presenter: mohammad javad hosseinishahi dastjerdi evaluation of blood coagulation and separation of...
TRANSCRIPT
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
Introduction
Experimental section
Conclusions
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Content
-Preparation sample
-Homeostasis test
-Separation methods
-Advantages of snake’s venoms
-Types of snake’s venoms
-Blood coagulation processes
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 drugs Variety of serums Variety of vaccines
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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.
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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/EchisRef: http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd
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Introduction
Taipan Snake
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Introduction
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Introduction
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Coagulation cascade in blood
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Introduction
Prothrombin to Thrombin Fibrinogen 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.
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Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3
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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
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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
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The PTT is functional determination of the intrinsic pathway of coagulation
Normal Values and Critical Limits 12 - 15 seconds
Experimental section
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Experimental section
Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3
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PT test prior to Echis carinatus venom protein separation
Presence of Echis carinatus venom---> Accelerate the coagulation
Experimental section
Ion Exchange Chromatography
Gel Filtration Chromatography
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Separation
Methods
HPLC
Electrophoresis
Experimental section
Ion Exchange Chromatography
Partition Chromatography
Gel Filtration Chromatography
HPLC
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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
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Experimental section
Gel Filtration Chromatography
Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3
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Experimental section
Ion Exchange Chromatography
Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3
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Experimental section
Ion Exchange Chromatography
Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3
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Experimental section
Partition Chromatography
Ref: Salmanizadeh et al. J Venom Anim Toxins incl Trop Dis. 2013, 19:3
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Conclusions
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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
Conclusions
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Echis carinatus venom can be used in operations and for analysis of blood of the patients who have liver disease
Isfahan University of Technology Department of Chemistry