Carinatus Venom Research Articles

Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib

Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.

Echicetin: A Snake Venom Protein That Inhibits Binding of von Willebrand Factor and Alboaggregins to Platelet Glycoprotein Ib

Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.

In Vivo Effect of Echis carinatus Venom Observed in a Woman in Mali

In Vivo Effect of Echis carinatus Venom Observed in a Woman in Mali

The Textarin/Ecarin Ratio: A Confirmatory Test for Lupus Anticoagulants

Lupus anticoagulants (LA) are immunoglobulins (IgG, IgM, IgA or a mixture) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, KCT, dilute Russell Viper Venom Time). LA are heterogeneous; consequently, the laboratory diagnosis is difficult and relies on multiple tests. We have developed a sensitive and relatively specific confirmatory test system based on fractions of two snake venoms. Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern brown snake), activates prothrombin in the presence of PL, factor V and calcium ions. Ecarin, a protein fraction of Echis carinatus venom, will activate prothrombin in the absence of any cofactors. The activation of prothrombin by Textarin yields thrombin while Ecarin yields meizothrombin. In the presence of LA, the Textarin time is prolonged and the Ecarin time is unaffected. The test results are reported as a ratio of Textarin/Ecarin times (abnormal greater than 1.3). We have evaluated this test system in the following patient populations: LA positive, therapeutically heparinized, stable oral anticoagulated, liver disease, routine preoperative, anticardiolipin antibody positive LA negative, hemophilia A, various other hereditary factor deficiencies or dysfunctional proteins, and specific inhibitors of factor V and factor VIII. The LA positive patients represented a mixed population of autoimmune disease, drug-induced and post-infectious states. Our findings indicate the sensitivity of the Textarin/Ecarin system in the confirmation of LA. In order to use the test system most effectively, it is recommended to incorporate polybrene with Textarin when evaluating heparinized samples. Factor V deficiency and specific inhibitors of factor V yielded, in some instances, false positive results.

Structure and function of snake venom metalloproteinase family

Venoms of snakes belonging to families Viperidae (viper) and Crotalidae (pit viper) produce striking local effects, consisting of hemorrhage, necrosis, and edema, and often induce marked alterations of blood coagulation system as well (Iwanaga and Suzuki, 1979). Among these pathological effects, hemorrhage is a most common occurrence in a victim bitten by crotalid and viperid snakes and various components, such as hemorrhagic factors and metalloproteinases, involved in these venoms have been isolated and characterized (Osaka, 1979). The hemorrhagic metalloproteinases cause localized hemorrhage by direct action on the blood vessel wall. Electron microscopic studies indicate that erythrocytes are leaked in a one-by-one fashion through widened inter-endothelial gaps when capillaries are exposed to these hemorrhagic proteins. Hemorrhagic metalloproteinases degrade basement membrane preparation, in addition to the isolated components including type IV collagen, laminin, nidogen, and fibronectin (Baramova et al., 1989). The enzymes may disrupt the pericellular basement membrane through a proteolytic activity and with subsequent damage to the integrity of the vessel wall after which hemorrhage occurs. On the other hand, crotalid and viperid venoms contain an abundance of metalloproteinases which are completely free from hemorrhagic activity (Iwanaga et al., 1976). Some of them have originally been reported as anticoagulant proteases with fibrinogenolytic or fibrinolytic activity (Markland, 1991). Additional group of venom metalloproteinases are procoagulants having very strict substrate specificities. Two well known examples of this type are Russell’s viper venom factor X activator (RVV-X) and Echis carinatus venom prothrombin activator (Morita and Iwanaga, 1981; Furie and Furie, 1976).

Heterogeneity of Echis carinatus venom: evidence for variation at the level of the gene: H. P. Desmond, J. M. Crampton and R. D. G. Theakston (Snake Venom Research Unit/Wolfson Molecular Genetics Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.).

Heterogeneity of Echis carinatus venom: evidence for variation at the level of the gene: H. P. Desmond, J. M. Crampton and R. D. G. Theakston (Snake Venom Research Unit/Wolfson Molecular Genetics Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.).

