BoaγPLI from Boa constrictor Blood is a Broad-Spectrum Inhibitor of Venom PLA2 Pathophysiological Actions.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

Dissection of phospholipases A2 reveals multifaceted peptides targeting cancer cells, Leishmania and bacteria.

Cationic peptides bio-inspired by natural toxins have been recognized as an efficient strategy for the treatment of different health problems. Due to the specific interaction with substrates from biological membranes, snake venom phospholipases (PLA2s) represent valuable scaffolds for the research and development of short peptides targeting parasites, bacteria, and cancer cells. Considering this, we evaluated the in vitro therapeutic potential of three biomimetic peptides (pCergo, pBmTxJ and pBmje) based on three different amino acid sequences from Asp49 PLA2s. First, short amino acid sequences (12-17 in length) derived from these membranolytic toxins were selected using a combination of bioinformatics tools, including AntiCP, AMPA, PepDraw, ToxinPred, and HemoPI. The peptide, from each polypeptide sequence, with the greatest average antimicrobial index, no toxicity, and no hemolysis predicted was synthesized, purified, and characterized. According to in vitro assays performed, pBmje showed moderate cytotoxicity specifically against MCF-7 (breast cancer cells) with an EC50 of 464.85 µM, whereas pBmTxJ showed an antimicrobial effect against Staphylococcus aureus (ATCC 25923) with an MIC of 37.5 µM, and pCergo against E. coli (ATCC 25922) with an MIC of 75 µM. In addition, pCergo showed antileishmanial activity with an EC50 of 93.69 µM and 110.40 µM against promastigotes of Leishmania braziliensis and L. amazonensis, respectively. Altogether, these results confirmed the versatility of PLA2-derived synthetic peptides, highlighting the relevance of the use of these membrane-interacting toxins as specific archetypes for drug design focused on public health problems.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

Evaluation of different Pakistani medicinal plants for inhibitory potential against Echis carinatus induced Phospholipase A2 toxicity.

Medicinal plants of Pakistan are known for their curative properties against snake bite as rural people have been using natural herbs for such injuries for hundreds to thousands of years. People of rural areas of Pakistan are prone to snakebite, and on the whole death due to snakebite has been increasing worldwide. The objective of this study was to test the neutralizing potential of 17 Pakistani medicinal plant extracts against phospholipase A2 activity in Echis carinatus venom. Plant material was extracted by simple maceration and fractionation of active plant extracts. Venom was collected by manual massage of the venom glands. The PLA2 enzymatic assay was performed to map out the venomous activity of Echis carinatus envenomation. Snake venom released fatty acids at different concentrations (0.1-5 mg/ml) of venom in a dose-dependent manner. Reduction of pH by 01 correlated with 133 µmol of fatty acids released at 5mg/ml of venom. All plants extract inhibited PLA2 activity, however, Curcuma longa, Citrullus colocynthis and Rubia cordifolia inhibited maximum of PLA2 activity (⁓78%) comparable to the standard antidote (p>0.5). Medicinal plants possess secondary metabolites and many active compounds that may have neutralizing or inhibiting properties against the PLA2 activity of Echis venom. Further studies such as compound analysis could provide an alternative against snakebites injuries resulting from Echis carinatus venom.

Combined Proteome and Toxicology Approach Reveals the Lethality of Venom Toxins from Jellyfish Cyanea nozakii.

Jellyfish are a type of poisonous cnidarian invertebrate that secrete lethal venom for predation or defense. Human beings often become victims of jellyfish stings accidentally while swimming or fishing and suffer severe pain, itching, swelling, inflammation, shock, and even death. Jellyfish venom is composed of various toxins, and the lethal toxin is the most toxic and hazardous component of the venom, which is responsible for deaths caused by jellyfish stings and envenomation. Our previous study revealed many toxins in jellyfish venom, including phospholipase A2, metalloproteinase, and protease inhibitors. However, it is still unknown which type of toxin is lethal and how it works. Herein a combined toxicology analysis, proteome strategy, and purification approach was employed to investigate the lethality of the venom of the jellyfish Cyanea nozakii. Toxicity analysis revealed that cardiotoxicity including acute myocardial infarction and a significant decrease in both heart rate and blood pressure is the primary cause of death. Purified lethal toxin containing a fraction of jellyfish venom was subsequently subjected to proteome analysis and bioinformation analysis. A total of 316 and 374 homologous proteins were identified, including phospholipase A2-like toxins and metalloprotease-like toxins. Furthermore, we confirmed that the lethality of the jellyfish venom is related to metalloproteinase activity but without any phospholipase A2 activity or hemolytic activity. Altogether, this study not only provides a comprehensive understanding of the lethal mechanism of jellyfish venom but also provides very useful information for the therapeutic or rescue strategy for severe jellyfish stings.

