生了抑制纖維母細胞在傷口階段時細胞移動的能力。

Most of cobra snakebites have been shown to induce significant local tissue necrosis and with a retarded wound healing process in addition to systemic neurotoxic paralysis.We used one step SPFF chromatography to purify atragin from the venom of Naja atra. On fibroblast migration in the wound closure assay ,40nM atragin inhibited~30% wound healing at 24 h without significant cytotoxicity. We also tested the effect of CTX A5 in the same assay, 5μM CTX A5 inhibited~40% of wound closure at 24 h compared to control without detectable cytotoxicity. Interesting, 5μM CTX A5 and 40nM atragin synergistically inhibited~65% of wound closure compared with 30% inhibition of 40nM atragin alone. However,this phenomena might come from cell cytotoxicity. Apoatragin, which lacked metalloprotease activity, is unable to inhibit fibroblast migration. We checked the secondary structure and αvβ3 integrin binding activity of apoatragin that ensure structural and functional stability. CHO cells expressing recombinant αvβ3 (β3-CHO) adhere to atragin-coated surface in a dose-dependent manner at the levels higher than the control or α5-CHO cells andβ3-CHO cells cells are more susceptible to atragin compared with wildtype- andα5- variants. Finally, atragin may impair fibroblast interaction with ECM protein via affecting cell surface receptor to binding to αvβ3 integrin ligand ,(fibronectin, vitronectin and fibrinogen ), but not to collagen. Taken together, atragin inhibited fibroblast migration through digesting ECM and cell surface receptor such asαvβ3 integrin.

1. Hung, D.Z., Liau, M.Y., Lin-Shiau, S.Y. (2003) The clinical significance of venom detection in patients of cobra snakebite. Toxicon .41, 409-415.
2 覃公平 (1998) 中國毒蛇學，第二版，廣西科學技術出版社。
3. Tu, A.T. (1996) Overview of snake venom chemistry .Adv.Exp.Med.
Biol. 391,37-62.

4. Ouyang, C., Teng, C.M., Huang,T.F. (1991) Characterization of snake venom Principles affecting blood coagulation and platelet aggregation. Adv.Exp.Med.Biol.281,151-163.

5. Pirkle, H. Theodor, I. (1990) Thrombin-like venom enzyme: structure and function. Adv.Exp.Med.Biol 281,165-175.

6. Pirkle, H. (1998) Thrombin-like enzyme from snake venoms : an updated inventory . Scientific and Standardization Committee’s Registry of Exogenous Hemostatic Factors.Thromb.Haemost.79, 675-683.

7. Evan, H.J. (1981) Cleavage of the A alpha-chain of fibrinogen and the alpha-polymer of fibrin by the venom of spitting cobra (Naja nigricollis). Biochim.Biophys.Acta 660, 219-226.

8. Alexander, J.S., and Elrod J.W. (2002) Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation. J.Anat. 200, 561-574.

9. Kini, R.M., and Evans, H.J. (1991) Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation. Biochim.Biophys.Acta 1095, 117-121.

10. Shoibonov, B.B., Osipov, A.V., Kryukova, E.V., Zinchenko, A.A., Lakhtin, V.M.,Tsetlin, V.I., and Utkin, Y.N. (2005) Oxiagin from the Naja oxiana cobra venom is the first reprolysin inhibiting the classical pathway of complement. Mol.Immunol. 42, 1141-1153.

11. Takeya, H., Miyata, T., Nishino, N., Omori-Satoh, T., and Iwanaga, S. (1993) Snake venom hemorrhagic and nonhemorrhagic metalloendopeptidases. Methods Enzymol. 223, 365-378.

12. Hite, L.A., Shannon, J.D., Bjarnason, J.B., and Fox, J.W. (1992) Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures .Biochemistry 31, 6203-6211.

13. Matsui, T., Fujimura, Y., and Titani, K. (2000) Snake venom proteases affecting hemostasis and thrombosis. Biochim. Biophys. Acta 1477, 146-156.

14. Jia, J.G., Shimokawa, K., Bjarnason, J.B., and Fox, J.W. (1996) Snake venom metalloproteinases: structure, function and relationship to the ADAMs family of proteins. Toxicon 34, 1269-1276.

