Purification of Therapeutic Serums of Snake Anti-Venom with Caprylic Acid.

Objectives: Antivenom serums have been used extensively for over a century and are the only effective treatment option for snake bites and other dangerous animal envenomations. In therapeutic serum centers, a wide range of antivenoms is made from animal serum, mainly equine and sheep, that are immunized with single or multiple venoms. This work aimed to use caprylic acid (CA) to purify therapeutic snake antivenom. Methods: Plasma was obtained from equine immunized with a mixture of venoms. Immunized plasma was obtained by precipitation of different concentrations (2-5%) of CA. This methodology was compared to that based on ammonium sulfate (AS) precipitation. Sediment plasma proteins were purified by ion-exchange chromatography. Protein assay, SDS-PAGE, and agar gel diffusion were performed. Results: The total protein precipitation with AS was higher than precipitation with CA, but the best results were obtained when CA was added to the plasma until a final CA concentration of 5% was reached. Chromatography and electrophoresis indicated a stronger band for the 5% CA, and the gel diffusion assay showed antigen-antibody interaction in the purified serum. Conclusion: The use of CA compared to the routine method for purifying hyperimmune serums is a practical and cost-effective method for preparing and producing therapeutic serums. It constitutes a potentially valuable technology for alleviating the critical shortage of antivenom in Iran.

Cytotoxic Effect of Bee (A. mellifera) Venom on Cancer Cell Lines.

OBJECTIVES: Nowadays cancer treatment is an important challenge in the medical world that needs better therapies. Many active secretions produced by insects such as honey bees used to discover new anticancer drugs. Bee venom (BV) has a potent anti inflammatory, anti cancer and tumor effects. The aim of present study is evaluation of anticancer effects induced by Apis mellifera venom (AmV) on cell Lines. METHODS: AmV was selected for study on cancer cell lines. Total protein, molecular weight and LD50 of crude venom were determined. Then, cells were grown in Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotics. The A549, HeLa and MDA-MB-231 cell Lines were exposed by different concentration of AmV. The morphology of cells was determined and cell viability was studed by MTT assay. Evaluation of cell death was determined by and DNA fragmentation. RESULTS: The results from MTT assay showed that 3.125 µg/mL of A549, 12.5 for HeLa and 6.25 µg/mL of MDA-MB-231 killed 50% of cells (p < 0.05). Morphological analysis and the results from hoescht staining and DNA fragmentation indicated that cell death induced by AmV was significantly apoptosis. CONCLUSION: The data showed that using lower dosage of AmV during treatment period cause inhibition of proliferation in time and dose dependant manner. Findings indicated that some ingredients of AmV have anticancer effects and with further investigation it can be used in production of anticancer drugs.

Biochemical, Hematological Effects and Complications of Pseudosynanceia Melanostigma Envenoming.

OBJECTIVES: Venomous fishes have different pharmacological effects and are useful. Among the venomous fish, stonefishes; especially Pseudosynanceia melanostigma has various pharmacological effects on the nervous, muscular and cardiovascular system of humans. In this study, toxicological characteristics, some blood effects, pharmacological and enzymatic properties of Pseudosynanceia melanostigma venom was investigated. METHODS: Crude venom purified by using gel filtration chromatography and the molecular weights of the venom and its fractions were estimated. The approximate LD values of this venom were determinedand the effects of LD50 dose on the blood of rabbits were studied. Hemolytic and Hemorrhagic activity of the venom sample was determined. In this case coagulation tests were performed. RESULTS: The LD50 of the Pseudosynanceia melanostigma crude venom was also determined to be 194.54 µg/mouse. The effect of two doses of LD50 showed a non-significant differences decrease in RBCs and MCV. In other cases, the results showed significant differences in WBC, Plt, Hb, MCH, MCHC and HCT; also it's showed a significant decrease. WBC count showed a significant increase with two doses of LD50 groups. The prothrombin time and partial prothrombin time were increased after venom treatment. As well as bleeding and clotting time were increased. According to the results, a minimum dose for Haemorrhagic effect 40 µg was obtained. CONCLUSION: Venom of Pseudosynanceia melanostigma has inhibitory effect on platelet aggregation that can be used to design and develop of anticoagulant drugs.