Systemic alterations induced by phospholipase A2 , BmooTX-I, isolated from Bothrops moojeni snake venom.

Ophidic accidents are among the problems of public health in Brazil. The components from bothropic venom are responsible for many systemic clinical complications resulting from envenomation. The present work aimed to analyse the systemic changes induced in mice after intraperitoneal administration of BmooTX-I, a myotoxic acidic phospholipase A2 isolated from Bothrops moojeni venom. Urinalysis was performed and the following plasma biochemical markers were documented: urea, creatinine and uric acid (renal function); glucose and amylase (pancreatic function); alanine aminotransferase, alkaline phosphatase and gamma-GT (intra- and extrahepatic function); creatine kinase and enzymatic lactate (muscle function). Our results showed that after the intraperitoneal injection of BmooTX-I the urine of these animals showed glycosuria, proteinuria, haematuria, bacteriuria, bilirubinuria, polyuria and nitrite. The plasma biochemical analysis showed alterations in levels of urea, creatinine and uric acid. Amylase concentration was not altered significantly, but the plasma glucose increased significantly compared to controls. The plasma levels of alanine aminotransferase and alkaline phosphatase decreased and increased, respectively, in these same animals. On the other hand, the plasma γGT concentration did not undergo significant modification compared to the control group. The plasma concentration of CK increased, while the enzymatic lactate concentration decreased after the injection of the BmooTX-I. Therefore, in mice BmooTX-I is capable of causing systemic alterations which manifest as renal, muscular, hepatic and pancreatic impairment.

Venomics: integrative venom proteomics and beyond.

Venoms are integrated phenotypes that evolved independently in, and are used for predatory and defensive purposes by, a wide phylogenetic range of organisms. The same principles that contribute to the evolutionary success of venoms, contribute to making the study of venoms of great interest in such diverse fields as evolutionary ecology and biotechnology. Evolution is profoundly contingent, and nature also reinvents itself continuosly. Changes in a complex phenotypic trait, such as venom, reflect the influences of prior evolutionary history, chance events, and selection. Reconstructing the natural history of venoms, particularly those of snakes, which will be dealt with in more detail in this review, requires the integration of different levels of knowledge into a meaningful and comprehensive evolutionary framework for separating stochastic changes from adaptive evolution. The application of omics technologies and other disciplines have contributed to a qualitative and quantitative advance in the road map towards this goal. In this review we will make a foray into the world of animal venoms, discuss synergies and complementarities of the different approaches used in their study, and identify current bottlenecks that prevent inferring the evolutionary mechanisms and ecological constraints that molded snake venoms to their present-day variability landscape.

Functional and structural studies of a Phospholipase A2-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism.

BACKGROUND: One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA2-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA2-like proteins and to advance the current knowledge of their action mechanism. METHODS: Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction. RESULTS: We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA2-like proteins undergo to exert their function. CONCLUSIONS: Our findings show that zinc ions are inhibitors of PLA2-like proteins and suggest two different mechanisms of inhibition for these ions. GENERAL SIGNIFICANCE: Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA2-like proteins.

PID15, a novel 6 kDa secreted peptide, mediates Naja naja venom phospholipase A2 induced apoptosis in isolated human peripheral lymphocytes.

BACKGROUND: Snake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A2 (NV-PLA2) using isolated human peripheral lymphocytes. RESULTS: Human peripheral lymphocytes when incubated with Naja naja venom phospholipase A2 (NV-PLA2) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA2 at 15th min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A2Induced DNA fragmentation factor secreted at 15th min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF). CONCLUSION: Results indicate that the secretion of PID15 is dependent on concentration of NV-PLA2 treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability.

