Abstract
Aim:
To study the enzymological characterization of a fibrinolytic enzyme (FIIa) from Agkistrodon acutus venom.
Methods:
The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FIIa was determined by atomic absorption spectroscopy.
Results:
After incubation with FIIa (0.25 g/L), Aα-, Bβ- and γ-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h, respectively. The molecular weights of major degradation products were 45 000 and 41 000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171±25 mg/kg), potassium (489±17 mg/kg) and calcium (319±13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA.
Conclusion:
FIIa can degrade the Aα-, Bβ- and γ-chains of fibrinogen. FIIa is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.
Similar content being viewed by others
Article PDF
References
Tseng YL, Lee CJ, Huang TF . Effects of a snake venom metalloproteinase, triflamp, on platelet aggregation, platelet-neutrophil and neutrophil-neutrophil interactions: involvement of platelet GPIbα and neutrophil PSGL-1. Thromb Haemost 2004; 91: 315–24.
Tatematsu R, Komori Y, Nikai T . A new thrombin-like enzyme, flavoviridiobin from the venom of Trimeresurus flavoviridis (habu). J Nat Toxins 2000; 9: 327–39.
Zhang Y, Wisner A, Xiong Y, Bon C . A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. J Biol Chem 1995; 270: 10 246–55.
Liang XX, Chen JS, Zhou YN, Qiu PX, Yan GM . Purification and biochemical characterization of FIIa, a fibrinolytic enzyme from Agkistrodon acutus venom. Toxicon 2001; 39: 1133–9.
Hahn BS, Chang IM, Kim YS . Purification and characterization of piscivorase I and II, the fibrinolytic enzymes from eastern cottonmouth moccasin venom (Agkistrodon piscivorus). Toxicon 1995; 33: 929–41.
Trikha M, Schmitmeier S, Markland FS . Purification and characterization of fibrolase isoforms from venom of individual southern copperhead (Agkistrodon contortrix) snakes. Toxicon 1994; 32: 1521–31.
Ouyang C, Hwang LJ, Huang TF . Alpha-fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom. Toxicon 1983; 21: 25–33.
Maruyama M, Sugiki M, Yoshida E, Shimaya K, Mihara H . Broad substrate specificity of snake venom fibrinolytic enzymes: possible role in haemorrhage. Toxicon 1992; 30: 1387–97.
Willis TW, Tu AT . Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom. Biochemistry 1988; 27: 4769–77.
Hung CC, Huang KF, Chiou SH . Characterization of one novel venom protease with beta-fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatus): purification and cDNA sequence analysis. Biochem Biophys Res Commun 1994; 205: 1707–15.
Siigur E, Siigur J . Purification and characterization of lebetase, a fibrinolytic enzyme from Vipera lebetina (snake) venom. Biochim Biophys Acta 1991; 1074: 223–9.
Ou-yang C, Huang TF . Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom. Biochim Biophys Acta 1976; 439: 146–53.
Chen JS, Liang XX, Qiu PX, Yan GM . Thrombolysis effect with FIIa from Agkistrodon acutus venom in different thrombosis model. Acta Pharmacol Sin 2001; 22: 420–2.
Gasmi A, Chabchoub A, Guermazi S, Karoui H, Elayeb M, Dellagi K . Further characterization and thrombolytic activity in a rat model of fibrinogenase from Vipera Lebetina venom. Thromb Res 1997; 86: 233–42.
Pinto AF, Dobrovolski R, Veiga AB, Guimaraes JA . Lonofibrase, a novel α-fibrinogenase from Lonomia obliqua caterpillars. Thromb Res 2004; 113: 147–54.
Swenson S, Markland, FS Jr . Snake venom fibrin(ogen)olytic enzymes. Toxicon 2005; 45: 1021–39.
Daoud E, Tu AT, el-Asmar MF . Mechanism of the anticoagulant, Cerastase F-4, isolated from Cerastes cerastes (Egyptian sand viper) venom. Thromb Res 1986; 41: 791–9.
Gasmi A, Karoui M, Benlasfar Z, Karoui H, el-Ayeb M, Dellagi K . Purification and characterization of a fibrinogenase from Vipera lebetina (desert adder) venom. Toxicon 1991; 29: 827–36.
Gatta D, Dong A, Witt J, Tu AT . Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus. Arch Biochem Biophys 1995; 317: 365–73.
Siigur J, Samel M, Tonismagi K, Subbi J, Siigur E, Tu AT . Biochemical characterization of lebetase, a direct-acting fibrinolytic enzyme from Vipera lebetina snake venom. Thromb Res 1998; 90: 39–49.
Gomis-Ruth FX, Kress LF, Bode W . First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinase/collagenase. EMBO J 1993; 12: 4151–7.
Zhang D, Botos I, Gomis-Ruth FX, Doll R, Blood C, Njoroge FG et al. Structural interaction of natural and synthetic inhibitors with the venom metalloproteinase, atrolysin c (from d). Proc Natl Acad Sci USA 1994; 91: 8447–51.
Assakura MT, Reichl AP, Asperti MC, Mandelbaum FR . Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca). Toxicon 1985; 23: 691–706.
De-Camargo-Goncalves LR, Chudzinski-Tavassi AM . High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom. Biochem Biophys Res Commun 2004; 318: 53–9.
Author information
Authors and Affiliations
Corresponding author
Additional information
Project supported by the Guangdong Science and Technology Commission (No 001365).
Rights and permissions
About this article
Cite this article
Liang, Xx., Zhou, Yn., Chen, Js. et al. Enzymological characterization of FIIa, a fibrinolytic enzyme from Agkistrodon acutus venom. Acta Pharmacol Sin 26, 1474–1478 (2005). https://doi.org/10.1111/j.1745-7254.2005.00204.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2005.00204.x
Keywords
This article is cited by
-
Isolation and characterization of two new non-hemorrhagic metalloproteinases with fibrinogenolytic activity from the mapanare (Bothrops colombiensis) venom
Archives of Toxicology (2013)
-
The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism
Acta Pharmacologica Sinica (2011)