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A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels. We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity. In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.
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Australia is the stronghold of the front-fanged venomous snake family Elapidae. The Australasian elapid snake radiation, which includes approximately 100 terrestrial species in Australia, as well as Melanesian species and all the world's sea snakes, is less than 12 million years old. The incredible phenotypic and ecological diversity of the clade is matched by considerable diversity in venom composition. The clade's evolutionary youth and dynamic evolution should make it of particular interest to toxinologists, however, the majority of species, which are small, typically inoffensive, and seldom encountered by non-herpetologists, have been almost completely neglected by researchers. The present study investigates the venom composition of 28 species proteomically, revealing several interesting trends in venom composition, and reports, for the first time in elapid snakes, the existence of an ontogenetic shift in the venom composition and activity of brown snakes (Pseudonaja sp.). Trends in venom composition are compared to the snakes' feeding ecology and the paper concludes with an extended discussion of the selection pressures shaping the evolution of snake venom.
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Sarafotoxins (SRTX) are endothelin-like peptides extracted from the venom of snakes belonging to the Atractaspididae family. A recent in vivo study on anesthetized and ventilated animals showed that sarafotoxin-b (SRTX-b), extracted from the venom of Atractaspis engaddensis, decreases cardiac output by inducing left ventricular dysfunction while sarafotoxin-m (SRTX-m), extracted from the venom of Atractaspis microlepidota microlepidota, induces right ventricular dysfunction with increased airway pressure. The aim of the present experimental study was to compare the respiratory effects of SRTX-m and SRTX-b. Male Wistar rats were anesthetized, tracheotomized and mechanically ventilated. They received either a 1 LD50 IV bolus of SRTX-b (n = 5) or 1 LD50 of SRTX-m (n = 5). The low-frequency forced oscillation technique was used to measure respiratory impedance. Airway resistance (Raw), parenchymal damping (G) and elastance (H) were determined from impedance data, before and 5 min after SRTX injection. SRTX-m and SRTX-b injections induced acute hypoxia and metabolic acidosis with an increased anion gap. Both toxins markedly increased Raw, G and H, but with a much greater effect of SRTX-b on H, which may have been due to pulmonary edema in addition to bronchoconstriction. Therefore, despite their structural analogy, these two toxins exert different effects on respiratory function. These results emphasize the role of the C-terminal extension in the in vivo effect of these toxins.
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Unlabelled: Sarafotoxin-m (24 amino acids) from the venom of Atractaspis microlepidota microlepidota was the first long-sarafotoxin to be identified, while sarafotoxin-b (21 aa) is a short-sarafotoxin from Atractaspis engaddensis. Despite the presence of three additional C-terminus residues in sarafotoxin-m, these two peptides display a high sequence homology and share similar three-dimensional structures. However, unlike sarafotoxin-b, sarafotoxin-m shows a very low in vitro affinity for endothelin receptors, but still has a very high in vivo toxicity in mammals, similar to that of sarafotoxin-b. We have previously demonstrated, in vitro, the crucial role of the C-terminus extension in terms of pharmacological profiles and receptor affinities of long- versus short-sarafotoxins. One possible hypothesis to explain the high in vivo toxicity of sarafotoxin-m could be that its C-terminus extension is processed in vivo, resulting in short-like sarafotoxin. To address this possibility, we investigated, in the present study, the in vivo cardiovascular effects of sarafotoxin-b, sarafotoxin-m and sarafotoxin-m-Cter (sarafotoxin-m without the C -terminus extension). Male Wistar rats were anaesthetised and mechanically ventilated. Invasive haemodynamic measurements and echocardiographic measurements of left and right ventricular function were performed. The rats were divided into four groups that respectively received intravenous injections of: saline, sarafotoxin-b (one LD50), sarafotoxin-m (one LD50) or sarafotoxin-m-Cter (one LD50). All measurements were performed at baseline, at 1 minute (+1) and at 6 minutes (+6) after injection. Results: Sarafotoxin-b and sarafotoxin-m-Cter decreased cardiac output and impaired left ventricle systolic and diastolic function, whilst sarafotoxin-m decreased cardiac output, increased airway pressures and led to acute right ventricular dilatation associated with a decreased tricuspid annulus peak systolic velocity. Sarafotoxin-b and sarafotoxin-m-Cter appear to exert toxic effects via impairment of left ventricular function, whilst sarafotoxin-m increases airway pressures and impairs right ventricular function. These results do not support the hypothesis of an in vivo processing of long sarafotoxins.
