BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Evidence for Existence of a Water-Extractable Anticoagulant in an Earthworm, Lumbricus rubellus

  • Woo, Jeong-Im (Department of Biochemistry, College of Medicine, Inha University) ;
  • Bahk, Yun-Kyung (Department of Biochemistry, College of Medicine, Inha University) ;
  • Yu, Kyoung-Hee (Department of Biochemistry, College of Medicine, Inha University) ;
  • Paik, Seung-R. (Department of Biochemistry, College of Medicine, Inha University) ;
  • Chang, Chung-Soon (Department of Biochemistry, College of Medicine, Inha University)
  • Received : 1996.07.16
  • Published : 1996.11.30
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Abstract

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.

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References

  1. Anal. Biochem. v.72 Bradford, M.M.
  2. Semin. Thromb. Hemostasis v.21 Callas, D.D.;Hoppensteadt, D.;Fareed, J. https://doi.org/10.1055/s-2007-1000393
  3. Biochem. J. v.93 Curragh, E.F.;Elmore, D.T. https://doi.org/10.1042/bj0930163
  4. Biochemistry v.30 Davie, E.W.;Fujikawa, K.;Kisel, W. https://doi.org/10.1021/bi00107a001
  5. J. Biol. Chem. v.265 Esmon, C.T.
  6. Can. J. Biochem. Physiol. v.37 Hummel, B.C.W. https://doi.org/10.1139/y59-157
  7. Semin. Hematol. v.17 Kelton, J.G.;Hirsch, J.
  8. J. Biochem. Mol. Biol. (formerly Korean Biochem. J.) v.28 Kim, Y.S.;Kim, J.E.;Byun, H.S.;Chang, C.S.;Suh, J.J.
  9. Nature v.227 Laemmli, U.K. https://doi.org/10.1038/227680a0
  10. Am. J. Med. Tech. v.48 Longberry, J.
  11. Blood v.76 Mann, K.G;Nesheim, N.E.;Church, W.R.;Haley, P.;Krishnaswamy, S.
  12. Thrombolytic agent Mihara, H.
  13. Anticancer Res. v.11 Nagasawa, H.;Sawaki, K.;Fuji, Y.;Kobayashi, M.;Segawa, T.;Suzuki, R.;Inatomi, H.
  14. Chem. Pharm. Bull. v.40 Noda, N.;Tsunefuka, S.;Tanaka, R.;Miyahara, K. https://doi.org/10.1248/cpb.40.2756
  15. J. Biochem. Mol. Biol. (formerly Korean Biochem. J.) v.29 Paik, S.R.;Kim, D.H.;Chang, C.S.
  16. Trends Biochem. Sci. v.20 Stubbs, M.T.;Bode, W. https://doi.org/10.1016/S0968-0004(00)88945-8
  17. J. Invertebr. Pathol. v.51 Valembois, P.;Roch, P.;Lassegues, M. https://doi.org/10.1016/0022-2011(88)90029-8
  18. Biochim. Biophys. Acta v.14 Van Thoai, N.;Robin, Y. https://doi.org/10.1016/0006-3002(54)90133-X
  19. Chem. Pharm. Bull. v.37 Wang, J.D.;Narui, T.;Kurata, H.;Takeuchi, K.;Hashimoto, T.;Okuyama, T. https://doi.org/10.1248/cpb.37.2236