Phospholipases A2: enzymatic assay for snake venom (Naja naja karachiensis) with their neutralization by medicinal plants of Pakistan.

Phospholipases A2 (PLA2) are the most lethal and noxious component of Naja naja karachiensis venom. They are engaged to induce severe toxicities after their penetration in victims. Present study was designed to highlight hydrolytic actions of PLA. in an egg yolk mixture and to encounter their deleterious effects via medicinal plants of Pakistan. PLA2 were found to produce free fatty acids in a dose dependent manner. Venom at concentration of 0.1 mg was found to liberate 26.6 pmoles of fatty acids with a decline in pH1 of 0.2 owing to the presence of PLA2 (133 Unit/mg). When quantity of venom was increased up to 8 mg, it caused to release 133 pmoles of free fatty acids with a decrease in 1.0 pH due to abundance in PLA, (665 Unit/mg). The rest of other doses of venom (0.3-4.0 mg) was found to liberate fatty acids between these two upper and lower limits. Twenty eight medicinal plants (0.1-0.6 mg) were tried to abort PLA, hydrolytic action, however, all were found useful (50-100%) against PLA,. Bauhinia variegate L., Citrus limon (L.). Burm.f. Enicostemnma hyssopifolium (Willd.) Verdoorn, Ocimum sanctum. Psoralea corylifolia L. and Stenolobium stans (L.) D. Don were found excellent in switching off 100% phospholipases A, at their lowest concentration (0.1 mg). Three plants extract were found useful only at lower concentration (0.1 mg), however, their higher doses were seemed to aggravate venom response. Eight medicinal plants failed to neutralize PLA, rather their higher doses were found effective. Standard antidote and rest of other plants extract were able to show maximum of 50% efficiencies. Therefore, it is necessary to identify and isolate bioactive constituent(s) from above cited six medicinal plants to eradicate the problem of snake bite in the future.

BthTX-I, a phospholipase A2-like toxin, is inhibited by the plant cinnamic acid derivative: chlorogenic acid.

Snakebite is a significant health concern in tropical and subtropical regions, particularly in Africa, Asia, and Latin America, resulting in more than 2.7 million envenomations and an estimated one hundred thousand fatalities annually. The Bothrops genus is responsible for the majority of snakebite envenomings in Latin America and Caribbean countries. Accidents involving snakes from this genus are characterized by local symptoms that often lead to permanent sequelae and death. However, specific antivenoms exhibit limited effectiveness in inhibiting local tissue damage. Phospholipase A2-like (PLA2-like) toxins emerge as significant contributors to local myotoxicity in accidents involving Bothrops species. As a result, they represent a crucial target for prospective treatments. Some natural and synthetic compounds have shown the ability to reduce or abolish the myotoxic effects of PLA2-like proteins. In this study, we employed a combination approach involving myographic, morphological, biophysical and bioinformatic techniques to investigate the interaction between chlorogenic acid (CGA) and BthTX-I, a PLA2-like toxin. CGA provided a protection of 71.8% on muscle damage in a pre-incubation treatment. Microscale thermophoresis and circular dichroism experiments revealed that CGA interacted with the BthTX-I while preserving its secondary structure. CGA exhibited an affinity to the toxin that ranks among the highest observed for a natural compound. Bioinformatics simulations indicated that CGA inhibitor binds to the toxin's hydrophobic channel in a manner similar to other phenolic compounds previously investigated. These findings suggest that CGA interferes with the allosteric transition of the non-activated toxin, and the stability of the dimeric assembly of its activated state.

Ascorbyl palmitate (ASC16) as a potential inhibitor of toxicity induced by Crotalus durissus terrificus venom.