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Endothelin (ET)-related peptides including ET-1 (1-39) were synthesized, and their constricting activity in rat pulmonary artery rings and pressor activity in unanesthetized rat were measured to elucidate their structure-activity relationship. The vasoconstrictor activities of ET-2, ET-3 and sarafotoxin S6b were one-half, one-60th and one-third that of ET-1, respectively. Such differences in biological activities should mainly arise from sequence heterogeneity at the N-terminal portion, especially at positions 4 to 7. All of the blocked ETs at the amino or carboxyl termini showed greatly decreased activities. A monocyclic analog, in which Cys3 and Cys11 were replaced by Ala, showed one-third the activity of ET-1; however, its deamino dicarba analog was almost completely inactive. Significant activities were retained even with replacement of amino acids at positions Ser4, Ser5, Leu6, Met7, Lys9, Tyr13, and Trp21 by Ala, Ala, Gly, Met(0), Leu, Phe, and Tyr or Phe, respectively. On the other hand, replacement of Asp8, Glu10 and Phe14 by Asn, Gln and Ala, respectively, resulted in complete loss of the biological activity. These results indicated that two disulfide bonds in ET molecule were not essential for the expression of vasoconstricting activity. Both terminal amino and carboxyl groups, carboxyl groups of Asp8 and Glu10, and the aromatic group of Phe14 seemed to be contributing, more or less, to the expression of the biological activities.
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Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) in up to 48 samples by monitoring the splitting of a fluorogenic substrate and comparing it to a constant known thrombin activity in a parallel, non-clotting sample. Thus, the non-linearity of the reaction rate with thrombin concentration is compensated for, and adding an excess of substrate can be avoided. Standard conditions were established at which acceptable experimental variation accompanies sensitivity to pathological changes. The coefficients of variation of the surface under the curve (endogenous thrombin potential) are: within experiment approximately 3%; intra-individual: <5% in PPP, <8% in PRP; interindividual 15% in PPP and 19% in PRP. In PPP, calibrated automated thrombography shows all clotting factor deficiencies (except factor XIII) and the effect of all anticoagulants [AVK, heparin(-likes), direct inhibitors]. In PRP, it is diminished in von Willebrand's disease, but it also shows the effect of platelet inhibitors (e.g. aspirin and abciximab). Addition of activated protein C (APC) or thrombomodulin inhibits thrombin generation and reflects disorders of the APC system (congenital and acquired resistance, deficiencies and lupus antibodies) independent of concomitant inhibition of the procoagulant pathway as for example by anticoagulants.
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By using a "slow" fluorogenic thrombin substrate and continuous comparison to a simultaneously run calibrator, thrombin generation can be monitored automatically, on line, in clotting PPP or PRP at a throughput of up to 100 samples per hour. The resulting "Thrombogram" in PPP measures hypocoagulability (haemophilias, oral anticoagulants, heparins (-likes), direct inhibitors) and hypercoagulabilities (AT deficiency, prothrombin hyperexpression, prot. C and S deficiency, factor V Leiden, oral contraceptives). In PRP it is diminished in thrombopathies, in von Willebrand disease, by antibodies blocking GPIIb-IIIa or GPIb, or by antiplatelet drugs like aspirin and clopidogrel. Lupus anticoagulant both retards and increases thrombin generation. The thrombogram thus appears to be a broad function test of the haemostatic-thrombotic mechanism of the blood.
