Blood Coagulation Induced by Iranian Saw-Scaled Viper (Echis Carinatus) Venom: Identification, Purification and Characterization of a Prothrombin Activator

Document Type : Original Article

Authors

1 Young Researches and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute, Karaj, Iran

Abstract

 

Objective(s): Echis carinatus is one of the venomous snakes in Iran. The venom of Iranian Echis carinatus
is a rich source of protein with various factors affecting the plasma protein and blood coagulation factor. Some of these proteins exhibit types of enzymatic activities. However, other items are proteins with no enzymatic activity.
 
Materials and Methods:
In order to study the mechanism and effect of the venom on human plasma proteins, the present study has evaluated the effect of crude venom and all fractions. A procoagulant factor (prothrombin activator) was isolated from the venom of the Iranian snake Echis carinatus with a combination of gel filtration (Sephadex G-75), ion-exchange chromatography (DEAE- Sepharose) and reverse phase HPLC. Furthermore, proteolytic activity of the crude venom and all fractions on blood coagulation factors such as prothrombin time (PT) was studied.
Results:
In the present study, the PT test was reduced from 13.4 s to 8.6 s when human plasma was treated with crude venom (concentraion of venom was 1 mg/ml). The purified procoagulant factor revealed a single protein band in SDS polyacrylamide electrophoresis under reducing conditions and its molecular weight was estimated at about 65 kDa. A single-band protein showed fragment patterns similar to those generated by the group A prothrombin activators, which convert prothrombin into meizothrombin independent of the prothrombinase complex.
Conclusion:
This study showed that the fraction which separated from Iranian snake Echis carinatus venom can be a prothrombin activators. It can be concluded that this fraction is a procoagulant factor.

Keywords


 
1. Gnanathasan A, Rodrigo C, Peranantharajah T, Coonghe A. Saw-scaled viper bites in Sri Lanka: is it a different subspecies. Clinical evidence from an authenticated case series. Am J Trop Med Hyg 2012; 86:254–257.
2. Matsui T, Fujimura Y, Titani K. Snake venom proteases affecting hemostasis and thrombosis. Biochim Biophys Acta 2000; 1477:146-156.
3. Roberto G, Alessio C, Nnadozie S, Hope O, Emiliano F, Helena C,
et al. In vitro effects of Echis carinatus venom on the human plasma proteome. Proteomics 2010; 10:3712-3722.
4. Kularatne SAM, Sivansuthan S, Medagedara SC, Maduwage K, de Silva A. Revisiting saw-scaled viper (
Echis carinatus) bites in the Jaffna Peninsula of Sri Lanka: distribution, epidemiology and clinical manifestations. Trans R Soc Trop Med Hyg 2011; 105:591–597l.
5. Fonseka CL, Jeevagan V, Gnanathasan CA. Life threatening intracerebral haemorrhage following saw-scaled viper (
Echis carinatus) envenoming-authenticated case report from Sri Lanka. BMC Emerg Med 2013; 13:13-15.
6. Morita T, Iwanga S. Purification and properties of prothrombin activator from the venom of
Echis carinatus. J Biochem 1978; 83:559-570.
7. Lövgren A. Recombinant snake venom prothrombin activators. Bioengineered 2013; 4:153–157
8. Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clotting. Science 1964; 145:1310–1312.
9. Macfarlane RG. An Enzyme Cascade in the Blood Clotting Mechanism, and it’s Function as a Biochemical Amplifier. Nature 1964; 202:498-499.
10. Jackson CM, Nemerson Y. Blood coagulation. Annu Rev Biochem 1980; 49:765-811.
11. Rosing J, Tans G. Meizothrombin, a major product of factor Xa-catalyzed prothrombin activation. Thromb Haemostasis 1988; 60:355-360.
12. Mann KG. The coagulation explosion. Ann NY Acad Sci 1994; 714:265-269.
13. Heldebrant CM, Noyes C, Kingdon HS, Mann KG. The activation of prothrombin. The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation. Biochem Biophys Res Commun 1973; 54:155-160.
14. Maruyama M, Kamiguti AS, Tomy SC, Antonio LC, Sugiki M, Mihara H. Prothrombin and factor X activating properties of
Bothrops erythromelas venom. Ann Trop Med Parasitol 1992; 86:549-556.
15. Casewell NR, Harrison RA, Wüster W, Wagstaff SC. Comparative venom gland transcriptome surveys of the saw-scaled vipers (Viperidae:
Echis) reveal substantial intra-family gene diversity and novel venom transcripts. BMC Genom 2009; 10:147-168.
16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275.
17. Ghorbanpur M, Zare Mirakabadi A, Zokaee F, Zolfagharian H, Rabiei H. Purification and partial characterization of a coagulant serine protease from the venom of the Iranian snake
Agkistrodon halys. J Venom Anim Toxins Incl Trop Dis 2009; 15:411-423.
18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage, T4. Nature 1970; 227:680-685.
19. Rizzo F, Papasouliotis K, Crawford E, Dodkin S, Cue S. Measurement of prothrombin time (PT) and activated partial thromboplastin time (APTT) on canine citrated plasma samples following different storage conditions. Res Vet Sci 2008; 85:166-170.
20. Kini RM, Rao VS, Joseph JS. Procoagulant proteins snake venoms. Thromb Haemostasis 2001; 31:218-224.
Blood Coagulation Induced by Echis carinatus Venom
 
Babaei et al
Iran J Basic Med Sci, Vol. 16, No. 11, Nov 2013
 
1150
 
21. Helene H, Cassian B. Blood coagulation induced by the venom of
Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator. Biochemistry 1986; 26:772-780.
22. Howes JM, Kamiguti AS, Theakston RDG, Wilkinson MC, Laing GD. Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper,
Echis ocellatus on blood coagulation and platelets. Biochim Biophys Acta 2005; 1724:194-202.
23. Yamada D, Sekiya F, Morita T. Isolation and characterization of carinactivase, a novel prothrombin activator in
Echis carinatus venom with a unique catalytic mechanism. J Biol Chem 1996; 271:5200-5207.
24. Morita T, Iwanaga S, Suzuki T. The mechanism of activation of bovine prothrombin by an activator isolated from
Echis carinatus venom and characterization of the new active intermediates. J Biochem 1976; 79:1089-1098.
25. Zhang Y, Lee WH, Gao R, Xiong YL, Wang WY, Zhu SW. Effects of
Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation and characterization of a prothrombin activator. Toxicon 1998; 36:143-152.
26. Hofmann H, Bon C. Blood coagulation induced by the venom of
Bothrops atrox. 1. Identification, purification and properties of a prothrombin activator. Biochemistry 1987; 26:772–780.
27. Paine MJ, Desmond HP, Theakston RD, Crampton JM. Purification, cloning and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin from
Bothrops jararaca venom. Insights into the disintegrin gene family. J Biol Chem 1992; 267:22869-22876.
28. Siigur E, Siigur J. Purification and characterization of lebetase, a fibrinolytic enzyme from
Vipera lebetina (snake) venom. Biochim Biophys Acta 1991; 1074:223-229.
29. Takeya H, Nishida S, Miyata T, Kawada S, Saisaka Y, Morita T,
et al. Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains. Biol Chem 1992; 267:14109-14117.