Background and purpose: Vipera lebetina lives in different areas in Iran, and its venom contains a variety of proteins with coagulant and anticoagulant activities. Fibrinolytic enzymes could have a therapeutic role in dissolution of blood clots, so, this study aimed at separating the venom components of Iranian V. lebetina and detecting its anticoagulant activity. Materials and methods: In this experimental study, crude venom components were isolated by gel filtration chromatography on sephadex G-100. We investigated the endopeptidase, arginine ester hydrolase, coagulant, anticoagulant, and fibrinolytic activities in crude venom and separated fractions. Results: The crude venom was separated into five fractions (PI-PV). 200 mg of crude venom contained 187 mg protein and 11. 75 mg protein was recovered from 187 mg protein used on the column. The venom showed coagulant activity at low concentrations and anticoagulant activity at high concentrations. Endopeptidase activity was detected in crude venom and all fractions except PV. Also, arginine ester hydrolase activity was seen in crude venom, PI, and PII. Fibrinolytic activity was found in crude venom and only in PIII. Conclusion: According to this study, the venom of Iranian V. lebetina has strong proteolytic activities including fibrinolytic that dissolve blood clots by lysis fibrin directly in laboratory conditions.

Objective: Envenomation by heamotoxic snakes constituted a critical health occurrence in the world. Bleeding is the most sever consequence following snake bite with viperid and crothalid snakes. It is believed that the degradation of vascular membrane caused hemorrhage; in contrast, some suggested that direct cytotoxicity has role in endothelial cell disturbances. This study was carried out to evaluate the direct toxicity effect of V. lebetina crude venom on Human Umbilical Vein Endothelial Cells (HUVECs). Methods: The effect of V. lebetina snake venom on HUVECs growth inhibition was determined by MTT assay and neutral red uptake assay. The integrity of cell membrane through LDH release was measured with the Cytotoxicity Detection Kit. Morphological changes of endothelial cells were also evaluated using a phase contrast microscope. Result: In MTT assay, crude venom showed a cytotoxic effect on endothelial cells which was confirmed by the effect observed with neutral red assay. Also, crude venom caused changes in the integrity of cell membrane by LDH release. The morphological alterations enhanced in high concentration results in total cells number reduced. Conclusion: V. lebetina venom showed potential direct cytotoxic effects on human endothelial cells in a manner of concentration-dependent inhibition.

Background and Objective: Snake venoms comprise complex mixtures of enzymatic and non-enzymatic proteins and small organic compounds. Hyaluronidase is a constant factor in the venom of the snake and is known as a spreading factor. This factor facilitate spread of toxins that harm into the blood circulation system.In this paper, we describe the determination of hyaluronidase activity in Iranian Vipera lebetina venom.Subjects and Methods: Hyaluronidase activity was assayed turbidometrically on hyaluronic acid. Turbidity reducing activity was expressed as a percentage of the hydrolysed hyaluronate, taking the absorbance of a tube as 100% in which no enzyme was added. Bovin testis hyaluronidase was used as standard.Results: The optima PH and temperature for hyaluronidase maximum activity was 6 and 37oC respectively.Conclusion: In conclusion the Iranian Vipera lebetina venom possessed considerable hyaluronidase activity.

Background and Objective: Snake venoms are complex mixtures containing many different biologically active proteins and peptides. Vipera lebetina is one of the most poisonous snakes in Iran. The aim of the present study was to test the existence of phospholipase A2 activity in the crude venom of the snake.Materials and Methods: One hundred mg of the crude venom of Vipera lebetina was resolved into five fractions using gel filtration chromatography on sephadex G-100, equilibrated with 20 mM ammonium acetate buffer pH 6.8. Protein concentration of crude venom and fractions was assayed by Bovin serum albumin as a standard. Phospholipase A2 (PLA2) activity was determined using suspension of egg yolk as substrate.Results: The preliminary results showed that the 89% of lyophilized venom was protein. Crude venom produced five fractions. These fractions were labeled as peak I to peak V (PI-P V), in order of their elution. Specific activity in crude venom, peak I, peak II and peak III were 2.25, 0.078, 2.76 and 1.04 U/mg.Conclusion: High PLA2 activity was detected in crude venom, which was more prominent in peak II and was not considerable in peak I of the fractions.