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Cases of snakebite envenomation are frequently presented to veterinary practitioners in southern Africa. Despite this, no published guidelines exist on how this medical emergency should be managed. Southern African snake venoms can be classified into 3 main types based on the main mechanism of venom action and clinical presentation. A polyvalent antivenom is manufactured in South Africa and contains antibodies against the most important southern African snake venoms. The cytotoxic venoms are represented mainly by the puff-adder (Bitis arietans), Mozambique spitting cobra (Naja mossabica), black-necked spitting cobra (Naja nigricollis) (in the Western Cape and Namibia) and the stiletto snake (Atractaspis bibronii). These venoms may cause dramatic local swelling, high morbidity and low mortality and infrequently require the use of antivenom for survival (the only cytotoxic venoms used to prepare the antivenom are the puff-adder and Mozambique spitting cobra). The neurotoxic venoms (represented chiefly by the non-spitting cobras and mambas) cause high mortality due to rapid onset of paresis and require antivenom and mechanical ventilatory support which is life-saving. The boomslang (Dispholidus typus) and the vine snake (coagulopathic venom) rarely bite humans but dogs may be bitten more frequently. These venoms cause a consumption coagulopathy and successful treatment of boomslang bites requires the use of snake species-specific monovalent antivenom. There is no antivenom available for treating vine snake (Thelotornis capensis), berg adder (Bitis atropos), night adder (Causus spp.), stiletto snake and other lesser adder bites. There are some important differences between the way snakebites are managed in humans and dogs.
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While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this wide-ranging snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.
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A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology.
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Venoms can deleteriously affect any physiological system reachable by the bloodstream, including directly interfering with the coagulation cascade. Such coagulopathic toxins may be anticoagulants or procoagulants. Snake venoms are unique in their use of procoagulant toxins for predatory purposes. The boomslang (Dispholidus typus) and the twig snakes (Thelotornis species) are iconic African snakes belonging to the family Colubridae. Both species produce strikingly similar lethal procoagulant pathologies. Despite these similarities, antivenom is only produced for treating bites by D. typus, and the mechanisms of action of both venoms have been understudied. In this study, we investigated the venom of D. typus and T. mossambicanus utilising a range of proteomic and bioactivity approaches, including determining the procoagulant properties of both venoms in relation to the human coagulation pathways. In doing so, we developed a novel procoagulant assay, utilising a Stago STAR Max analyser, to accurately detect real time clotting in plasma at varying concentrations of venom. This approach was used to assess the clotting capabilities of the two venoms both with and without calcium and phospholipid co-factors. We found that T. mossambicanus produced a significantly stronger coagulation response compared to D. typus. Functional enzyme assays showed that T. mossambicanus also exhibited a higher metalloprotease and phospholipase activity but had a much lower serine protease activity relative to D. typus venom. The neutralising capability of the available boomslang antivenom was also investigated on both species, with it being 11.3 times more effective upon D. typus venom than T. mossambicanus. In addition to being a faster clotting venom, T. mossambicanus was revealed to be a much more complex venom composition than D. typus. This is consistent with patterns seen for other snakes with venom complexity linked to dietary complexity. Consistent with the external morphological differences in head shape between the two species, CT and MRI analyses revealed significant internal structural differences in skull architecture and venom gland anatomy. This study increases our understanding of not only the biodiscovery potential of Toxins 2017, 9, 171 2 of 20 these medically important species but also increases our knowledge of the pathological relationship between venom and the human coagulation cascade.
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Venom is a key evolutionary trait, as evidenced by its widespread convergent evolution across the animal kingdom. In an escalating prey-predator arms race, venoms evolve rapidly to guarantee predatory or defensive success. Variation in venom composition is ubiquitous among snakes. Here, we tested variation in venom activity on substrates relevant to blood coagulation amongst Pseudonaja (brown snake) species, Australian elapids responsible for the majority of medically important human envenomations in Australia. A functional approach was employed to elucidate interspecific variation in venom activity and all nine currently recognised species of Pseudonaja. Fluorometric enzymatic activity assays were performed to test variation in whole venom procoagulant activity among species. Analyses confirmed the previously documented ontogenetic shift from non-coagulopathic venom in juveniles to coagulopathic venom as adults, except for the case of P. modesta, which retains non-coagulopathic venom as an adult. These shifts in venom activity correlate with documented ontogenetic shifts in diet among brown snakes from specialisation on reptilian prey as juveniles (and throughout the life cycle of P. modesta), to a more generalised diet in adults that includes mammals. The results of this study bring to light findings relevant to both clinical and evolutionary toxinology.