Gene expression in Echis carinatus (carpet viper) venom glands following milking

RNA was extracted from the venom glands of Echis carinatus at different times after milking, and the temporal pattern and nature of mRNA transcribed during venom regeneration was examined by in vitro translation. Venom products were immunoprecipitated with E. carinatus venom polyclonal antiserum. Maximum transcriptional activity occurred 3 days after milking. Electrophoretic analysis of the translation products showed minimal differences in the banding patterns at each time interval. Analysis of the translation products from Kenyan, Nigerian and Saudi Arabian carpet vipers, however, revealed differences which suggest that the observed heterogeneity in E. carinatus venom occurs at the level of the genome.

A Novel Enzyme Immunoassay for Quantitation of Rat Prothrombin in Microsomal Subfractions

An enzyme linked immunoassay (ELISA) for quantitation of rat prothrombin, based on a biotin-streptavidin alkaline phosphatase system is described. The assay utilizes rabbit antiserum raised against purified rat prothrombin. The assay was twenty fold more sensitive than a rat prothrombin assay based on amidolytic activity following activation by Echis carinatus venom. Results obtained with the two assays show good correlation. The ELISA is a valuable tool for quantitation of minute amounts of prothrombin in subcellular fractions and large series of plasma samples.

Prothrombin Himi; An abnormal prothrombin characterized by a defective thrombin activity

An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured using either the classical one-stage assay or the assay with Echis carinatus venom, whereas prothrombin antigen level was normal. In keeping with current nomenclature practices, the abnormal prothrombin was designated “Prothrombin Himi”. The electrophoretic behavior and calcium binding properties of Prothrombin Himi did not differ significantly from normal. Prothrombin Himi was isolated by chromatography on Q-Sepharose. Electrophoretic migration of the purified abnormal prothrombin on SDS -PAGE was normal. Upon prothrombin activation by Echis carinatus venom, the clotting activity produced from Prothrombin Himi was only 37% of the normal level after 90 minutes of the activation time, where as the amidolytic activity was almost the same as normal. The cleavage patterns of Prothrombin Himi by factor Xa or Echis carinatus venom investigated by SDS-PAGE, were found to be normal. These results indicate that Prothrombin Himi was characterized by a defective thrombin enzymatic activity.

Prolonged Hypofibrinogenemia and Protein C Activation after Envenoming by Echis Carinatus Sochureki

Following envenomization by Echis carinatus sochureki, a professional snake handler developed a profound coagulopathy manifested by hemorrhage from the bite site, venipuncture sites and gums; coagulation testing revealed prothrombin and partial thromboplastin times greater than 150 seconds, a fibrinogen of 0 mg%, and marked elevation of fibrin degradation products. In addition, protein C antigen levels were undetectable. The coagulopathy was treated with cryoprecipitate; two different antivenoms were also administered with uncertain benefit. Subsequently, the properties of the venom and antivenoms were studied. Venom did not directly clot fibrinogen; however, venom concentrations as low as 0.2 micrograms/ml caused significant prothrombin activation. In addition, venom activated protein C in the absence of thrombomodulin, and this activity was inhibited by hirudin. The ability of four commercial antivenoms to neutralize the venom prothrombinase and hemorrhagic activity was measured. Three of the four antivenoms partially neutralized venom-induced prothrombin activation. Extreme differences in efficacy were found among the four antivenoms in neutralizing venom hemorrhagic activity in mice. This case illustrates the difficulty in managing the complex coagulopathy that can result from exotic snake envenomization, and identifies a new coagulant property of Echis carinatus venom (protein C activation).

Prediction of Vitamin K Response Using the Echis Time and Echis-Prothrombin Time Ratio

Echis carinatus venom contains proteases capable of activating both normal and descarboxy prothrombin. We showed this venom (Sigma) principally activates prothrombin with almost no factor X activation. Echis time in combination with prothrombin time can predict vitamin K responsiveness since the Echis time is usually normal in the presence of descarboxy prothrombin associated with vitamin K deficiency. 38 patients with abnormal routine prothrombin times (PT) had both coagulant and immunogenic factor II assays along with Echis times done before and after vitamin K. Of 22 patients responding to vitamin K, based on correction of PT, 21 had normal initial Echis times and of 16 not responding, 11 had abnormal Echis times, giving a sensitivity of 95.4% and specificity of 68.8% for vitamin K responsiveness. 90% of patients with a PT/Echis time ratio less than 1.3 and a prolonged Echis time did not correct their PTs with vitamin K therapy. The 5 non-responders with normal Echis times all showed normal initial coagulant and antigenic prothrombin, but 3 had low F V and/or F VII.