The Effects of Buthotus schach Scorpion Venom on Electrophysiological Properties of Magnocellular Neurons of Rat Supraoptic Nucleus.

Bothutous Schach (BS) scorpion venom consists of several polypeptides that could modulate ion channels. In this study, the effects of BS crude venom on passive and active electrophysiological properties of rat neurons in supraoptic nucleus (SON) of hypothalamus was investigated using whole-cell patch clamp technique. The results showed that bath application of BS venom produced significant change in passive properties of SON neurons, namely a decrease in resting membrane potential and an increase in input resistance of the cells. Also, significant change in active properties of SON neurons was shown after bath application of BS venom; including a decrease in the number of evoked action potential along with an increase in half width and decay time of action potential and a significant decrease in after-hyperpolarization amplitude. Finally, a decreased latency to the first spike accompanied by a lower current threshold to elicit the first spike was shown compared with the values before venom application. These effects are possibly through blocking different ion channels including potassium channels. Further experiments using different fractions of the venom is required to specify venom effects on various ion channels.

Cell-Mediated and Humoral Immune Responses to Bordetella pertussis Inactivated Whole-Cells Encapsulated Alginate Microspheres as a New Vaccine Candidate.

Effectiveness of the whole-cell pertussis vaccine is apparent, but improvement in the quality of the vaccine is necessary to achieve strong immunogenicity with a low bacterial number content. METHOD: Inactivated Bordetella pertussis (B. pertussis) cells entrapped microspheres were prepared via an emulsification method and analyzed for morphology, size, size distribution, loading efficiency, loading capacity, release kinetic, in vivo cytokines and antigen specific antibody subclasses. RESULTS: Bordetella pertussis encapsulated microspheres exhibited a smooth surface and spherical shape, mean particle size 151.1 µm, size distribution index 0.43, loading efficiency 89.6%, loading capacity 36.3% and release kinetic similar to the Korsmeyer-Peppas model. Splenocytes of animals immunized with new microsphere-based whole-cell vaccine produced greater quantities of IFN-γ and higher amounts of IL-4 and IL-5 cytokines compared to conventional adjuvant-adsorbed vaccines. Conventional adjuvant-adsorbed vaccines produced smaller quantities of IL-4 and IL-5. Bordetella pertussis entrapped microspheres induced both cell-mediated and humoral antibody in mice, evidenced by high levels of IgG2a and IgG1. IgG2a levels in mice were enhanced using the common aluminum phosphate-adsorbed B. pertussis whole-cell vaccine, and IgG1 levels did not increase significantly. Bordetella pertussis entrapped microspheres and common B. pertussis whole-cell vaccine samples enhanced total IgG levels in mice; however, B. pertussis-entrapped microspheres produced significantly higher levels of total IgG than other test samples. CONCLUSION: Encapsulation of inactive B. pertussis cells in microspheres appears to be a suitable approach for improving the wP vaccine quality, in particular by decreasing its toxicity to obtain good cell-mediated and humoral immunogenicity with a low bacterial number content.

A New Vaccine Delivery Vehicle and Adjuvant Candidate: Bordetella pertussis Inactivated Whole Cells Entrapped in Alginate Microspheres.

There is no doubt about the whole cell pertussis vaccine efficacy, but it is necessary to improve the vaccine quality specially to decrease its toxicity by obtaining good immunogenicity with low bacterial content. In this work, under optimum condition inactivated B. pertussis bacteria cells entrapped with alginate microparticles were fabricated and in vivo immunogenicity and ptency of new microparticle based vaccine were evaluated in mice. Microspheres loaded with inactive B. pertussis bacterium cells were prepared via an emulsification method and analyzed for morphology, size, polydispersity index, loading efficiency, loading capacity, release profile and in vivo potency. The inactivated bacterial suspension mixture prepared in this work was nontoxic and showed potent ED50 (1:333 of human dose) and preserved agglutinins 1, 2, 3. The optimum conditions for the preparation of microparticles were achieved at alginate concentration 3.8% (w/v), CaCl2 8% (w/v), PLL 0.1% (w/v), lipophilic surfactant 0.22 (%w/v), hydrophilic surfactant 3.6 (%w/v), cross linking time 3min, homogenization rate 600 rpm, and alginate to CaCl2 solution ratio 4. Both empty and B. pertussis loaded microparticles exhibited smooth surface texture and relatively spherical shape. The B. pertussis encapsulated microspheres fabricated under optimized conditions showed mean particle size 151.1 µm, polydispersity index 0.43, loading efficiency 89.6%, loading capacity 36.3%, and relatively constant release rate lasted to 15 days. In vivo immunogenicity and protection study against wild type challenge showed strongly higher potency (approximately 2.5 fold) of encapsulated B. pertussis organisms than non-encapsulated conventional aluminum hydroxide adsorbed vaccine. It can be concluded that microencapsulation of inactive B. pertussis cells appears to be a suitable approach for improving the wP vaccine quality, specially by obtaining good immunogenicity with low bacterial content.

