Partial Purification and Characterization of Anticoagulant Factor from the Snake (Echis carinatus) Venom

Document Type: Original Article

Authors

1 Payam Noor University of Tehran, Tehran, Iran

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

Abstract

 




Objective(s): Snake venoms contain complex mixture of proteins with biological activities. Some of these proteins affect blood coagulation and platelet function in different ways. Snake venom toxin may serve as a starting material for drug design to combat several pathophysiological problems such as cardiovascular disorders. In the present study, purification of anticoagulation factor from venom of snake (Echis carinatus) was studied. Anticoagulation activity of crude venom, fractions and purified peptide were determined by using prothrombin time (PT) and thrombin time (TT). Three fractions were partially purified from the venom of E. Carinatus by gel filtration on sephadex G-75 and final purification was performed by high-performance liquid chromatography (HPLC) with C18 column. A purified anticoagulant factor was derived which showed a single protein band in SDS-PAGE electrophoresis under reducing condition. Results of PT and TT tests for purified peptide (EC217) were found to be 102±4.242 and < 5 min. respectively. Determination of molecular weight revealed that the active purified peptide (EC217) was about 30 KD. In conclusion, the present study showed that the venom of E. carinatus contains at least one anticoagulant factor.

Keywords


1

. Julian W. Snake venoms and coagulopathy. Toxicon 2005; 45:951–967.

2. Kini RM

. Serine proteases affecting blood coagulation and fibrinolysis from snake venoms. Pathophysiol Haemost Thromb 2005; 34:200– 204.

3. Pirkle H, Markland Jr. FS. Hemostasis and Animal Venoms. Marcel Decker, New York:1987.p.655-94.

4. Hutton RA, Warrell DA. Action of snake venom components on the haemostatic system. Blood Rev 1993; 7:176–189.

5. Lu Q, Clemetson JM, Clemetson KJ. Snake venoms and hemostasis. J Thromb Haemostasis 2005; 3:1791–1799.

6. Markland FS. Snake venoms and the hemostatic system. Toxicon 1998; 36:1749–1800.

7. Scarborough RM, Naughton MA, Teng W, Rose JW, Phillips DR, Nannizzi L,

et al. Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J Biol Chem 1993; 268: 1066–1073.

8. Phillips DR, Scarborough RM. Clinical pharmacology of eptifibatide. Am J Cardiol 1997; 80

:11–20.

9. Kornalik F. The influence of snake venom proteins on blood coagulation. In: Harvey AL, editor. Snake Toxins. New York: Pergamon Press; 1991.p. 323–383.

10. Gan ZR, Gould RJ, Jacobs JW, Friedman PA, Polokoff MA. Echistatin. A potent platelet aggregation inhibitor from the venom of the viper. J Biol Chem 1988; 263:19827-19832.

11. Jasti J, Paramasivam M, Srinivasan A, Singh TP. Structure of an acidic phospholipase A2 from Indian saw-scaled viper (

Echis carinatus) at 2.6 A resolution reveals a novel intermolecular interaction. Acta Crystallogr D Biol Crystallogr 2004; 60:66-72.

12. Navdaev A, Clemetson KJ. GPIb cross-linking/ligation on echicetin-coated surfaces or echicetin/IgMκ in stirred suspension activates platelets by cytoskeleton modulated calcium release. J Biol Chem 2002

; 277: 45928- 95434.

13

. Navdaev A, Dormann D, Clemetson JM, Clemetson KJ. Echicetin, a GPIb- binding Snake C-type lectin from Echis carinatus, also contains a binding site for IgMκ, responsible for platelet agglutination in plasma and inducing signal transduction. Blood 2001; 97: 2333-2341.

14. Shebuski RJ

, Ramjit DR, Sitko GR, Lumma PK, Garsky VM. Citation: Prevention of canine coronary artery thrombosis with echistatin, a potent inhibitor of platelet aggregation from the venom of the viper, Echis carinatus. Thromb Haemoss 1990; 64:576-581.

15. Kemparaju K, Nijaguna Prasad B, Veerabasappa Gowda T. Purification of a basic phospholipase A2 from Indian saw-scaled viper (

Echis carinatus) venom:

Anticoagulant from Echis carinatus Amrollahi Byoki and Zare Mirakabadi

Iran J Basic Med Sci, Vol. 16, No. 11, Nov 2013

 

1144

 

Chracterization of antigenic, catalytic and pharmacological properties. Toxicon 1994; 32:1187-1196.

16. Ali G, Kak M, Kumar M, Bali SK, Tak SI, Hassan G, Wadhwa MB. Acute renal failure following E

chis carinatus (saw–scaled viper) envenomation. Ind J Neph?? 2004; 14:177-181.

17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193, 265–275.

18. Quick AJ. The prothrombin time in haemophilia and in obstructive jaudice. J Biol Chem 1935; 109:73–74.

19. Bajwa SS, Markland FS, Russel FE. Fibrinolytic enzyme(s) in western diamondback rattlesnake (

Crotalus atrox) venom. Toxicon 1980; 18:185– 290.

20. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685.

21.Peichoto ME, Teibler P, Mackessy SP, Leiva L, Acosta O, Gonçalves LR,

et al. Purification and characterization of patagonfibrase, a metalloproteinase showing α-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom. Biochim Biophys Acta 2007; 1770:810-819.

22. Ghorbanpur M, Zare Mirakabadi A, Zokaee F, Zolfagharian H. Identification and partial purification of an anticoagulant factor from the venom of the Iranian snake

Agkistrodon halys. J Venom Anim Toxins incl Trop Dis 2009; 16:96-106, 1678-9199.

23. Ouyang CH, Ma YH, Jih HC, Teng CM. Characterization of the platelet aggregation inducer and inhibitor from

Echis carinatus snake venom. Biochim Biophys Acta 1985; 841:1, 1-7.

24. Peng M, Lu W, Beviglia L, Niewiarows ki, Kirby EP. Echicetin a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib. Blood 1993; 81:2321-2328.

25. Peng M, Emig FA, Mao A. Interaction of echicetin with a high affinity thrombin binding site on platelet glycoprotein GPIb. Thromb Haemost 1995; 74:954-957.

26. Fujimura YikedaY, Miura S. Isolation and characterization of jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib. Thromb Haemost 1995; 74:743-750.

27. Gomes MS, de Queiroz MR, Mamede CC, Mendes MM, Hamaguchi A, Homsi-Brandeburgo MI,

et al. Purification and functional characterization of a new metalloproteinase from Bothrops leucurus snake venom. Comp Biochem Physiol C Toxicol Pharmacol 2011; 153:290-300.