Evaluation of blood coagulation and separation of proteins in Echis carinatus venom

2. Isfahan University of Technology Department of Chemistry M.Sc. Seminar Evaluation of blood coagulation and separation of proteins in Echis carinatus venom Supervisor: Prof.M.Saraji Presenter: Mohammad Javad Hosseinishahi Dastjerdi

3. Isfahan University of Technology Department of Chemistry Content Introduction -Advantages of snake’s venoms -Types of snake’s venoms - Blood coagulation processes Experimental section -Preparation sample -Homeostasis test -Separation methods Conclusions 1

4. Isfahan University of Technology Department of Chemistry Introduction Snakebite affects around 2.5 million humans annually, accounting for more than 100,000 deaths Snake’s venom used for produce Variety of drugs Variety of serums Variety of vaccines 2 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

5. Isfahan University of Technology Department of Chemistry Introduction Snake’s venom is oily and acidic liquid Depending on the type of snake’ white to dark yellow transparent or opaque Snake’s venom is a complex mixture of proteins and enzymes and non-proteins (Types of lipids, Carbohydrates, Salts of various metal and metalloid, Riboflavin and water) 3 Ref: White J: Snake venoms and coagulopathy. Toxicon 2005, 45(8):951–967. Ref:Sajevic T, Leonard A, Križaj I: Haemostatic ally active proteins in snake venoms. Toxicon 2011, 57(5):627–645.

6. Isfahan University of Technology Department of Chemistry Introduction Classification of snake’s toxins Neurotoxin Myotoxin Vasculotoxin Viperidae Echis carinatus (Saw-scaled) Species of this genus are found in Pakistan, India, Sri Lanka, parts of the Middle East, Africa and north of the equator 4 Ref: http://en.wikipedia.org/wiki/Echis Ref: http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd

7. Isfahan University of Technology Department of Chemistry Introduction Taipan Snake 5

8. Isfahan University of Technology Department of Chemistry Introduction 6

9. Isfahan University of Technology Department of Chemistry Coagulation cascade in blood Introduction 7

10. Isfahan University of Technology Department of Chemistry Introduction Prothrombin to Thrombin Fibrinogen to Fibrin Clot Formation 8 Ref: 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(3):411–423.

11. Isfahan University of Technology Department of Chemistry Experimental section Separation methods Evaluation of blood coagulation 9 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

12. Isfahan University of Technology Department of Chemistry Experimental section Prothrombin time (PT) test Evaluation of blood coagulation Partial thromboplastin time (PTT) test 10 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

13. Isfahan University of Technology Department of Chemistry Experimental section Prothrombin time (PT) test The PT is functional determination of the intrinsic pathway of coagulation Specimen Requirements: thromboplastin, citrated or oxalate, platelet-poor plasma and Calcium is used for the PT Normal Values and Critical Limits  12 - 15 seconds Partial thromboplastin time (PTT) test The PTT is functional determination of the intrinsic pathway of coagulation Specimen Requirements: cephalin, citrated or oxalate, platelet-poor plasma andcalcium is used for the PTT Normal Values and Critical Limits  60 - 75 seconds 11 Ref: Hirsh J, Bates S: The multiples faces of the partial thromboplastin time APTT. J Thromb Haemost 2:2254-2256, 2004

14. Isfahan University of Technology Department of Chemistry Experimental section 12

15. Isfahan University of Technology Department of Chemistry Experimental section PT test prior to Echiscarinatusvenom protein separation Presence of Echiscarinatus venom---> Accelerate the coagulation 13 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

16. Isfahan University of Technology Department of Chemistry Experimental section Electrophoresis Separation Methods Gel Filtration Chromatography Ion Exchange Chromatography HPLC 14

17. Isfahan University of Technology Department of Chemistry Experimental section HPLC Gel Filtration Chromatography Ion Exchange Chromatography Partition Chromatography 15

18. Isfahan University of Technology Department of Chemistry Experimental section Preparative Ec venom for separation of proteins Lyophilization Ec venom Ammonium acetate buffer (pH 6.8) Centrifuging Eluted with buffer 16 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

19. Isfahan University of Technology Department of Chemistry Experimental section Gel Filtration Chromatography 17 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

20. Isfahan University of Technology Department of Chemistry Experimental section Ion Exchange Chromatography 18 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

21. Isfahan University of Technology Department of Chemistry Experimental section Ion Exchange Chromatography 19 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

22. Isfahan University of Technology Department of Chemistry Experimental section Partition Chromatography 20 Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3

23. Isfahan University of Technology Department of Chemistry Conclusions • This research provide a effective and repeatable method for separation of coagulation factors of Echis carinatus • With respect of coagulation test advantages, specially PT test it was shown that Echis carinatus venom has a considerable considerable coagulation specification at all • Echis carinatusvenom can directly affect the prothrombin and coagulation blood without completing coagulation cascade steps , whereas blood coagulation will be disturbed if any co-factors disrupts in body for blood coagulation • HPLC is a efficient technique for separation of available proteins in Echis carinatus venom 21

24. Isfahan University of Technology Department of Chemistry Conclusions • Echis carinatusvenom can be used in operations and for analysis of blood of the patients who have liver disease 22

25. Thanks a lot for attention

26. Isfahan University of Technology Department of Chemistry