15. white, J.W. (2003) ADAMs: modulators of cell-cell and cell-matrix interactions. Curr. Opin. Cell. Biol. 15, 598-606.

16. Hite, L.A., Jia, L.G., Bjarnason, J.B., Fox, J.W. (1994) cDNA sequences for four snake venom metalloproteinases: structure, classification, and their relationship to mammalian reproductive proteins. Arch Biochem. Biophys. 308, 182-91.

17. Bjarnason, J.B., and Fox, J.W. (1994) Hemorrhagic metalloproteinase from snake venoms. Pharmacol. Ther. 62, 325-372.

18. McLane, M.A., Marcinkiewicz C., Vijay-Kumar S., Wierzbicka-Patynowski, I., Niewiarowski, S. (1998) Viper venom disintegrins and related molecules. Proc. Soc. Exp. Biol. Med. 219, 109-119.

19. Kamiguti, A.S., Gallagher P., Marcinkiewicz C., Theakston, R.D., Zuzel, M., Fox, J.W. (2003) Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function. FEBS Lett. 549, 129-134.

20. De Luca, M., Dunlop, L.C., Andrews, R.K., Flannery, J.V., Jr, Ettling, R., Cumming, D.A., Veldman, G.M., and Berndt, M.C. (1995) A novel cobra venom metalloproteinase, mocarhagin, cleaves a 10-amino acid peptide from the mature N terminus of P-selection glycoprotein ligand receptor, PSGL-1, and abolishes P-selectin binding. J.Biol.Chem. 270, 26734-26737.

21. Gardiner, E.E., De Luca, M., McNally, T., Michelson, A.D., Andrews, R.K., Berndt M.C. (2001) Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-Selectin glycoprotein ligand-1 by neutrophil elatase and cathepsin G. Blood 98, 1440-1447.

22. O’Keefe, M.C., Caporale, L.H., and Vogel, C.W. (1998) A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor. J.Biol. Chem. 263, 12690-12697.

23. Kock, M.A., Hew, B.E., Bammert, H., Fritzinger, D.C., and Vogel, C.W. (2004) Structure and function of recombinant cobra venom factor. J.Biol. Chem. 279, 30836-30843.

24. Vogel, C.W., Bredehorst, R., Fritzinger, D.C., Grunwald, T., Ziegelmuller, P., and Kock, M.A. (1996) Structure and function of cobra venom factor, the complement-activating protein in cobra venom. Adv. Exp.Med.Biol. 391, 97-114.

25. Hamako, J., Matsui, T., Nishida, S., Nomura, S., Fujimura, T., Ito, M., Ozeki, Y., Titani, K. (1998) Purification and characterization of kaouthigin, a von Willebrand factor-binding and cleaving metalloproteinase from Naha kaouthia cobra venom. Thromb. Haemost. 80, 499-505.

26. Miura, S., Sakurai, Y., Takatsuka, H., Yoshioka, A., Matsumoto, M., Yagi, H., Kokubo T., Ikeda, Y., Matsui, T., Titani, K., Fujimura Y.(1999) Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments. Br. J. Haematol. 105, 1092-1100.

27. Clark, R.A.F. Wound repair Overview and General Consideration. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 3.

28. Nanney, L.B., King, L.E.J. (1996) Epidermal Growth Factor and Transforming Growth Factor-α. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 171.

29. Abraham, J.A., Klagsbrum, M. (1996) Modulation of Wound Repair by Members of the Fibroblast Growth Factor Family. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 195.

30. Heldin, C-H., Westermarck, B. (1996) Role of platelet-derived growth factor in vivo. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 249.

31. Roberts, A.B., Sporn, M.B. (1996) Transforming Growth factor-beta. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 275.

32. Singer, A.J., Clark, R.A. (1999) Mechanisms of Disease: Cutaneous Wound Healing. N. Engl. J. Med. 341, 738-746.

33. Reibman, J., Meixler, S., Lee, T.C. (1991) Transforming growth factor beta 1, a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways. Proc.Natl.Acad.Sci. U.S.A. 88,6805-6809.

