Ecarin is a snake venom (Echis carinatus) that directly activates prothrombin to meizothrombin This action is not dependent on phospholipid membranes and . Objective(s): Echis carinatus is one of the venomous snakes in Iran. The venom of Iranian Echis carinatus is a rich source of protein with various factors affecting . In this research, the effects of Echis carinatus crude venom and its fractions on mice were analyzed. Moreover, the results of coagulation tests.
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The venom of the family Viperidae, including the saw-scaled viper, is rich in serine proteinases and metalloproteinases, which affect the nervous system, complementary system, blood coagulation, platelet aggregation and blood pressure. One of the most prominent effects of the snake venom of Echis carinatus Ec is its coagulation activity, used for killing prey.
Blood samples were taken before and after the administration of these subfractions.
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Times for prothrombin, partial thromboplastin and fibrinogen were recorded. Snaks of the prothrombin time before and after F1A and F1B administrations showed that time for blood coagulation after injection is shorter than that of normal blood coagulation and also reduced coagulation time after Ec crude venom injection.
This difference in coagulation time shows the intense coagulation activity of these subfractions that significantly increase the coagulation cascade rate and Causes to snnake blood coagulation.
The LD50 of the Ec crude venom was also determined to be Different crude venom doses were prepared with physiological serum and injected into four mice.
In vivo evaluation of homeostatic effects of Echis carinatus snake venom in Iran
Comparison of the prothrombin times after injection of subfractions F1A and F1B showed that the rate of mouse blood coagulation increases considerably. Comparing the partial thromboplastin times after injecting these subfractions with this normal test time showed that the activity rate of intrinsic blood coagulation system vennom sharply in mice.
Carinatud, by comparing the fibrinogen time after subfraction sname and normal test time, we can infer intense activation snakw coagulation cascade and fibrin production.
Snakebite affects around 2. Coagulopathy is a significant cause of both morbidity and mortality in these patients, either directly, or indirectly. Clinical concerns in carinwtus include types of coagulopathy procoagulant, anticoagulant, fibrinogen clotting, fibrinolytic, platelet-active, thrombotic, hemorrhagicdiagnosis and treatment [ 1 – 3 ].
Where available, antivenom is the most effective treatment against snakebites, while standard treatments for other forms of coagulopathy, such as factor replacement therapy and heparin, are either ineffective or dangerous, except in specific situations. Interference with aspects of the human hemostatic system is a common theme that encompasses all four families of venomous snakes including Echis carinatus from Viperidae family in Iran [ 4 ].
Among the many potential effects of envenoming by snakes in humans, only a few broad categories are of major clinical significance veonm paralysis and mild stroke; systemic myolysis; coagulopathy and hemorrhage; renal damage and failure; cardiotoxicity; and local tissue injury at the bite site [ 45 ]. Any single snake species may possess venpm that act in carinatu or more of these categories, though rarely all six. In this research, the effects of Echis carinatus crude venom and its fractions on mice were analyzed.
Moreover, the results of coagulation tests on its venom were recorded. The other reagents and chemicals of fchis grade were purchased from Fluka and Merck.
Mouse blood samples were centrifuged for ten minutes at 3, rpm. The thromboplastin-D vial was brought to the laboratory and equilibrated to room temperature.
The process of plasma clotting was observed and the time recorded [ 1011 ]. The process of plasma clotting was observed and the time recorded genom 11 ]. Half an hour before conducting the test, the reagents were taken out of the refrigerator in order to equilibrate their temperature to room temperature. Two minutes after incubation, 0. As soon as the first signs of clotting were observed, time was recorded and the fibrinogen level determined [ 1213 ]. For measuring the Iranain Echis carinatus crude venom coagulation activity, 10 mg of the crude venom was initially used to prepare different concentrations 1, 0.
These concentrations were them exposed in the PT test. Isolation and purification of coagulation factors were performed using 50 mg of Ec crude venom using a combination of gel chromatography and ion exchange chromatography. Ec crude venom was primarily isolated using gel chromatography Sephadex G column which initially gained equilibrium using 20 mM ammonium acetate buffer pH 6.
That is, the column input and output pH became the same. Eechis milligrams of Ec crude venom was dissolved in 4 mL of ammonium acetate cariatus. The absorption of the resulting solution was read using a spectrophotometer at nm and relevant absorption curve was drawn in terms of the tube number [ 101415 ].
For taking the ammonium acetate buffer out of the solutions, each of the peaks was dialyzed for 24 h with distilled water. The ion exchange chromatography 1. For the peaks obtained by gel chromatography, the fraction that exhibited coagulation activity was exposed to ion exchange chromatography for further echos and subfractionation. Subsequently, the column was eluted with Tris—HCl 0. The absorption of collected tubes was read using a spectrophotometer at nm and relevant optical absorption curve was shake in terms of the tube number [ 1014 – 16 echiss.