Bee Venom (Apis Mellifera) an Effective Potential Alternative to Gentamicin for Specific Bacteria Strains: Bee Venom an Effective Potential for Bacteria.

OBJECTIVES: Mellitine, a major component of bee venom (BV, Apis mellifera), is more active against gram positive than gram negative bacteria. Moreover, BV has been reported to have multiple effects, including antibacterial, antivirus, and anti-inflammation effects, in various types of cells. In addition, wasp venom has been reported to have antibacterial properties. The aim of this study was to evaluate the antibacterial activity of BV against selected gram positive and gram negative bacterial strains of medical importance. METHODS: This investigation was set up to evaluate the antibacterial activity of BV against six grams positive and gram negative bacteria, including Staphylococcus aureus (S. aureus), Salmonella typhimurium, Escherichia coli (E. coli) O157:H7, Pseudomonas aeruginosa, Burkholderia mallei and Burkholderia pseudomallei. Three concentrations of crude BV and standard antibiotic (gentamicin) disks as positive controls were tested by using the disc diffusion method. RESULTS: BV was found to have a significant antibacterial effect against E. coli, S. aureus, and Salmonella typhyimurium in all three concentrations tested. However, BV had no noticeable effect on other tested bacteria for any of the three doses tested. CONCLUSION: The results of the current study indicate that BV inhibits the growth and survival of bacterial strains and that BV can be used as a complementary antimicrobial agent against pathogenic bacteria. BV lacked the effective proteins necessary for it to exhibit antibacterial activity for some specific strains while being very effective against other specific strains. Thus, one may conclude, that Apis mellifera venom may have a specific mechanism that allows it to have an antibacterial effect on certain susceptible bacteria, but that mechanism is not well understood.

Study on Toxicity Reduction and Potency Induction in Whole-cell Pertussis Vaccine by Developing a New Optimal Inactivation Condition Processed on Bordetella pertussis.

BACKGROUND: Whooping cough is caused by Bordetella pertussis, and it remains a public health concern. Whole-cell pertussis vaccines have been commonly employed for expanded immunization. There is no doubt of the efficacy of whole cell pertussis vaccine, but it is necessary to improve the vaccine to decrease its toxicity. OBJECTIVES: In this study, an inactivation process of dealing with pertussis bacteria is optimized in order to decrease the bacteria content in human doses of vaccines and reduce the vaccine's toxicity. MATERIALS AND METHODS: The bacterial suspensions of pertussis strains 509 and 134 were divided into 21 sample parts from F1 to F21 and inactivated under different conditions. The inactivated suspensions of both strains were tested for opacity, non-viability, agglutination, purity, and sterility; the same formulation samples that passed quality tests were then pooled together. The pool of inactivated suspensions were analyzed for sterility, agglutination, opacity, specific toxicity, and potency. RESULTS: The harvest of both bacterial strains showed purity. The opacity of various samples were lost under different treatment conditions by heat from 8% to 12%, formaldehyde 6% to 8%, glutaraldehyde 6% to 8%, and thimerosal 5% to 8%. Tests on suspensions after inactivation and on pooled suspensions showed inactivation conditions not degraded agglutinins of both strains. The samples of F2, F4, F8, F12, F15, and F17 passed the toxicity test. The potency (ED50) of these samples showed following order F17 > F12 > F8 > F15, F4 > F2, and F17 revealed higher potency compared to other formulations. CONCLUSIONS: It can be concluded that F17 showed desirable outcomes in the toxicity test and good immunogenicity with a low bacterial number content. Consequently, lower adverse effects and good immunogenicity are foreseeable for vaccine preparation with this method.