34. Clark, R.A.F. (1990) Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin. J. Invest. Dermatol. 94,128-134.

35. Larjava, H., Salo, T., Haapasalmi, K., Kramer, R.H., Heino, J. (1993) Expression of integrin and basement membrane components by wound keratinocyte. J.Clin. Invest. 92, 1425-1435.

36. Clark, R.A.F., Ashcroft,G.S., Spencer, M-J., Larjava, H., Ferguson, M.W.J. (1996) Reepithelialization of normal human excisional wounds is associated with a switch from αvβ5 toαvβ6 integrins. Br.J.Dermatrol. 135, 46-51.

37. Pilcher, B.K., Dumin, J.A., Sudbeck, B.D., Krane, S.M., Welgus, H.G., Parks, W.C. (1997) The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix. J.Cell Biol. 137, 1445-1457.

38. Mignatti, P., Rifkin, D.B., Welgus, H.G., Parks, W.C. (1996) Proteinases and tissue remodeling. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 427.

39. Hunt, T.K. ed. (1980) Wound healing and wound infection : theory and surgical practice. New York: Appleton-Century-Crofts.

40. Tonnesen, M.G., Feng, X., Clark, R.A. (2000) Angiogenesis in wound healing . J.Investig.Dermatol.Symp.Proc. 5, 40-46.

41. Gray, A.J., Bishop, J.E., Reeves, J.T., Laurent, G.J. (1993) Aαand Bβ chains of fibrinogen stimulate proliferation of human fibroblast. J.Cell.Sci. 104, 409-413.

42. Xu, J., Clark, R.A.F. (1996) Extracellular matrix alters PDGF regulation of fibroblast integrins. J.Cell.Biol. 132, 239-249.

43. Clark, R.A.F., Lanigan, J.M., DellaPelle, P., Mansean, E., Dvorak, H.F., Colvin, R.B. (1982) Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization. J.Invest.Dermatol. 79, 264-269.

44. Greiling, D., Clark, R.A.F. (1997) Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix. J.Cell.Sci. 110, 861-870.

45. Toole, B.P. (1991) Proteolycans and hyaluronan in morphogenesis and differentiation. In: Hay, E.D., ed. Cell biology of extracellular matrix. 2nd ed. New York : Plenum Press. 305.

46. Clark, R.A.F., Nielsen, L.D., Welch, M.P. McPherson, J.M. (1995) Collagen matrices attenuate the collagen-synthetic response of cultured fibroblasts to TGF-β. J.Cell.Sci. 108, 1251-1261.

47. Welch, M.P., Odland, G.F., Clark, R.A.F. (1990) Temporal relationships of F-actin bundle formation,collagen and fibronectin matrix assembly,and fibronectin receptor expression to wound contraction. J.Cell.Biol. 110, 133-145.

48. Greenhalgh, D.G. (1998) The role of apotosis in wound healing . Int.J.Biochem.Cell.Biol. 30,1019-1030.

49. Clore, J.N., Cohen, I.K., Diegelmann, R.F. (1979) Quantitation of collagen types I and III during wound healing in rat skin. Proc.Soc.Exp.Biol.Med. 161, 337-340.

50. Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell. 48, 549-554.

51. Hynes, R.O. (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69, 11-25.

52. Porter, J.C., Hogg ,N. (1998) Integrins take partners: cross-talk between integrins and other membrane receptors. Trends.Cell.Biol. 8, 390-6.

53. Ruoslahti, E. (1996) RGD and other recognition sequences for integrins. Annu Rev.Cell.Dev.Biol. 12, 697-715.

54. Hu, D.D., Barbas, C.F., Smith, J.W. (1996) An allosteric Ca2+ binding site on the beta3-integrins that regulates the dissociation rate for RGD ligands. J Biol Chem. 271, 21745-21751.

55. Greiling, D., Clark, R.A. (1997) Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix. J.Cell.Sci. 110, 861-870.

56. Gailit, J., Clark, R.A. (1996) Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen. J. Invest.Dermatol. 106, 102-8.