The subfractions were pooled and dialyzed like in gel chromatography. This test was conducted according echiw the method by Meier ecyis Theakston [ 17 ]. The doses were chosen was so that no mouse would die at the lower dose, and all mice would die at the higher dose. Mouse mortality within 24 h was recorded and each sample LD 50 was calculated.
Upon recording of mortality, the Spearman-Karber statistical method was used for LD 50 calculation [ 161819 ]. Echis carinatus crude venom decreases coagulation time of mouse plasma in relation to its normal levels. Thus, the venom shows coagulation properties. Therefore, as the venom concentration increases, its coagulation properties will also augment.
The existence of coagulation factors in Ec venom was then established. As per the existing standards on gel chromatography in which protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions, F 1 was considered the peak with the highest amount of protein.
Regarding the gel chromatography isolation process based on molecular weight, peaks or fractions respectively containing less total protein will exit from the gel chromatography column. Fractions F 2 to F 5 contain proteins with molecular weights lower than that of F 1.
Gel chromatography Sephadex G of 50 mg cehis Echis carinatus crude venom. Prothrombin time test on mouse plasma by using fractions obtained from gel chromatography.
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Among the fractions obtained from gel chromatography fraction F 1 was selected for furhter isolation because of its lower coagulation time, and was taken to the DEAE-Sepharose ion exchange column.
Ion exchange chromatography of F 1 fraction. Compared venpm the normal time, this interval is carinaatus, showing the intense coagulation properties of these subfractions. For more investigation into the coagulation activity, these subfractions were selected for injection into mice.
The Spearman-Karber statistical method was used to calculate LD 50 as follows:. It has been important for scientists to identify and study the compounds in snake venom.
Echjs, there are different manners to isolate and purify snake venom enzymes and proteins and study their effects. Chromatography is the most commonly used echls for crude venom isolation. Echis carinatus crude venom fractions isolated by chromatography showed that this method is useful for fraction separation.
Viperidae venoms, including that of Ec, are rich in compounds that may be useful for medicine and pharmaceutics [ 20 ]. At lower concentrations, small clots are formed and coagulation time is longer, whereas at higher concentrations, larger clots are found and coagulation time is shorter [ 21 cairnatus.
When compared with normal PT If the normal PT is equal to For isolation, identification and investigation of the properties of Ec crude venom coagulation factors, a combination of gel chromatography and ion exchange chromatography was employed. Fifty milligrams wchis crude venom were subjected to gel chromatography Sephadex G and five fractions were obtained F 1 to F 5. The isolation of subfractions was performed according to gel chromatography standards based on molecular weight. F 1 showed the highest level of proteins among the fractions.
The total time of PT was obtained for fraction F 1with a mean of Coagulation tests were performed with fraction F 1 and the coagulation cascade decreased, which could be due to venom toxic properties on the hemostatic system. PT test showed that F 1 was a coagulation fraction whereas other fractions were considered to be anticoagulation fractions. F 1 ion exchange chromatography led to the formation of eight subfractions F 1 A to F 1 H. The PT test was edhis conducted carihatus mouse plasma using these subfractions.
Regarding the PT test results, subfractions F 1 A mean 6. Thus, they were selected for injection into mice. Another study, similar to ours, was conducted on snake venoms.
The purification phases were similar to our work. In another work, Agkistrodon acutus snake venom was exposed to ion exchange chromatography with Sepharose DEAE followed by gel carinaatus on Sephacryl S to isolate fractions with coagulation activities [ 25 ]. In our research, to further study in vivo the coagulation properties of these two subfractions, F 1 A and F 1 B were administered to male NIH mice.
The mean PT before the F 1 A injection was The mean PTT before administration was About carinstus hour after injection, blood samples were collected. The mean PT test after the F 1 A injection was 6. This rapid response of the coagulation cascade occurs in the animal body and generates clinical effects such as coagulopathy, which may vemom death. The mean PTT after injection was equal to This value compared with the one before injection The mean FT before injection was This difference in coagulation time can be indirectly attributed to failure and dysfunction in blood coagulation factors by the presence of procoagulation compounds like prothrombin activators.
The mean PT before carinatuus was An hour after the injection of F 1 B, blood samples were collected. The mean PT after injection was 3.
The mean PTT before injection was This coagulation difference may be indirectly attributed to failure and dysfunction in blood coagulation factors by the presence of procoagulation compounds, such as prothrombin activators. Statistical results suggest that H 0 is rejected by both subfractions F 1 A and F 1 B and, hypothetically, H 1 is accepted by both. Other similar studies have also been conducted.
For example, Gao et al.