Background: Snake venom is a complex mixture of different components including proteins and peptides that mostly has enzymatic properties. In this study, the alkaline phosphatase (ALP) activity is measured in Iranian Vipera lebetina, venomous snake on sephadex G-100 to investigate the suitability of this procedure.Methods: The method of this study is experimental.100 mg of the crude venom of Vipera lebetina by gel filtration chromatography on sephadex G-100, that equilibrated with 20mM ammonium acetate buffer pH 7.6, was fractionated into 6 fractions. ALP enzyme activity was determined using Sulkowski et al method and using paranitrophenylphosphate as substrate.Results: Ninety mg (approximately 79%) of crude venom of Iranian Vipera lebetina is protein. Crude venom produced 6 distinct fractions.These fractions were labeled peak I to peak VI (PI-PVI), in order of their delution.Specific activity in crude venom and PI were 4x10-4 and 1x10-3 unit/mg, respectively. Other peaks didn’t show any activity of ALP enzyme.Conclusion: The results indicated that the Iranian Vipera lebetina venom has ALP enzyme activity and that their total activity belongs to PI. Other peaks didn’t show any ALP activity.

Background and Objective: Snake venom is a complex of several toxic elements and enzymes. It has the agents with the ability to destroy cellular and subcellular membrane and to bring about hemolysis of red blood cells (RBC). Two types of direct and indirect hemolytic activity are known in snake venom in that phospholipase A2 is responsible for the indirect lysis. The aim of this study was to investigate the effect of a-lipoic acid on hemolytic activity of Iranian Vipera lebetina venom.Material and Methods: Protein concentration of the crude venom of Vipera lebetina was determined using bovine serum albumin as a standard. Direct hemolytic activity of venom was determined by using the Human RBC and Indirect hemolytic activity was assayed on RBC in the presence of egg yolk. Then, a-lipoic acid with different concentrations in 100 mM Tris-HCL buffer was applied and its effect on hemolysis of RBC was studied.Results: direct hemolytic activity on RBC was not observed while its indirect activity was detected to be increased proportional to different concentration of a-lipoic acid. The range of indirect hemolysis was increased up to 60% by 60µm a-lipoic acid.Conclusion: Not only has a-lipoic acid no inhibitory effects on the hemolytic activity of Iranian Vipera lebetina venom but also has the positive effects on it.

Background and Objective: The venom of many Viperidae snake appear to contain proteins that affect blood coagulation. The aim of this study was to identify and isolate a blood coagulation factor V activator present in the venom from Vipera lebetina.Material and Methods: Factor V activator was purified from 200 mg of crude venom by three steps of chromatorgraphy which included: gel filtration on sephadex G100, Ion exchange chromatography on DEAE and cellulose and affinity chromatography on heparin agarose. The effect of factor V activator on human factor V was studied by measuring the amidiolytic activity of the produced thrombin by activated factor V (Va).Results: The results of SDS – PAGE identified an activator of factor V as a single protein band with a molecular weight of 29 KDa. This compound was converted, in the presence of calcium ions, to active factor Va. It also had arginine esterase activity toward substrate BAEE (NX-benzoyl arginine ethyester) and a weak amidase activity on S-2222 (benzoylle-glu-Gly-Arg-p- nitroanilide).Conclusion: The results of this study showed that the isolated factor is a specific activator on factor V.

Background and Objective: Snake venom particulary those belonging to Crotalidae and viperidae families, are known to contain number of components affecting blood coagulation.The Aim of this paper is to describe the isolation a specific blood coagulation factor X activator from the crude venom of Iranian Vipera lebetina.Material and Methods: Factor X activator was purified from two hundred mg of crude venom by gel filtration on sephadex G-100 and two step ion-exchange chromatography on DEAE-cellulose (DEAE-52).Results: It showed a single band in sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE) in non-reducing condition. The mol. wt was estimated to be 78000Da by SDS-PAGE the activator, activated factor X to Xa in presence of calcium ions. It could not activate prothrombin, no had effect an fibrinogen and thus appeared to act specifically on factor X.The activator no amidolytic activtiy on factor Xa substrate benzoyl-Ile-glu-Gly-Arg-p-nitracnilide (S-2222).It had no arginine esterase activity toward substrate benzoyl-agrinine ethylester (BAEE) while crude venom hydrolyzes this substrate.Conclusion: The results of this study showed that The activator do act specifically on factor X.