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Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 [mu]g/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom). Copyright
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Hypofibrinogenemia is an important clinical consequence following envenomation by Crotalus species, usually attenuated or prevented by administration of antivenom. It has been determined that iron and carbon monoxide (CO) enhance fibrinogen as a thrombin substrate, likely secondary to conformational changes in molecular structure. We tested the hypothesis that pretreatment of plasma with iron and CO could attenuate the effects of exposure to Crotalus atrox venom. Human plasma was exposed to 0 to 10 μmol/l ferric chloride (iron source) and 0 to 100 μmol/l CO-releasing molecule-2 (CO source) followed by exposure to 0 to 0.5 μg/ml venom for 5 to 20 min. Changes in coagulation kinetics were determined with thrombelastography. Iron and CO significantly attenuated venom-mediated degradation of plasmatic coagulation in terms of onset time, velocity of clot growth and final clot strength. Further preclinical investigation of iron and CO administration as a 'bridge-to-antivenom' to preserve plasmatic coagulation is justified.
Article
Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. It has been demonstrated that iron and carbon monoxide change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, the present investigation sought to determine if pre-treatment of plasma with iron and carbon monoxide could attenuate venom-mediated catalysis of fibrinogen obtained from Agkistrodon species with fibrinogenase activity. Human plasma was pre-treated with ferric chloride (0-10 μM) and carbon monoxide releasing molecule-2 (CORM-2, 0-100 μM) prior to exposure to 0.5-11 μg/ml of six different Agkistrodon species' venom. The amount of venom used for experimentation needed to decrease coagulation function of one or more kinetic parameters by at least 50% of normal values for (e.g., half the normal speed of clot formation). Coagulation kinetics were determined with thrombelastography. All six snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially prolonging the onset to clot formation and diminishing the speed of clot growth. Pre-treatment of plasma with iron and carbon monoxide attenuated these venom-mediated coagulation kinetic changes in a species-specific manner, with some venom effects markedly abrogated while others were only mildly decreased. Further in vitro investigation of other pit viper venoms that possess fibrinogenolytic activity is indicated to identify species amenable to or resistant to iron and carbon monoxide-mediated attenuation of venom-mediated catalysis of fibrinogen. Lastly, future preclinical investigation with animal models (e.g., rabbit ear bleed model) is planned to determine if iron and carbon monoxide can be used therapeutically after envenomation. This article is protected by copyright. All rights reserved.
Article
Objective To investigate hemostatic changes in dogs envenomed by cytotoxic (African puffadder) and neurotoxic snakes (snouted cobra) using thromboelastography (TEG) and plasma-based coagulation assays.DesignProspective observational clinical study.SettingUniversity teaching hospital.AnimalsEighteen client-owned dogs; 9 envenomed by African puffadder (Bitis arietans) and 9 by snouted cobra (Naja annulifera). Ten healthy dogs served as controls.InterventionsNone.Measurements and Main ResultsBlood was collected at presentation and 24 hours post envenomation. Platelet count, TEG, prothrombin time, activated partial thromboplastin time (aPTT), antithrombin activity, and fibrinogen (Fib) and C-reactive protein (CRP) concentrations were measured. Outcomes were analyzed using linear mixed models at 5% significance. At presentation, R time was significantly prolonged in the puffadder group compared to the cobra (P = 0.01) and control groups (P = 0.05). Platelet count was significantly lower in the puffadder compared to the cobra (P = 0.04) and control groups (P = 0.001), respectively. Antithrombin activity was significantly decreased in the puffadder (P = 0.002) and cobra groups (P = 0.004) compared to the control group. Both prothrombin time and activated partial thromboplastin time were significantly prolonged in the cobra group compared to the control group (P = 0.03 for both). The TEG variables, maximum amplitude (MA) and G, were significantly increased 24 hours post envenomation in the puffadder group compared to their values at presentation (P = 0.05 for both). Fib and CRP concentrations were significantly increased 24 hours post envenomation in both snake-envenomed groups.Conclusions Prolonged clot initiation was a common feature in puffadder-envenomed dogs at presentation and this was likely venom induced. Snouted cobra-envenomed dogs were normo- to hypercoagulable at presentation. Dogs from both puffadder and cobra groups progressed to a more hypercoagulable by 24 hours post envenomation, most likely due to marked inflammation as indicated by the increased Fib and CRP concentrations. TEG proved a sensitive tool for detecting abnormal hemostasis in snake-envenomed dogs.
