Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms

Phospholipases A₂ from three elapid snake venoms were studied in order to determine if differences in toxicity correlate with differences in pattern or level of phospholipid hydrolysis. The comparatively toxic basic phospholipase A₂ isolated from Naja nigricollis venom exhibits a cardiotoxic action following iv administration in mice that is not exhibited by the less toxic neutral phospholipase A₂ from Hemachatus haemachatus venom or the acidic phospholipase A₂ from Naja naja atra venom. This cardiotoxic action correlates with high levels of phosphatidylserine hydrolysis in heart. Levels and patterns of phospholipid hydrolysis in heart, lung and kidney following iv administration suggest that only the N. nigricollis enzyme has the ability to penetrate permeability barriers in the heart. No cardiotoxic effects are seen following intraventricular injection of a lethal dose of the phospholipases A₂. All three phospholipases A₂ (12 μg/ml) abolish the directly and indirectly elicited muscle twitches of the rat phrenic nerve-diaphragm preparation. This block, in normal or altered bathing media, appears to correlate with the level of phospholipid hydrolysis for the N. naja atra enzyme, but not for the N. nigricollis enzyme. These results suggest that N. nigricollis phospholipase A₂ acts by another mechanism in addition to phospholipid hydrolysis.     

Temperature stability of phospholipase A activity. II. Variations in optimum temperature of phospholipases A2 from various snake venoms.

Maximum phospholipase A₂ activities with Naja nigricollis, N. melanoleuca, N. n. siamensis, N. n. annulifera, Naja naja and Agkistrodon piscivorus venoms were observed at 65°C. Bungarus fasciatus and Bitis gabonica venoms showed optimum temperature for phospholipase A₂ activity at 55°C and Echis carinatus at 50°C. Crotalus scutulatus, Crotalus scutulatus salvini and Crotalus adamanteus venoms lost their phospholipase activity above 45°C. Purified phospholipase A from Crotalus scutulatus salvini venom also showed optimum temperature of activity at 45°C. Under the same substrate conditions, the variations in the rate of hydrolysis at elevated temperatures, with origin of the PLA₂, may be the result of changes in the conformation of the enzyme molecules due to the increased temperature.     

Effects of phospholipase A2 from cobra and bee venom on the presynaptic membrane

L. G. Magazanik, I. M. Gotgilf, T. I. Slavnova, A. I. Miroshnikov and U. R. Apsalon. Effects of phospholipase A₂ from cobra and bee venom on the presynaptic membrane. Toxicon17, 477–488, 1979.—Phospholipases A₂ from bee venom and cobra venom have been isolated and studied. A parallelism was found between enzymatic activity and the ability to block spontaneous miniature end-plate potentials (m.e.p.p.'s) or end-plate potentials (e.p.p.'s) induced by nerve stimulation in the frog sartorius muscle. Different experimental procedures affected both enzymatic activity and blocking ability in qualitatively the same way. Thus, modification of the histidine residue in cobra venom phospholipase by bromophenacyl bromide or the removal of Ca-ions from the medium abolished both activities. Replacement of Ca²⁺ by Sr²⁺ inhibited both the enzymatic and presynaptic effects of cobra venom phospholipase, but did not inhibit the presynaptic action of bee venom phospholipase and decreased its enzymatic activity only 6-fold. Irreversible binding of cobra and bee venom phospholipase to the presynaptic membrane was found in Ca-free solution but Ca-ions were essential for the presynaptic blocking effect induced by these phospholipases. A reduction in the effect of high K⁺ on m.e.p.p. frequency was observed after cobra venom phospholipase treatment. The similar effects of hypertonic sucrose solution and the mitochondrial poison TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) were changed only slightly by bee and cobra venom phospholipase pretreatment. It is concluded that the mechanism of presynaptic blockade induced by bee venom and cobra venom phospholipase consists mainly of damage to sites of release at the presynaptic membrane. There are also some signs of disturbances of depolarization-secretion coupling and of the process of formation of new quanta. The possible functional role of enzymatic activity in the presynaptic effect is discussed.     