Study on development of Vipera lebetina snake anti-venom in chicken egg yolk for passive immunization.

Chicken egg yolk antibodies against Vipera lebetina venom were evaluated for their antivenom potential. White leghorn hens were immunized with detoxified V. lebetina venom (γ-irradiated venom). The detoxified venom (200 µg) was mixed with an equal volume of complete Freund's adjuvant and was injected intramuscularly into the hens. The antibodies showed high activity (1.6 LD50/mL) in egg yolks after 12 d of venom injection. The eggs were collected after 12 days, and the egg yolks were removed and washed with purified water to remove any contamination with egg whites. The purification was performed using a method described by Maya Devi et al., followed by gel filtration (Sephadex G-50). The purity and molecular weight of antivenom antibodies (IgY) were determined using electrophoresis, and the molecular weight was found to be approximately 185 kDa. The potency of IgY was 6 LD50/mL (mice), i.e., 1 mL of IgY could neutralize 43.8 µg of standard V. lebetina venom). Our results showed that chicken egg yolk antibodies were effective in neutralizing the lethality and several pharmacological effects of V. lebetina venom and could be used for developing effective antivenom.

Honey Bee Venom (Apis mellifera) Contains Anticoagulation Factors and Increases the Blood-clotting Time.

OBJECTIVES: Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. METHODS: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. RESULTS: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs) = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The LD50 of the crude BV was found to be 177.8 µg/mouse. CONCLUSION: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2) and melittin, and that can increase the blood clotting times in vitro.

Study on camel IgG purification: a new approach to prepare Naja Naja Oxiana antivenom as passive immunization for therapy.

A combined process of ammonium sulfate precipitation (salting out) and ion-exchange chromatography on DEAE-Sepharose CL-6B was used to prepare camel antivenom (IgG) against Naja Naja Oxiana for therapy. In the ammonium sulfate precipitation, the best condition for fractionation of IgG from the other proteins in camel serum was 55% precipitate. The camel IgG presented as 2 bands with molecular masses of 250 and 100 kDa, the latter corresponding to heavy chain IgG, on 10% gel electrophoresis. A trace amount of non-IgG proteins was not isolated and remained in this precipitate. Therefore in order to effectively separate albumin and the other nonspecific proteins from the IgG, the 25% precipitate of ammonium sulfate precipitation of serum was subjected to DEAE-Sepharose CL-6B column chromatography. A peak of antibody (IgG) could be obtained by elution with sodium phosphate buffer. In this stage, 2 bands of molecular masses of 150 and 75 kDa were observed on 7% gel electrophoresis. A comparative study was performed between camel IgG and conventional horse F(ab) 2 antivenoms in term of potency (serum neutralization test and ELISA). Our results showed that the potency of camel antivenom was 4-fold higher than that of horse. It is suggested the combined ammonium sulfate precipitation and ion-exchange chromatography process effectively removed residual proteins in the final camel IgG preparation and can be a suitable method for large-scale refinement of therapeutic camel antivenoms.

In vivo evaluation of homeostatic effects of Echis carinatus snake venom in Iran.

BACKGROUND: 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. MATERIALS AND METHODS: Subfractions F1A and F1B were isolated from Ec crude venom by a combination of gel chromatography (Sephadex G-75) and ion exchange chromatography on a DEAE-Sepharose (DE-52). These subfractions were then intravenously (IV) injected into NIH male mice. Blood samples were taken before and after the administration of these subfractions. Times for prothrombin, partial thromboplastin and fibrinogen were recorded. RESULTS AND CONCLUSIONS: Comparison 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 quick blood coagulation. The LD50 of the Ec crude venom was also determined to be 11.1 µg/mouse. Different crude venom doses were prepared with physiological serum and injected into four mice. 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 rose sharply in mice. Finally, by comparing the fibrinogen time after subfraction injections and normal test time, we can infer intense activation of coagulation cascade and fibrin production.