Article
The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A(2) (PLA(2)), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA(2)s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey.
Article
Preparation of an antivenom against Atractaspis had been done for the first time in the year 2007 in NAVPC in Riyadh, however, it was a lengthy and expensive process. In this study, an alternative treatment was tested using: 1- Nitroglycerin to antagonize the coronary vasospasm induced by the venom, 2- Bosentan to block the endothelin receptors since there is a similarity in structure and effect between the toxic fraction of venom (Sarafotoxins) and endothelins and 3- The specific Antivenom in comparison to nitroglycerin and bosentan. Pretreatment of rabbits with nitroglycerin, antivenom or bosentan completely protected all rabbits from the minimal lethal doses of venom or its toxic fraction. On the other hand, injecting any of the three drugs a few minutes (min) after injecting one minimal lethal dose (MLD) of the venom or the toxic fraction and at the first signs of ischemia, just before death, showed that bosentan completely saved all rabbits. In case of nitroglycerin all rabbits died and in case of antivenom, only 5 out of 10 rabbits were rescued. It is clear that bosentan is superior to the specific antivenom in protecting rabbits; this may be due to its higher affinity to endothelin receptors than sarafotoxins. This preclinical study shows a good potential in using bosentan as a selective antidote for atractaspis envenomation, especially in the African continent.
Article
Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p<0.0001), Hoplocephalus stephensii (Stephen's-banded snake; p<0.0001) and Notechis scutatus (tiger snake; p=0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy.
Article
The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).
Article
This report provides a brief description of the venomous snakes encountered in Southwest Asia, as well as a brief review of the clinical implications of envenomation from each animal. Specific therapy for snake envenomation in the United States is somewhat controversial, and it is no less controversial with animals from this region. The most logical approach probably combines medical management with antivenom when available, and surgical intervention when clearly indicated due to elevated compartment pressure or massive tissue necrosis. Antivenom is available for all species except W aegyptia (in vitro only) and Atractaspis species. Antivenom for V palaestinae may be used for Atractaspis envenomation (Tables 2 and 3).
Article
In endothelial cells of the blood vessels and in other cell types by the cleavage of precursor-molecules 21 amino acids containing peptides--the endothelins--are formed. There exist 3 isoforms. The synthesis of endothelin 1 in the endothelial cells is stimulated by adrenaline, noradrenaline, angiotensin II, arginine-vasopressin, thrombine, interleukin 1 and hypoxia. Receptors for endothelins are distributed in most tissues. A significance of the endothelins apparently exists in the stimulation of the activity of the heart and in the vasoconstriction in the case of a decrease of the blood pressure (when blood is lost) and of hypoxia. Endothelins are also formed in the lungs, the kidneys, the brain and the eye. In experimental animals already a low dose of endothelin has a lethal effect by inducing disturbances in the cardiovascular system. By the snake species Atractaspis engaddensis formed sarafotoxins have a similar structure as the endothelins and are effective by binding to their receptors.