Geographical variation of Indian Russell’s viper venom and neutralization of its coagulopathy by polyvalent antivenom

Abstract

Indian Russell’s viper venoms of four different geographical locations were found to vary in composition, coagulopathy and phospholipase A₂ (PLA₂) activity. Venom from Kerala showed highest procoagulant activity followed by Tamil Nadu, West Bengal and Karnataka whereas PLA₂ activity was highest in venom from West Bengal. The commercial polyvalent antivenom differentially neutralized the aforesaid activities of the crude venoms. Antivenomics study showed the presence of non-immunodepleted and partially immunodepleted proteins in the crude venoms. Thus, Indian Russell’s viper venom from different region varies in composition and accentuates the need to design regiospecific antivenoms to confront the problem of envenomation more effectively.     

A new method for the detection of phospholipase A2 variants: identification of isozymes in the venoms of newborn and adult Bothrops asper (terciopelo) snakes

A new method for the identification of phospholipase A2 isozymes in snake venoms is described. The technique is based on the separation of the venom components by isoelectric focusing in agarose gels, transfer of the protein bands by diffusion onto nitrocellulose paper and detection of the phospholipolytic activity of the enzymes by a hemolytic assay either in agarose gels or by benzidine reaction on a solid matrix. Striking differences in the electrophoretic patterns of the phospholipase A2 isozymes between the Atlantic and Pacific venoms and between the newborn and adult venoms from Bothrops asper specimens were observed. The method allowed the detection of 9 different phospholipase A2 isozymes in the venom of adult Atlantic, 7 isozymes in the venom of adult Pacific, and 2-3 isozymes in the venoms of newborn specimens. Horse polyvalent antivenom varied in its capacity to neutralize the phospholipolytic activity of the different isozymes in the same venom and among different venoms.     

Molecular weight variations in the diversity of phospholipase A2 forms in reptile venoms

Reptile venoms exhibit a wide diversity of phospholipase A2 forms when examined by electrophoretic and chromatographic techniques which separate on the basis of net charge. In principle, diversity in charge among the enzyme forms could result from two types of structural modifications: (i) pretranslational modifications, such as differences in amino acid sequences; (ii) post-translational modifications, such as partial proteolysis or hydrolysis of amide functions of asparagine and glutamine residues. Some types of modifications alter both charge and molecular weight. A radiolabeling technique has been developed which allows detection of phospholipase A2 enzymes after electrophoretic separation on the basis of molecular weight in polyacrylamide gels containing sodium dodecylsulfate. Venom preparations were radiolabeled by incubation with p-dodecylphenacylbromide, which was prepared radiolabeled to high specific activity with tritium. The labeling agent was dispersed in phosphatidylcholine liposomes and incubated with venom preparations under conditions of optimal enzyme activity. The preparations were then extracted with ether and hexane to remove substrate and unbound label and subjected to polyacrylamide gel electrophoresis under standard conditions. Protein-bound radiolabel was detected by fluorography and autoradiography. Patterns of labeled species obtained by gel electrophoresis of selected venoms using separation on the basis of charge were similar to the patterns of phospholipase A2 variants obtained under the same electrophoretic conditions using enzymatic detection in phosphatidylcholine-containing gels. Examination of a variety of snake and lizard venoms using this technique revealed the presence of multiple molecular weight forms of labeled enzymes, but the extent of diversity was less than that observed with the same venoms using separation of phospholipase electrophoretic variants on the basis of charge. The results are consistent with diversity in reptile venom phospholipase A2 forms being derived from differences in both molecular weight and charge.     

Coagulant effects of black snake (Pseudechis spp.) venoms and in vitro efficacy of commercial antivenom