Biochemical and pharmacological investigation of Echis carinatus venoms from three different geographical regions: Jürg Meier1 and Götz Nowak2 ( 1Pentapharm Ltd, CH-4002 Basle, Switzerland, and 2Institute of Pharmacology and Toxicology, Med. Academy, DDR-50 Erfurt, G.D.R.)

Biochemical and pharmacological investigation of Echis carinatus venoms from three different geographical regions: Jürg Meier1 and Götz Nowak2 ( 1Pentapharm Ltd, CH-4002 Basle, Switzerland, and 2Institute of Pharmacology and Toxicology, Med. Academy, DDR-50 Erfurt, G.D.R.)

Snake venoms in science and clinical medicine 2. Applied immunology in snake venom research

Enzyme-linked immunosorbent assay (ELISA) is a very important tool for studying both the epidemiology and clinical effects of snake bite in man. For epidemiology ELISA depends on the development and persistence of specific humoral venom antibody in previous snake bite victims. In the Nigerian savanna 63% of previous bite victims possessed specific venom antibodies against Echis carinatus venom; in Ecuador, where there is a 5% annual mortality due to snake bite in a population of Waorani Indians, venom antibodies against a wide range of different venoms were identified in previous bite victims using ELISA. In certain areas it is often not possible, using the symptoms of envenoming, to determine which species of snake has bitten the patient. Field studies using ELISA in Nigeria and Thailand have been successful in establishing the species responsible for envenoming. Current studies are in progress on the development of a rapid immunoassay which should be capable of detecting the biting species within 5–10 min of sampling from the admission patient. This will be useful for the clinician as it will enable the rapid detection of the species responsible for envenoming and, therefore, the use of the correct antivenom. Experimental work on the development of new methods of antivenom production includes immunization of experimental animals with venom/ liposome preparations, the preparation of venom antigens using monoclonal antibodies on affinity columns, and recombinant deoxyribonucleic acid technology. Liposomal immunization requires only a single injection of venom to obtain a rapid, high level and protective immune response. Venom liposomes may also be given orally resulting in a serum immunoglobulin G immune response in experimental animals. Use of such a system may eventually result in immunization of man in areas of high snake bite incidence and mortality.

Preliminary study of an extract from Diodia scandens on some toxic effects of Echis carinatus venom

An extract of Diodia scandens is prepared, and its toxicity is evaluated in mice. The effects of the extract on venom-induced effects are studied in mice and in the anaesthetised cat, by measure of mortality in mice, and by changes in blood pressure and respiration in anaesthetised animals

Isolation and purification of a fibrinogenolysin from the venom of the saw-scaled viper (Echis carinatus) by high-performance liquid chromatography

This paper describes the successful fractionation of crude snake venoms by hydrophobic interaction HPLC, with retention of biological activity. A fibrinogenolysin, with its biological activity intact, was isolated from crude E. carinatus venom with use of a hydrophobic interaction column (TSK-Phenyl 5PW). After further purification by ion-exchange and reserved-phase HPLC, fourteen amino acid residues of its N-terminal sequence were determined

Preliminary in vitro studies on the antagonistic effects of an ethanolic extract from Diodia scandens on Echis carinatus venom‐induced changes in the clotting of human blood

AbstractThe water soluble fraction of an ethanolic extract from Diodia scandens was found to have no effect on the in vitro clotting time of normal human blood. However, it increased to 2.8 min the in vitro blood clotting time previously reduced from 4.7 min to 1.8 min following treatment with Echis carinatus venom. The plant extract was shown to possess definite anti‐thromboplastin activity against brain thromboplastin and venom thromboplastin activity. The extract did not significantly inhibit the thrombin‐like activity of the venom. It is suggested that the antagonistic effect of the extract on venom‐induced coagulation is due to inhibition of thromboplastin/Factor VII activation of Factor X.

Antithrombin III in <i>Echis</i><i>carinatus</i> Envenomation in Northern Nigeria

Antithrombin III was evaluated in 21 patients admitted with Echis carinatus envenomation. Although there was evidence of incoagulability in all patients and elevated fibrin degradation products, mean antithrombin III levels were found to be significantly greater than in controls. This result suggests that Echis carinatus venom (echarin) may bypass the consumption of antithrombin III molecules.