Article
Sarafotoxin S6a, S6b and S6c are chemically related vasoconstrictor polypeptides obtained from the venom of the snake, Atractaspis engaddensis. Each contains twenty one amino acid residues, two intrachain cysteine linkages and a long hydrophobic tail. Structurally these polypeptides resemble endothelin. Binding studies with 125I-endothelin showed that 125I-endothelin bound to rat ventricular membranes is totally displaceable by sarafotoxin S6b and endothelin, with IC50 values of 0.21 and 0.16 nM, respectively. Sarafotoxin S6c, which differs from sarafotoxin S6b in containing threonine instead of serine at residue 2, arginine instead of lysine at residue 4, and glutamic acid instead of lysine at residue 9, only weakly displaced bound 125I-endothelin (IC50, 854 nM). These results indicate that the ability of the sarafotoxins to interact with the endothelin binding site is not solely dependent on the long hydrophobic tail or the cysteine linkages.
Article
We have characterized and purified the two components of the venom of Bothrops atrox that activate the coagulation factor X. Activator 1 and activator 2 were separated by ion-exchange chromatography but otherwise presented similar characteristics. They consist of a heavy polypeptide of Mr 59,000 and either one or two light chains forming a doublet of Mr 14,000-15,000. They are inactive on synthetic substrates and on prothrombin or fibrinogen and thus appear to act specifically on factor X. They are not sensitive to inhibitors of serine proteases or thiol esterases. The activation of factor X is activated by Ca2+ ions with a Hill coefficient of 2.4 and is inhibited by Hg2+, Ba2+, and Cd2+. Its pH dependency suggests that the activity depends on the ionization of a group with an apparent pK of 6.9. We studied the cleavage of purified bovine factor X by B. atrox activators and compared it to that obtained with the factor X activator from Vipera russelli venom. Like the physiological activators, the venom's activators cleave the heavy chain of factor X, producing the activated factor Xa alpha. They produce however two other cleavages: one near the N-terminal end of the heavy chain of factor X, generating factor Xmu, and a second one located at one extremity of the heavy chain of factor Xa alpha, generating factor Xav.
Article
The venom from the snake Atractaspis engaddensis has been shown to be cardiotoxic in anesthetized mice. The effects of the venom were further tested on both atrial and Langendorff heart preparations of rats, in addition to its cardiovascular effects in anesthetized mice. The venom (0.1 mg/kg, i.v.) produced a transient hypertension followed by fluctuation of arterial blood pressure, leading to cardiac failure within 20 min. Various kinds of ECG changes, including S-T depression and A-V block were observed within 10 sec after injection. A dose as low as 1 microgram of venom injected into the perfusion system produced a marked coronary vasospasm in the Langendorff heart preparation, whereas no deleterious effect was found in the atrial preparation at a concentration as high as 10 micrograms/ml. It is concluded that the cardiotoxic effects of the venom are primarily due to coronary vasospasm.
Article
The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.
Article
The effects of Malayan pit viper (Calloselasma rhodostoma) venom on human blood coagulation and fibrinolysis were studied in vitro using computerized thromboelastography. At low concentrations the venom had a coagulant effect shown by faster onset of the coagulation process (shortened SP and R), faster progress of the clot (increased angle and shortened K), and increased coagulation (TEG) index. The maximum amplitude (MA) was not affected, suggesting that the venom had no apparent effect on platelet function; and clot lysis was similar to that in the controls, suggesting that there was no primary fibrinolytic activity. At higher concentrations the venom had anticoagulant effects, SP and R were progressively shortened, but there was poor/no progress in the clot formed, evident from prolonged or absent K, diminished MA and reduced angle. These results show that C. rhodostoma venom has both coagulant and anticoagulant actions. The coagulant action may be due to Factor X activator predominance at low concentrations, while the anticoagulant action could be due to ancrod action. TEG is able to demonstrate the dual effect of this venom, previously described as a paradox, and may be a useful tool in the diagnosis and monitoring of envenomation patients.