The coagulant effects of Australasian black snakes (Pseudechis spp.) are poorly understood and differ to the procoagulant venoms of most dangerous snakes in Australia. This study aimed to investigate in vitro coagulant effects of Pseudechis venoms and the efficacy of commercial black snake antivenom (BlSAV), tiger snake antivenom (TSAV) and specific rabbit anti-snake IgG to neutralise these effects. Using a turbidimetric assay, all six Pseudechis venoms had anticoagulant activity, as well as phospholipase A₂ (PLA₂) activity. Inhibition of PLA₂ activity removed anticoagulant effects of the venoms. Pseudechis porphyriacus was unique and had procoagulant activity independent of PLA2 activity. Both BlSAV and TSAV completely inhibited the coagulant and PLA2 activity of all Pseudechis venoms. PLA2 activity was also inhibited completely by p-Bromophenacyl bromide (pBPB) and partially by specific anti-N. scutatus IgG antibodies. Anti-N. scutatus IgG also completely inhibited anticoagulant activity of Pseudechis venom. All Pseudechis venoms showed immunological cross reactivity with specific anti-snake IgG antibodies to P. porphyriacus, Pseudechis australis and Notechis scutatus. Pseudechis venoms have in vitro anticoagulant activity that appears to be attributable to PLA₂ activity. Both antivenoms inhibited anticoagulant and PLA₂ activity at concentrations below those occurring in patients treated with one vial of antivenom. There was cross-neutralisation of Pseudechis venoms and N. scutatus antibodies that might be attributable to immunological similarities between the venoms.     

A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venom

Phospholipase A₂ (PLA₂, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A₂ from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA₂s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA₂s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.     

Specificity of antibodies to the reconstituted crotoxin complex,from the venom of South American rattlesnake (Crotalus durissus terrificus), using enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion

Some differences between the reaction of antiserum to reconstituted crotoxin complex, to ‘native’ crotoxin and to whole Crotalus durissus terrificus (South American rattlesnake) venom were detected by enzyme-linked immunosorbent assay (ELISA) and by immunodiffusion. ELISA showed the presence of antibodies to the components of the crotoxin complex, phospholipase A₂ and crotapotin. When the antiserum was tested against North American rattlesnake venoms some species gave a positive reaction (C. horridus atricaudatus and C. basiliscus), whilst the venoms of other species were virtually negative (C. durissus totonacus, C. horridus horridus, C. viridis viridis and C. atrox). Confirmation of antigenic similarities between crotoxin and Mojave toxin (from the venom of C. scutulatus scutulatus) was obtained with ELISA and some implications of these species differences in antigen-antibody reaction are discussed. No paraspecificity was observed with heterologous snake venoms from other Crotalidae, Elapidae, Hydrophiidae or Viperidae. ELISA also showed a lack of cross-reactivity of the antiserum to heterologous purified phospholipases A₂ from Enhydrina schistosa, Naja nigricollis or Apis mellifera venoms or from porcine pancreas. The antiserum reacted to the homologous phospholipase A₂ inactivated with p-bromophenacyl bromide as well as to the detoxified crotoxin complex reconstituted with this modified phospholipase A₂. This may be useful in the raising of high titres of antibody to crotoxin.     

Antibodies to sea anemone nematocyst venom--II. Neutralization of the hemolytic, phospholipase A2 and lethal activities by purified antibodies produced in response to attenuated venom.

D. A. Hessinger and R. I. Grove. Antibodies to sea anemone nematocyst venom—II. Neutralization of the hemolytic, phospholipase A₂ and lethal activities by purified antibodies produced in response to attenuated venom. Toxicon17, 109–120, 1979.—The IgG class of antibodies was purified from rabbit serum following the primary immune response to attenuated nematocyst venom from the sea anemone, Aiptasia pallida. This immune IgG fraction was judged to be pure immunoglobulin class G by serological and electrophoretic criteria. The amounts of immune IgG which specifically inhibited the hemolytic, phospholipase A₂ and lethal activities of the venom were determined and related to the possible mechanisms of hemolysis and mouse lethality. The i.v. mouse ld₅₀ of active anemone venom was 136 μg/kg, making it the most toxic of the reported cnidarian venoms. The implications of the rapid onset of toxic symptoms and the very steep dose-response curve are discussed in terms of the possible mechanism of lethality.     

Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2

Carbamylation of 9 out of 10 lysine residues in the toxic phospholipase A₂ from N. nigricollis venom decreased its lethality at least 8-fold and abolished its direct hemolytic and anticoagulant activities, while the enzymatic activity, as measured on purified substrates, decreased only about 50%. Likewise, carbamylation of 3 out of 5 lysines in the relatively less toxic N. naja atra phospholipase induced detoxification and caused a loss of its blocking activity on the phrenic nerve-diaphragm preparation, while its enzymatic activity on purified substrates was unaltered. Results obtained when 7.4 out of 10 lysines in N. nigricollis phospholipase were carbamylated indicate that basicity is not an absolute requirement for high lethal potency, hemolytic activity or cardiotoxicity.The extent of phospholipid hydrolysis induced in erythrocytes, rabbit plasma, phrenic nerve-diaphragm preparation, brain minces and brain synaptic plasma membranes by incubation with the carbamylated enzymes was in agreement with their enzymatic activities as measured on purified substrates. Levels of phospholipid hydrolysis in heart, lung and kidney of mice given phospholipase intravenously, and in brain synaptic plasma membranes from rats given phospholipase intraventricularly, showed that carbamylated derivatives of N. nigricollis phospholipase A₂ lost their ability to reach and/or hydrolyze substrates in vivo. However, the decrease in in vivo phospholipid hydrolysis did not correlate with the decrease in toxicity since, at comparably low levels of phospholipid hydrolysis, some phospholipases were lethal and others were not. Moreover, when intraventricularly administered, both lethal amounts of the native N. naja atra enzyme and its nonlethal carbamylated derivatives produced equally low hydrolysis of synaptic membrane phospholipids.By means of lysine carbamylation, a dissociation between hydrolytic activity and pharmacological properties of phospholipases A₂ has been achieved. We suggest, therefore, that the toxicity of pure phospholipases is primarily due to a direct effect which does not correlate with levels of phospholipid hydrolysis and that this direct effect is prominent in the relatively toxic phospholipases while it is less manifest in the relatively non-toxic enzymes.     

Interactions of Venoms and Venom Components with Blood Platelets

Abstract

Many venoms demonstrate their main toxicity on systems other than blood. Despite this, many venoms also have effects on blood and blood components such as platelets. Crude venoms of snakes, scorpions and spiders have been shown to have indirect (through stimulation of coagulation or epinephrine release, e.g., Echis carinatus and Gentruroides sculpturatus venoms, respectively) and/or direct effects on platelets. For coagulation-dependent effects on platelets, thrombin- or prothrombin-like action (Agkistrodon rhodostoma or contortrix, Bothrops atrox, Crotalus species) may not affect platelets, while those acting at earlier steps may yield normal thrombin and thus activate platelets (Vipera russelli, Echis carinatus). Crude venom effects can be stimulatory (promote or cause aggregation) or inhibitory, with either predominating as a function of dose. Such variability, if observed, often indicates the presence of opposing activities in crude venom, but may also indicate biphasic activity of a single component (e.g., phospholipase A₂). Fractionation of crude venoms of several snakes, particularly pit vipers, has yielded compounds with direct platelet stimulation activity: e.g., aggregoserpentin (Trimeresurus mucrosquamatus), convulxin (Crotalus durissus), crotalocytin (Crotalus horridus horridus) and thrombocytin (Bothrops atrox). These stimulators are polypeptides or glycoproteins, with molecular weights between 35000-80000: one, crotalocytin, is a serine protease. A direct-acting inhibitor has been isolated from Agkistrodon halys venom. Direct aggregatory activity has also been found in a fraction of Loxoscleles reclusa spider venom, specifically in the fraction representing sphingomyelinase D. Centruroides sculpturatus scorpion venom and one fraction of it may induce a low level aggregation which can proceed rapidly to a maximal response on additional stimulation. Since platelets can facilitate coagulation, effects of venoms on platelets may explain in part the ability of the venoms to induce thrombosis and consumption coagulopathies such as disseminated intravascular coagulation. Venoms, and particularly their active fractions, provide unique additional tools for platelet and coagulation research.     

Inhibitory effects of ascorbic acid,vitamin E,and vitamin B-complex on the biological activities induced by Bothrops venom

Context: Natural compounds have been widely studied with the aim of complementing antiophidic serum therapy.

Objective: The present study evaluated the inhibitory potential of ascorbic acid and a vitamin complex, composed of ascorbic acid, vitamin E, and all the B-complex vitamins, on the biological activities induced by snake venoms.

