Purification and partial characterization of a coagulant serine protease from the venom of the Iranian snake Agkistrodon halys

Agkistrodon halys is one of several dangerous snake species in Iran. Among the most important signs and symptoms in patients envenomated by this snake is disseminated intravascular coagulation. A thrombin-like enzyme, called AH143, was isolated from Agkistrodon halys venom by gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a DEAE-Sepharose and high performance liquid chromatography (HPLC) on a C18 column. In the final stage of purification, 0.82 mg of purified enzyme was obtained from 182.5 mg of venom. The purified enzyme showed a single protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, and its molecular mass was found to be about 30 kDa. AH143 revealed clotting activity in human plasma, which was not inhibited by EDTA or heparin. This enzyme still demonstrated coagulation activity when exposed to variations in temperature and pH ranging, respectively, from 30 to 40°C and from 7.0 to 8.0. It also displayed proteolytic activities on synthetic substrate. The purified enzyme did not show any effect on casein. We concluded that the venom of the Iranian snake Agkistrodon halys contains about 0.45% single procoagulant protein which appears to be a thrombin-like enzyme

An in vitro Comparative study upon the Hemolytic, Thrombogenic, Coagulation parameters and Stability properties of the Hemiscorpius lepturus Venom

Hemiscorpius lepturus belonging to Hemiscorpiidae family is the most venomous of all types of scorpion existing in south west of Iran causing hemoglobinuria and dermal lesions by envenomation. We compare the hemolytic pattern upon time in different domestic animals upon time according to their different sphingomyelin contents. In addition other in vitro hematologic parameters, platelet lysis, coagulation changes and finally preservative factors (temperature, pH, protases) are discussed. The hemolytic activity was inhibited significantly by heating at 100 °C for 60 minutes (26%) and reached 38% via incubation with papain (10U/ml) while retained over a pH range of 4-11. Horses and sheep have the lower (61%) and upper (100%) rate of hemolysis. Calcium and magnesium ions could increase rate of hemolysis and EDTA solution had significantly decresing effect. The venom significantly changed in vitro coagulation factors (PT and APTT) from base line levels and had no effect on platelet lysis. It seems that our venom belongs to metalloproteinases due to potentiation effects of bivalent cations (calcium and magnesium) and ghost cell formation in our study indicatiing hemoglobin efflux

Cardiotoxic and Arrhythmogenic Effects of Hemiscorpius lepturus Scorpion Venom in Rats

Background: Envenomation by Hemiscorpius lepturus is not painful and the clinical manifestations include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions,cardiac arrhythmia and in minority of cases acute renal failure which may lead to death following disseminated intravascular coagulation in infants. Cardiac effects of envenomation by H. lepturus venom including inotropic, chronotropic and arrhythmogenic properties are not studied as now in rat hearts with Langendorff apparatus. Methods: Rat hearts were allowed to equilibrate in its buffer and cardiotropic plus arrhythmogenic effects induced by injection of different doses of H. lepturus venom were detected and recorded by computer acquisition based data in Langendorff apparatus. The neutralizing effects of Razi Institute antivenom and autonomic drugs were assayed in parallel studies. Results: Hemiscorpius lepturus venom (25 μg/100 l) treatment caused a negative inotropic (65.4 ± 3.2 versus 110.2 ± 3.4) and chronotropic effects (186.3 ± 4.2 versus 302 ± 6.3) in comparison to normal saline. Arrhythmogenic aspects including bradycardia, QRS widening and ST depression were induced by venom injection. Pre venom treatment (20 min) of Razi Institute antivenom (10 μl) neutralized cardiotropic effects but post venom injection (15 min later) had no therapeutic role. Pre (10 min before) and post (15 min after) injection of adrenaline (10 μl) neneutralized cardiotropic effects while pre venom injection (20 min) of propanolol (10 μl) had aggravating effects. Conclusion: Our study paves the way for further in vivo investigation of cardiovascular effects of this venom for finding suitable treatments instead of its ordinary antivenom medication in cardiogenic shock induced by the veno

Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (

Hemiscorpius lepturus envenomation: Manifestations and management with specific antivenom

Scorpionism is a known significant problem of medical and social importance in many tropical and subtropical regions including the Middle East. In Iran, highest prevalence of scorpion sting about 60% of all the stings has been reported from Khuzestan province. Among the 21,000 cases of reported scorpion stung patients, 12% were caused by H. lepturus, but contributed to 95% of all moralities in scorpion stung patients. The sting of H. lepturus does not produce an immediate pain as does the sting of other scorpions, rather cause delayed swelling that may diffuse and is often accompanied by late necrosis at the sting site suggestive of less significant role of the nervous system stimulation. Since the venom from H. lepturus is cytotoxic in nature and the renal response and blood toxicity are normally simultaneously manifested, it is suggested that the toxin binds to kidney tissue and potentially induce acute renal failure in stung patients. Pharmacokinetic analysis revealed that Intramuscular) i.m) injection of antivenoms is ineffective in neutralizing the action of venoms. Although some reports mention the slow distribution rate of H. lepturus venom following sting, but since the cytotoxic effect of venom from this scorpion is irreversible by antivenom once it occurs, it is recommended to use antivenom through intravascular (i.v) route. Antivenoms of F(ab)2 fraction are the best choice of treatment for their fast extravasation, their ample distribution into the extracellular space, and their prolonged retention time

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

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

Final 3.indd

Cell culture The HUVEC cell line was cultured in DMEM:F12 supplemented with 7% heat-inactivated FBS along with 100 IU/mL penicillin G and 100 g/mL streptomycin. The culture was incubated at 37°C with humidi ed air that contained 5% CO 2 . Abstract Background: Agiogenesis is the development of new blood vessels from pre-existing vasculatures. Although essential in the physiological process, it becomes pathological in various diseases including cancer. Preventing the formation of new blood vessels causes reductions in tumor size and metastasis. This study has been undertaken to elucidate the anti-angiogenesis effects of ICD-85 (derived peptides from venom). Methods: We evaluated the ICD-85 anti-angiogenesis activity by the in vivo CAM assay and in vitro tube formation assay of human umbilical vein endothelial cells (HUVECs). The anti-proliferative activity of ICD-85 was also determined through MTT assay on HUVECs. Results: Results of this study revealed the anti-proliferative activity of ICD-85 on the HUVEC cell line with an IC50 of 12 g/mL. The in vivo CAM assay also clearly showed the prevention of new vascular formation when the chick embryos were exposed to 0.15 g/disc of ICD-85. In vitro tube formation assay of HUVECs also showed the complete prevention of capillary tube formation on 18 g/mL. Conclusion: Based on the results obtained in this study, ICD-85 has anti-angiogenesis activity as shown by the prevention of capillary tube formation and the CAM assay

Biomacromolecular Journal Acute Effects of the Iranian Snake (Naja Naja Oxiana) Venom on Heart

ABSTRACT The myocardial effect of snake venoms is considered as one of the most common pathogenesis in many cases of snake envenomation. This study was undertaken to investigate the effects of the Iranian cobra (Naja naja oxiana) venom on cardiac function in experimental animals. The blood samples from all the rabbits were collected before venom injection, and then 140 µg kg -1 venom of snake (Naja naja oxiana) was injected intramuscularly to the rabbits. Following venom injection the blood was collected again at 1, 3 and 24 h. Electrocardiogram (ECG) was recorded during the experiment. The levels of serum enzymes lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and creatine phosphokinase (CPK) were determined. Statistical analyses were carried out by SPSS version 21 software. CPK enzyme showed a significant increase at 1 and 3 h after venom injection. The level of CK-MB also rose significantly after 1 h following venom injection. However, even at 24 h the level of CPK was not changed significantly, and the rise in CK-MB at 3 and 24 h following venom injection was not significant statistically. Although there was a rise in LDH level following venom injection but it was not significant. The ECG also confirmed changes in heart rhythmic and showed bradycardia and T tall. Based on the results obtained in the present study, it seems that the Naja naja oxiana venom has acute effect on cardiac system during the first few hours of snake bite