Article
1H NMR studies on the nonselective endothelin receptor agonist sarafotoxin SRTb have identified a helix between residues Asp 8 and His 16, and a beta-turn involving residues Cys 3 to Met 6; however, the biologically important C-terminal five residues were found to be conformationally variable. The average RMSD, measured for the final 43 refined structures to the average structure over residues 1-16, was 0.78 +/- 0.18 A for the backbone atoms and 1.39 +/- 0.22 A for all atoms. The torsion angles Cys 3 psi/Lys 4 theta, Thr 7 psi/Asp 8 theta and Gln 17 theta were identified as sites of conformational variability. Differences were found between the structures in the bicyclic loop region for SRTb and those published for ET1, another nonselective receptor agonist, which may explain the observed differences in potency of these peptides. The conformation of an ETB receptor-specific agonist, IRL 1620, which lacks the N-terminal seven residues and the two intrachain disulfides, was found by NMR and circular dichroism spectroscopy to be predominantly random coil, despite the fact that its affinity for the ETB receptor almost equals that of ET1. However, close analysis of the NMR results indicated the presence of turn-like structures, or a nascent helix, in the part of the sequence corresponding to the helical region in the parent peptides. These results suggest that the helical conformation may be required for ligand binding to the ETB receptor as well as to the ETA receptor.
Article
Research into venoms have been extensively developed during the last years, and some groups of venomous animals seldom studied have been recently the subject of new important results, such as the Cones (Gasteropodes, Molluscs) and Spiders, in the venom of which new types of neurotoxic molecules have been described. Concerning the most studied groups, new toxic molecules have been identified in snake (Dendroaspis sp., Atractaspis sp.) and scorpion venoms. The scorpion venoms contain a large variety of toxins acting on various ionic channels, Na+, K+, Ca++ or Cl-. Many structural relations between venomous toxins (Cones and Najas) or between toxins and physiological molecules (sarafotoxins and endothelins, scorpion toxins and Arthropod defensins) have been described and raise important phylogenetic problems which might be also the starting point of new domains of applied research.
Article
Among the components in snake venom are a number which have profound effects (either stimulatory or inhibitory) on haemostatic mechanisms, including coagulation, fibrinolysis, platelet function and vascular integrity. As a consequence, human victims of snakebite may suffer severe and sometimes fatal haemorrhagic and/or thrombotic sequelae. Many of these venom components have been isolated and their precise mechanisms of action established. Apart from direct fibrinolysins, procoagulants predominate, most of these exerting their effect late in the clotting cascade, activating factor X or prothrombin or directly converting fibrinogen to fibrin. Some of the procoagulants are, or have the potential to be, used as therapeutic agents. Some venom components have been put to use as laboratory reagents for diagnostic purposes or for characterising molecular defects of haemostasis, although because they often have unphysiological actions, results must be interpreted with caution. These and other useful constituents e.g. protein C activator and platelet aggregating agents are discussed.
Article
The crude venom of Pseudechis australis exhibited a dose-dependent anticoagulant action on human blood in vitro using computerized thromboelastography. Clot progress parameters (K and alpha) were affected at low dose levels which had no effect on onset of coagulation parameters (SP, R). At high dose levels there was total anticoagulant effect, but in all cases there was no evidence of fibrinolytic activity. These results generally agree with the known effects of this venom on coagulation in vivo, and further support our earlier suggestion that TEG may be a useful, one-step tool in the diagnosis and monitoring of the progress of envenomation patients.