Material and methods: The effect of vitamins was evaluated on the phospholipase, proteolytic, coagulant, and fibrinogenolytic activities induced by Bothrops moojeni (Viperidae), B. jararacussu, and B. alternatus snake venoms, and the hemagglutinating activity induced by B. jararacussu venom.

Results: The vitamin complex (1:5 and 1:10 ratios) totally inhibited the fibrinogenolytic activity and partially the phospholipase activity and proteolytic activity on azocasein induced by the evaluated venoms. Significant inhibition was observed in the coagulation of human plasma induced by venoms from B. alternatus (1:2.5 and 1:5, to vitamin complex and ascorbic acid) and B. moojeni (1:2.5 and 1:5, to vitamin complex and ascorbic acid). Ascorbic acid inhibited 100% of the proteolytic activities of B. moojeni and B. alternatus on azocasein, at 1:10 ratio, the effects of all the venoms on fibrinogen, the hemagglutinating activity of B. jararacussu venom, and also extended the plasma coagulation time induced by all venoms analyzed.

Discussion and conclusion: The vitamins analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms, suggesting their interaction with toxins belonging to the phospholipase A₂, protease, and lectin classes. The results can aid further research in clarifying the possible mechanisms of interaction between vitamins and snake enzymes.     

广西眼镜王蛇毒一种新的酸性磷脂酶A_2的基因克隆和性质

通过SephadexG 75 ,CM SepharoseCL 6B和DEAE SephadexA 5 0连续的柱层析 ,从广西眼镜王蛇 (Ophiophagushannah)蛇毒中分离到一种新的酸性磷脂酶A2 (pI 4 .0 ) ,命名为APLA2 2 .它的分子量约为 14 0 0 0 ,具有较高的磷脂酶活力 (4 2 9mol·g- 1·min- 1) .它对小鼠没有致死性 (LD50 >2 0mg·kg- 1) ,但具有抗血小板聚集功能及诱导水肿形成能力 .通过cDNA测序解决了它的完全的一级结构 .由cDNA推导的APLA2 2蛋白质全序列与福建眼镜王蛇和台湾眼镜王蛇蛇毒酸性磷脂酶A2 的同源性分别为73.9% ,64.7% ,它不存在 62～ 66位的“胰腺环” ,因而它可能是一种新的眼镜王蛇毒酸性磷脂酶A2 .APLA2 2基因克隆及其生化药理性质的研究 ,为探讨蛇毒活性蛋白结构与功能关系及蛇伤中毒机理创造了良好的条件 .     

Characterization of a basic phospholipase A2-homologue myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A₂ activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A₂ myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A₂-homologues with myotoxic, cytolytic, and edema-inducing activities.     

Organization and role of platelet membrane phospholipids as studied with phospholipases A2 from various venoms and phospholipases C from bacterial origin

Phospholipases A₂ from various snake or bee venoms and phospholipases C secreted as exotoxins by several bacteria have been used to study the transverse distribution of phospholipids in the platelet plasma membrane and their role in platelet activation. An asymmetric distribution was described for phospholipids, characterized by a preferential localization of sphingomyelin and phosphatidylcholine in plasma membrane outer leaflet, whereas the inner half contains almost all of the anionic procoagulant phosphatidylserine and phosphatidylinositol. Such a distribution might explain the latency of procoagulant activity in resting platelets and implies an intracellular localization of arachidonic acid, the precursor of prostaglandins and thromboxanes. The external arachidonic acid is involved in phospholipase A₂-induced aggregation, whereas phospholipase C from Clostridium welchii stimulates platelets through a thromboxane-independent pathway. The latter one is directly linked to the formation of phosphatidic and lysophosphatidic acids, which are able to activate cells through calcium mobilization. So, phospholipase C represents an interesting tool for studying the biochemical processes accompanying stimulation, since it is shown that it mimicks the effects of an intracellular phospholipase C, the role of which in platelet activation is discussed.     

Expression of Human Recombinant Antibody Fragments Capable of Partially Inhibiting the Phospholypase Activity of Crotalus durissus terrificus Venom

Abstract: Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A₂ with high enzymatic activity. The phospholipases A₂ are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema‐inducing, hypotensive, platelet‐aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A₂. In this study, we used a non‐immune human single‐chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema‐inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single‐chain fragment variable libraries in the production of antivenoms.     