It is well known that C. d. terrificus venom causes pathophysiological effects such as neuropathies, coagulopathies, and even death. Previous studies have reported that ASC16 can interact with monomeric phospholipases A2 from the venom of various snake species (e.g., Vipera russelli and Echis carinatus). As a result, ASC16 has been proposed as an inhibitor of the toxic effects induced by the heterodimeric complex (crotoxin) and other components of the venom of C. d. terrificus. To investigate this further, in silico studies were designed using the crotoxin (CTX) protein complex as a model, and experimental assays were conducted to evaluate the inhibitory effect of ASC16 on CTX, as well as on other venom enzymes such as thrombin-like enzyme (TLE), phosphodiesterase (PDE) and l-aminoxidase (LAAO). For in vitro assays, specific substrates were used, and lethal activity was measured over 48 h using an in vivo murine experimental model (CF01). In silico studies have indicated that the hydrophilic portion of ASC16 adopts a stable conformation while interacting with the catalytic site of crotoxin. At the highest concentrations, ASC16 significantly inhibited the activities of PLA2 (40.89 ± 0.09 %), TLE (11.03 ± 0.69 %), PDE (51.33 ± 2.83 %), and LAAO (56.79 ± 2.91 %). Furthermore, ASC16 neutralized the 2 LD50 lethality of crotalic venom. These findings lay the groundwork for designing promising adjuvants that can facilitate the incorporation of a larger quantity of proteins in immunization schemes. Consequently, this approach aims to achieve higher antibody titers, reduce the number of required immunizations, and minimize local damage in the producer animal.

The Toxin Diversity, Cytotoxicity, and Enzymatic Activity of Cape Cobra (Naja nivea) Venom.

"True" cobras (genus Naja) are among the venomous snakes most frequently involved in snakebite accidents in Africa and Asia. The Cape cobra (Naja nivea) is one of the African cobras of highest medical importance, but much remains to be learned about its venom. Here, we used a shotgun proteomics approach to better understand the qualitative composition of N. nivea venom and tested its cytotoxicity and protease activity as well as its effect on intracellular Ca2+ release and NO synthesis. We identified 156 venom components representing 17 protein families, with the dominant ones being three-finger toxins, mostly of the short-chain type. Two-thirds of the three-finger toxin entries identified were assigned as cytotoxins, while the remainder were categorized as neurotoxins, including short-chain, long-chain, and ancestral three-finger toxins. We also identified snake venom metalloproteinases and members of CRISP, l-amino acid oxidase, and other families. Protease activity and its effect on intracellular Ca2+ release and NO synthesis were low. Phospholipase A2 activity was surprisingly high, despite this toxin family being marginally recovered in the analyzed venom. Cytotoxicity was relevant only at higher venom concentrations, with macrophage and neuroblastoma cell lines showing the lowest viability. These results are in line with the predominantly neurotoxic envenomation symptoms caused by Cape cobra bites. The present overview of the qualitatively complex and functionally intriguing venom of N. nivea may provide insights into the pathobiochemistry of this species' venom.

Effect of oxidation and in vitro intestinal hydrolysis on phospholipid toxicity towards HT29 cell line serving as a model of human intestinal epithelium.

Oxidation of food-derived phospholipids (PLs) can influence nutrient digestion and induce oxidative stress in gastrointestinal epithelium. In this study, hen egg yolk PL fraction was used to evaluate the effect of lipoxygenase (LOX)-induced PL oxidation on the rate of PL hydrolysis catalyzed by pancreatic phospholipase A2 (PLA2) in the presence of bile salts (BSs). Then, PL/BS solutions containing native or oxidized PLs were used in in vitro intestinal digestion to assess the effect of PL oxidation and hydrolysis on the toxicity towards HT29 cell line. Based on the obtained results, we suggest that hexanal and (E)-2-nonenal, formed by the decomposition of PL hydroperoxides, inhibited PLA2 activity. The cell exposure to simulated intestinal fluid (SIF) containing BSs decreased HT29 cell viability and significantly damaged cellular DNA. However, the genotoxic effect was reversed in the presence of all tested PL samples, while the protective effect against the BS-induced cytotoxicity was observed for native non-hydrolyzed PLs, but was not clearly visible for other samples. This can result from an overlap of other toxic effects such as lipotoxicity or disturbance of cellular redox homeostasis. Taking into account the data obtained, it was proposed that the PLA2 activity decline in the presence of PL oxidation products may be a kind of protective mechanism against rapid release of oxidized FAs characterized by high cytotoxic effect towards intestinal epithelium cells.