Characterisation of a monoclonal antibody capable of neutralising the haemorrhagic activity of West African Echis carinatus (carpet viper) venom

D. Iddon, M. Hommel and R.D.G. Theakston. Characterisation of a monoclonal antibody capable of neutralising the haemorrhagic activity of West African Echis carinatus (carpet viper) venom. Toxicon 26, 167 – 179, 1988. — A murine monoclonal antibody (SV-1/F10) was highly specific immunologically for West African E.carinatus venom both by ELISA and immunoblotting. In cross-protection tests in vivo, it possessed strong antihaemorrhagic activity. This IgGl monoclonal antibody recognised an epitope present in a protein band of 124,000 mol. wt using immunoblotting of non-reduced Nigerian and Ghanaian E.carinatus venoms, as well as in a second protein band of 105,000 mol. wt in Ghanaian E.carinatus venom. The SV-1/F10 monoclonal antibody is of potential use for the isolation of West African E.carinatus venom haemorrhagin from whole venom, allowing the possibility of elucidation of the mechanism of its actions as well as its interactions with antibody.

An investigation of the coagulant activity of the venom of the saw-scaled viper (Echis carinatus) from Saudi Arabia.

Unlike the venom of Echis carinatus from India, Pakistan, Nigeria, Kenya, Iran and Oman, Saudi Arabian E. carinatus venom is a poor activator of prothrombin. However, it possesses similar defibrinogenating activity to the other venoms. This is because the venom from Saudi Arabian snakes contains a calcium-dependent factor X activator. It is suggested that in future studies of the coagulant activity of venoms, the determination of plasma coagulant activity should be carried out in the presence of added calcium ions. This applies particularly to those venoms which do not act on plasma or fibrinogen, but which do cause in vivo defibrinogenation.

Pulmonary fibrin deposition and increased microvascular permeability to protein following fibrin microembolism in dogs: A structure-function relationship

The effects of fibrin microembolism were examined using an infusion of a prothrombin activator ( Echis carinatus venom, ECV; 30 min, 0.5 NIH thrombin equivalent units/kg) in acute mongrel dogs prepared with a pulmonary lymph cannula ( n = 6, 12.3–21.5 kg). Lymph flow increased approximately 2.5-fold after 1-1.5 hr of elevated left atrial pressure ( P la = 20 cm H 2O; 26 ± 7 to 63 ± 16 μl/min, P < 0.01) and the plasma to lymph protein concentration ratio ( C P C L ) declined from 0.66 ± .04 to 0.54 ± .16 ( P < 0.01, x ± SE). After P la was reduced to control levels, the initiation of fibrin microembolism was associated with an approximate 2.7-fold elevation of lymph flow (62 ± 8 μl/min, P < 0.01) and the C P C L was not changed (0.56 ± 0.04, P = ns). When P la was increased following microembolism, lymph flow more than doubled to 117 ± 24 μl/min ( P < 0.01) and the C P C L remained unaltered (0.56 ± 0.03, P = ns). These changes were associated with afibrinogenemia and the appearance of fibrin degradation products (FDP) in plasma (150 ± 50 μg/ml) and lymph (80 μg/ml) in three of the animals tested. No consistent pattern was seen in the C L C P of separate endogenous plasma proteins after each intervention. These data support the view that pulmonary fibrin microembolism without inhibition of the fibrinolytic system was associated with an early increased pulmonary microvascular permeability to protein. In a separate group of similarly prepared animals ( n = 8, 13–21.5 kg) without a lymph catheter, scanning electron microscopic observations showed branching fibrin microemboli to partially occlude some pulmonary arterioles. Mixed thrombus formations in larger precapillary blood vessels were also seen. Ultrastructural observations revealed the deposition of fibrin strands (periodicity = 220–230 Å) within the pulmonary capillaries. Some of these deposits were overlaid by lamellar pseudopodia from endothelial cells and the fibrin appeared to be within these cells. Although plasmalemmal vesicles seemed to be more numerous in the endothelial cells with adjacent fibrin deposits, no gaps or breaks were seen in the densely stained interendothelial cell junctions and/or the endothelial cell membrane of the affected lung capillaries. Activated neutrophils and platelets were more numerous in the pulmonary capillaries following EVC. These data suggest that the presence of FDP and/or fibrin deposits within the pulmonary microvasculature may influence the early functional integrity of pulmonary endothelial cells at sites of fibrin accumulation.