Inhibition of Snake Venoms and Phospholipases A2 by Extracts from Native and Genetically Modified Eclipta alba: Isolation of Active Coumestans

Abstract: We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A₂ and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high‐performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A₂ activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A₂ isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX‐I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.     

Identification of presynaptic neurotoxin complexes in the venoms of three Australian copperheads (Austrelaps spp.) and the efficacy of tiger snake antivenom to prevent or reverse neurotoxicity

The venom of the Australian lowlands copperhead, Austrelaps superbus, produces significant and potentially lethal neurotoxic paralysis in cases of clinical envenomation. However, little is known about the neurotoxic components within this venom or venoms from the related alpine copperhead (Austrelaps ramsayi) or pygmy copperhead (Austrelaps labialis). Using the isolated chick biventer cervicis nerve-muscle preparation, all Austrelaps venoms were found to exhibit potent and rapid inhibition of nerve-evoked twitch contractions and block of contractures to nicotinic agonists, consistent with postsynaptic neurotoxic activity. Following separation by size-exclusion liquid chromatography under non-denaturing conditions, all Austrelaps venoms were found to also contain a high molecular mass fraction with only weak phospholipase A₂ (PLA₂) activity that caused a slow inhibition of twitch contractions, without inhibiting contractures to nicotinic agonists. These actions are consistent with the presence of additional snake presynaptic PLA₂ neurotoxin (SPAN) complexes in all three Austrelaps venoms. However, there was no evidence of direct muscle damage produced by any Austrelaps venom or SPAN complex. Monovalent tiger snake antivenom was effective in neutralising the neurotoxicity of both whole venom and the SPAN complex. However antivenom was unable to effectively reverse whole venom neurotoxicity, or prejunctional SPAN neurotoxicity, once established. Given the strong neurotoxicity of all Austrelaps venoms, particularly A. ramsayi and A. labialis, effective bites from these copperhead species should be considered potentially lethal. Furthermore, clinicians need to be aware of possible irreversible presynaptic neurotoxicity following envenomation from all copperhead species and that early antivenom intervention is important in preventing further development of toxicity.     

Basic phospholipase A2 from Naja nigricollis snake venom: phospholipid hydrolysis and effects on electrical and contractile activity of the rat heart

To clarify the mechanism of the cardiotoxic action of the basic phospholipase A₂ from Naja nigricollis snake venom, its effects were tested on the rat heart, including hydrolysis of cardiac phospholipids, electrocardiogram, (Na + K)-ATPase activity, resting and action potentials, and cardiac contractility. Its effects contrasted with those of a noncardiotoxic acidic phospholipase from Naja naja atra snake venom. Only the N. nigricollis enzyme exhibited cardiotoxic effects in isolated perfused rat hearts. The cardiotoxic effects occurred with low levels of phospholipid hydrolysis, phosphatidylserine being hydrolyzed to the greatest extent. Although high concentrations of either phospholipase decreased (Na + K)-ATPase activity in vitro, this reaction did not account for the cardiotoxic effects observed in isolated perfused hearts. In rat atrial preparations the cardiotoxic enzyme depolarizes the cells, decreases action potential amplitude and overshoot, decreases time to 50 and 90% repolarization of action potential, and prolongs the latency to initiation of the action potential. Simultaneously recorded contractile activity showed prolonged latency to initiation of contraction, increased time to peak force of contraction, and decreased amplitude of contraction. These effects were mimicked by caffeine and 7.5 mm Ca²⁺ in the bathing medium. The cardiotoxic effects were blocked by Mn²⁺ (Mn²⁺ also reversed cardiotoxic actions) and 4.0 mm Ca²⁺, but not by lanthanum, tetrodotoxin, lidocaine, acetylcholine, or cyanide. Manganese added to rat atrial preparations before N. nigricollis phospholipase decreased hydrolysis of phosphatidylserine. These results suggest that the N. nigricollis enzyme exerts its cardiotoxic action by increasing intracellular Ca²⁺ levels. The cardiotoxic effects correlate with, but may not be related to, phosphatidylserine hydrolysis.