57. Woodard, A.S., Garcia-Cardena, G., Leong, M., Madri, J.A., Sessa, W.C., Languino, L.R. (1998) The synergistic activity of alphavbeta3 integrin and PDGF receptor increases cell migration. J.Cell.Sci. 111, 469-478.

58. Wu, P.L., Lee, S.C., Chuang, C.C., Mori, S., Akakura, N., Wu, W.G., Takada, Y. (2006) Non-cytotoxic cobra cardiotoxin A5 binds to alpha(v)beta3 integrin and inhibits bone resorption. Identification of cardiotoxins as non-RGD integrin-binding proteins of the Ly-6 family. J. Biol. Chem. 281 ,7937-7945.

59. 施秉辰 (2005) 1.比較東岸和西岸台灣眼鏡蛇蛇毒的高分子蛋白組成。2.N-鍵結的醣化作用對於台灣眼鏡蛇的蛇毒金屬蛋白水解酵素之活性影響。清華大學碩士論文。

59. Saarialho-Kere, U., Kerkela, E., Jahkola, T., Suomela, S., Keski-Oja, J., Lohi, J. (2002) Epilysin (MMP-28) expression is associated with cell proliferation during epithelial repair. J Invest Dermatol. 119, 14-21.

60. Sacco, O., Silvestri, M., Sabatini, F., Sale, R., Defilippi, A.C., Rossi, G.A. (2004) Epithelial cells and fibroblasts: structural repair and remodelling in the airways. Paediatr Respir Rev. 5, 35-40.

61. Asmussen, P.D., Sollner, B. (1993) Principles of wound healing . J.Am.Podiatr.Med.Assoc. 92, 12-18.

62. Hynes, R. O. (2002). "Integrins: bidirectional, allosteric signaling machines." Cell 110, 673-687.

63. 吳柏龍 (2006) 台灣眼鏡蛇毒與整合素作用之非RGD毒蛋白的鑑定與生物活性探討。清華大學博士論文。

64. You,W.K., Seo, H.J., Chung, K.H., Kim, D.S. (2003) A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis through its protease and disintegrin-like domains. J Biochem (Tokyo). 134, 739-749.

65. You, W.K., Jang, Y.J., Chung, K.H., Jeon, O.H., Kim, D.S.(2006) Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation. Biochem Biophys Res Commun. 2339, 964-970.

66. Gao, Z., Sasaoka, T., Fujimori ,T., Oya, T., Ishii, Y., Sabit, H., Kawaguchi, M., Kurotaki, Y., Naito, M., Wada, T., Ishizawa, S., Kobayashi, M., Nabeshima ,Y., Sasahara, M. (2005) Deletion of the PDGFR-beta gene affects key fibroblast functions important for wound healing. J Biol Chem. 280, 9375-9389.

67. Li, W., Fan, J., Chen, M., Guan, S., Sawcer, D., Bokoch, G.M., Woodley, D.T. (2004) Mechanism of human dermal fibroblast migration driven by type I collagen and platelet-derived growth factor-BB. Mol Biol Cell. 215, 294-309.

68. Wu, P.L., Lee, S.C., Chuang, C.C., Mori, S., Akakura, N., Wu, W.G., Takada, Y. (2006) Non-cytotoxic cobra cardiotoxin A5 binds to alpha(v)beta3 integrin and inhibits bone resorption. Identification of cardiotoxins as non-RGD integrin-binding proteins of the Ly-6 family. J Biol Chem. 281, 7937-45.

69. Wu, W.B., Chang, S.C., Liau, M.Y., Huang, T.F. (2001) Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells. Biochem J. 357, 719-28.

70. Clark, R.A., Lin, F., Greiling, D., An, J., Couchman, J.R. (2004) Fibroblast invasive migration into fibronectin/fibrin gels requires a previously uncharacterized dermatan sulfate-CD44 proteoglycan. J Invest Dermatol. 2122, 266-277.

71. Bridges, L.C., Bowditch, R.D. (2005) ADAM-Integrin Interactions: potential integrin regulated ectodomain shedding activity. Curr Pharm Des. 11, 837-847.