Sixteen healthy native dogs aged about 2 years with average body weight of 22.5±1.5 kg of both sexes were selected for this study. Dogs were divided randomly into four groups (4 dogs/group). Clinical signs were recorded and blood samples were collected for laboratory examination before injection of venom. The control group was injected with 1ml of saline solution and the groups 2-4 respectively received 0.1,0.5 and 1mg/kg of the venom powder of Vipera lebetina dissolved in 1ml saline solution, injected into the hind limb (biceps femoris) of each dog. Clinical signs were recorded and blood samples were collected at different time (10, 20, 30 and 60 minutes and 2, 4, 8 and 24 hours) after injection of venom examined for sedimentation rate, WBC count, number of neutrophils, RBC and platelet count, HCT, Hb concentration, MCV, MCH, MCHC, bleeding and clotting time, PT and PTT. The results were analyzed statistically.Results indicated that the main clinical signs were pain, pawing, weakness, whimpering, leash chewing, subcutaneous hemorrhage, hypotension and death in dogs in high doses. Heart and respiratory rate and body temperature in dogs of groups 2, 3 and 4 increased after injection of snake venom (P<0.05) which could be related to pain, stress and tissue damage at the site of venom injection. This resulted in an increase in body temperature due to release of pyrogenic substances especially in dogs of group 4. Number of white blood cells (WBC) decreased significantly (P<0.05) which could be related to stress and accumulation of white blood cells in the foci of exogenic factor and consumption of WBC and tissues damage. Comparison of means of platelets, indicated a significant difference in dogs of the group 3 (P<0.05). Primary decrease in the platelets of dogs is due to consumption of platelets in blood circulation at the site of venom injection and hemorrhage then increased due to body replacement. A significant increase (P<0.05) in coagulation, prothrombin and partial thromoplastin time in dogs of groups 3 and 4 was observed. This increase could be related to anticoagulant factor of the venom of this snake, which showed its effects in high doses. It is concluded that in addition to clinical signs and hematological changes, coagulopathy and hemolysis in high doses of Vipera lebetina venom were the main effects on hematological parameters in this respect.

Background: The hemotoxic and neurotoxic factors of snake venoms is the main responsible for necrosis and tissue sloughing. Envenomations are common in rural areas in all provinces of Iran caused by snake species which causes local swelling, ecchymosis and alterations in blood profile in case of hemotoxic venom. In this study some in vivo and in vitro properties (Hemorrhagic, edematogenic and coagualant) of Iranian Vipera lebetina venom in addition to neutralizing capacity of pepsin derived Razi Institute polyvalent antivenin were assayed.Material and Methods: Escalating doses of Vipera lebetina venom dissolved in Normal saline (2.5-50 mg/ml) were injected (100 ml) subcutaneously to dorsal area of rats (n=3) to investigate mean hemorrhagic amount after 24 hours.Groups of three mice were injected subcutaneously in the right foodpad with various amounts of venom (10-150 mg).The left foodpad received the same amount (100 ml) of normal saline alone (negative control) to evaluate the edematogenic property of this venom. To determine the coagulant activity, various amounts of venom dissolved in normal saline (50 ml) were added to human plasma (200 ml) and coagulation time was measured. Razi Institute antivenom was used for neutralization of all three measured biological parameters.Results: Mean hemorrhagic, procoagulant and edematous amounts (increasing 30% in hind paw edema) were 8.5, 1.1 and 70 microgram, respectively. Preincubation with polyvalent antibody (30 and 200 microliter) decreased hemorrhagic and procoagulant activity. Edematogenic property of this venom decreased significantly by incubation with antivenom (78% to 38% by incubation with 1000 microliter of polyvalent antivenom). Intra peritoneal injection of this remedy following envenomation had no effect in relieving symptoms. Myonecrotic effects were seen by intramuscular injection of Vipera lebetina venom in rats.Conclusion: Our study shows that Iranian antivenom could neutralize some in vivo and in vitro hazardous effects of envenomation by this snake like hemorrhagic, edematogenic and procoagulant properties that paves the way for separation and purification of multiple enzymes present in this venom to investigate the neutralizing capacity from Razi Institute polyvalent antivenom.