In vivo evaluation of homeostatic effects of Echis carinatus snake venom in Iran

Background: 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. Materials and methods: Subfractions F1A and F1B were isolated from Ec crude venom by a combination of gel chromatography (Sephadex G-75) and ion exchange chromatography on a DEAE-Sepharose (DE-52). These subfractions were then intravenously (IV) injected into NIH male mice. Blood samples were taken before and after the administration of these subfractions. Times for prothrombin, partial thromboplastin and fibrinogen were recorded. Results and conclusions: Comparison 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 quick blood coagulation. The LD50 of the Ec crude venom was also determined to be 11.1 µg/mouse. Different crude venom doses were prepared with physiological serum and injected into four mice. 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 rose sharply in mice. Finally, by comparing the fibrinogen time after subfraction injections and normal test time, we can infer intense activation of coagulation cascade and fibrin production.(AU)

In vivo evaluation of homeostatic effects of Echis carinatus snake venom in Iran

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. Materials and methods Subfractions F1A and F1B were isolated from Ec crude venom by a combination of gel chromatography (Sephadex G-75) and ion exchange chromatography on a DEAE-Sepharose (DE-52). These subfractions were then intravenously (IV) injected into NIH male mice. Blood samples were taken before and after the administration of these subfractions. Times for prothrombin, partial thromboplastin and fibrinogen were recorded. Results and conclusions Comparison 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 quick blood coagulation. The LD50 of the Ec crude venom was also determined to be 11.1 μg/mouse. Different crude venom doses were prepared with physiological serum and injected into four mice. 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 rose sharply in mice. Finally, by comparing the fibrinogen time after subfraction injections and normal test time, we can.

Isolation and partial purification of anticoagulant fractions from the venom of the Iranian snake Echis carinatus.

Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic activity. The molecular weights of these anticoagulants (F2C, F2D) were estimated by SDS/PAGE electrophoresis. F2C comprises two protein bands with molecular weights of 50 and 79 kDa and F2D a single band with a molecular weight of 42 kDa.

Blood coagulation induced by Iranian saw-scaled viper (echis carinatus) venom: identification, purification and characterization of a prothrombin activator.

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 the plasma protein and blood coagulation factor. Some of these proteins exhibit types of enzymatic activities. However, other items are proteins with no enzymatic activity. MATERIALS AND METHODS: In order to study the mechanism and effect of the venom on human plasma proteins, the present study has evaluated the effect of crude venom and all fractions. A procoagulant factor (prothrombin activator) was isolated from the venom of the Iranian snake Echis carinatus with a combination of gel filtration (Sephadex G-75), ion-exchange chromatography (DEAE- Sepharose) and reverse phase HPLC. Furthermore, proteolytic activity of the crude venom and all fractions on blood coagulation factors such as prothrombin time (PT) was studied. RESULTS: In the present study, the PT test was reduced from 13.4 s to 8.6 s when human plasma was treated with crude venom (concentraion of venom was 1 mg/ml). The purified procoagulant factor revealed a single protein band in SDS polyacrylamide electrophoresis under reducing conditions and its molecular weight was estimated at about 65 kDa. A single-band protein showed fragment patterns similar to those generated by the group A prothrombin activators, which convert prothrombin into meizothrombin independent of the prothrombinase complex. CONCLUSION: This study showed that the fraction which separated from Iranian snake Echis carinatus venom can be a prothrombin activators. It can be concluded that this fraction is a procoagulant factor.

Partial Fractionation of Venoms from Two Iranian Vipers, Echis carinatus and Cerastes persicus Fieldi and Evaluation of Their Antiplatelet Activity.

Platelet aggregation inhibitory effect and anticoagulant properties of fractions separated from the venoms of Cerastes persicus fieldi and Echis carinatus were investigated. The partial fractionation was performed on a Sephadex G-100 column. Two fractions separated from Cerastes persicus fieldi showed anti platelet aggregation activity on ADP (200 µM)-induced platelet aggregation (ca 80% inhibition). Attempts to measure the antiplatelet aggregation activity of crude Echis carinatus venom and its fractions were not successful due to the protein coagulation of the plasma samples after the addition of venom. Anticoagulant activities of venoms were also evaluated. Total venom of Echis carinatus showed anti coagulant activity in PT test, while its fractions showed procoagulant activity.