Article
During routine milking of a group of Burrowing Asps Atractaspis engaddensis, one of the authors was bitten in the index finger by one fang, as is characteristic of bites by snakes of the genus. Local effects, oedema, erythema and numbness appeared within minutes, followed by systemic effects, including general weakness, sweating, pallor, fluctuations in the level of consciousness, vomiting and watery non-bloody diarrhoea. Gross oedema of the hand developed and extended up to the forearm. Two hours after admission to the hospital, blood pressure rose to 180/110, the ECG showed normal sinus rhythm and no signs of atrioventricular conduction block. An ECG obtained 24 h after the bite showed new T-wave inversions in leads V5 + 6, which gradually returned to baseline within several days. The local effects healed during the following weeks, but some discoloration and tenderness remained even 10 months after the bite. A maximal exercise (SPECT) study carried out five months after the bite was normal and a multigated radionuclear ventriculogram (MUGA) showed normal left-ventricular function. It may be assumed that the rise in blood pressure observed in this case reflects a systemic vasoconstrictive effect of the sarafotoxins, while the ST changes may have been caused by the direct effect of the toxins on the heart or indirectly by vasoconstriction of the coronary arteries. However, ischaemia secondary to a rise in blood pressure or to excitement could also explain the observed ECG-changes.
Article
Ten species of venomous snakes belonging to three families occur in Israel and in Jordan, some of which may pose a serious threat to humans. Specific, local antivenins are available against only two of the species, while against others regional or European preparations are used. It is suggested that in addition to the monospecific anti-Vipera palaestinae, a polyspecific antivenin be prepared against the clinically most important venomous snakes of the region, namely, Echis coloratus, Pseudocerastes fieldi, Cerastes cerastes, Walterinnesia aegyptia, and Atractaspis engaddensis.
Article
The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.
Article
Sarafotoxins are peptides isolated from the Atractaspis snake venom, with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer. In the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris, and secreted to the culture medium. Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5x10(-10)M and 5x10(-11)M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release.
Article
The two deadly snakes, Walterinnesia aegyptia (black desert cobra) and Atractaspis microlepidota (mole viper) share a common habitat in the central, eastern and western provinces of Saudi Arabia. Bites by either snake were characterized by rapid death, sometimes before reaching any medical facility. Confusing reports of "a black snake bite" are frequently found. The NAVPC had succeeded in preparing a highly effective antivenom against W. aegyptia venom which is now available in the market, but no antivenom against Atractaspis venom is found worldwide. This is probably because of the low molecular weight of sarafotoxins in the venom and hence their poor antigenic properties. At the NAVPC, sarafotoxins were separated by sequential gel filtration of A. microlepidota venom, while toxin T(III) of W. aegyptia venom obtained by cation exchange chromatography and gel filtration. Conjugation of the two toxins was carried out using glutaraldehyde in a two-step procedure followed by exhaustive dialysis. The conjugate was utilized to hyperimmunize 3-years old horses for 10 months, applying a low-dosage protocol and immunostimulants; the crude venoms of both snakes being added during the last 2 months. The F(ab')2 fraction of the antivenom was obtained by pH-guided salt precipitation, enzyme digestion and tangential desalting and filtration. The bivalent antivenom obtained protected mice and rats against the lethal effects of both venoms and rescued the rats challenged with lethal doses of the venoms in recovery experiments. It also neutralized the haemorrhagic, necrotizing and the cardiotoxic effects of A. microlepidota venom and the neuromuscular blocking effect of W. aegyptia venom. The antivenom offers a good rescue potential to those who are bitten by "a black snake" in Saudi Arabia.
Signs, symptoms and treatment of envenomation
  • L Boyer
  • A Alagón
  • B G Fry
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Boyer, L., Alagón, A., Fry, B.G., Jackson, T.N.W., Sunagar, K., Chippaux, J.P., 2015. Signs, symptoms and treatment of envenomation. In: Fry, B.G. (Ed.), Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery. Oxford University Press, New York, pp. 32-60.
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Koludarov, I., Jackson, T.N., Brouw, B.O.D., Dobson, J., Dashevsky, D., Arbuckle, K., Clemente, C.J., Stockdale, E.J., Cochran, C., Debono, J., Stephens, C., Panagides, N., Li, B., Manchadi, M.R., Violette, A., Fourmy, R., Hendrikx, I., Nouwens, A., Clements, J., Martelli, P., Kwok, H.F., Fry, B.G., 2017. Enter the dragon: the dynamic and multifunctional evolution of Anguimorpha lizard venoms. Toxins (Basel) 9.