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

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

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

5. 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) 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. Isfahan University of Technology Department of Chemistry 3

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

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

8. Introduction Isfahan University of Technology Department of Chemistry 6

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

10. Introduction Prothrombin to ThrombinFibrinogen to Fibrin Clot Formation 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. Isfahan University of Technology Department of Chemistry 8

11. Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3 Isfahan University of Technology Department of Chemistry 9

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

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

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

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

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

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

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

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

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

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

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

23. Conclusions Isfahan University of Technology Department of Chemistry 21 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 carinatus venom 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

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

26. Isfahan University of Technology Department of Chemistry