A Review of Rattlesnake Venoms.

Venom components are invaluable in biomedical research owing to their specificity and potency. Many of these components exist in two genera of rattlesnakes, Crotalus and Sistrurus, with high toxicity and proteolytic activity variation. This review focuses on venom components within rattlesnakes, and offers a comparison and itemized list of factors dictating venom composition, as well as presenting their known characteristics, activities, and significant applications in biosciences. There are 64 families and subfamilies of proteins present in Crotalus and Sistrurus venom. Snake venom serine proteases (SVSP), snake venom metalloproteases (SVMP), and phospholipases A2 (PLA2) are the standard components in Crotalus and Sistrurus venom. Through this review, we highlight gaps in the knowledge of rattlesnake venom; there needs to be more information on the venom composition of three Crotalus species and one Sistrurus subspecies. We discuss the activity and importance of both major and minor components in biomedical research and drug development.

Proteomic characteristics of six snake venoms from the Viperidae and Elapidae families in China and their relation to local tissue necrosis.

Patients envenomed by snakes from the Viperidae and Elapidae families in China often have varying degrees of local tissue necrosis. Due to the relative clinical characteristics of local tissue necrosis and ulceration following envenoming, this study has analyzed the proteome of six snake venoms from the Viperidae and Elapidae family, and the toxin profiles of each snake were compared and correlated with the clinical manifestations that follow cytotoxic envenoming. Deinagkistrodon acutus and Naja atra envenomation induce severe ulceration, which is absent in Bungarus multicinctus envenomation and mild in the other three vipers. It is interesting to note that the proportion of c-type lectins (CTL) (20.63%) in Deinagkistrodon acutus venom was relatively high, which differs from the venom of other vipers. In addition, three-fingered toxin (3FTx) (2.15%) is present in the venom of Deinagkistrodon acutus, but has not been detected in the remaining three vipers. Snake venom metalloprotease (SVMP) (34.4%-44.7%), phospholipase A2 (PLA2) (9.81%-40.83%), and snake venom serine protease (SVSP) (9.44%-16.2%) represent the most abundant families of toxin in Viperidae venom. The Elapidae venom proteome was mainly composed of neurotoxins and cytotoxins, including 3FTx (39.28%-60.08%) and PLA2 (8.24%-58.95%) toxins, however, the proportion of CRISPS (26.36%) in Naja atra venom was relatively higher compared to Bungarus multicinctus venom. Significant differences in SVMP, SVSP, and 3FTx expression levels exist between the Viperidae and the Elapidae family. The main toxins responsible for the development of tissue necrosis and ulcerations following Viperidae envenoming are hematotoxins (SVSMP, SVSP) and myotoxins (PLA2). Deinagkistrodon acutus venom contains high levels of CTL and traces of 3FTx, leading to more severe local necrosis. However, Naja atra venom can also cause severe local necrosis through the effects of myotoxin (3FTx, CRISP, PLA2). Bungarus multicinctus venom does not contain myotoxins, resulting in pure systemic neurological manifestations no obvious necrosis of local tissue in patients.

Biochemical and computational approaches to understand venom toxin-toxin interaction.

Snake venoms are mainly composed of proteins and peptides (venom toxins). The venom transcriptomes and proteomes have been extensively investigated; however, venom toxin-toxin interactions remain poorly characterized. We detected the interaction of venom Asp49-PLA2 and 3FTx using biochemical and computational approaches. A stable structure of Asp49-PLA2-3FTx was identified, and the interface of Asp49-PLA2 and 3FTx was analyzed. The approaches will shed light on understanding the venom complexity and deciphering the synergistic effects of venom toxins.

The edematogenic effect of Micrurus lemniscatus venom is dependent on venom phospholipase A2 activity and modulated by non-neurogenic factors.

Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.

Diversity of Phospholipases A2 from Bothrops atrox Snake Venom: Adaptive Advantages for Snakes Compromising Treatments for Snakebite Patients.

The evolution of snake venoms resulted in multigene toxin families that code for structurally similar isoforms eventually harboring distinct functions. PLA2s are dominant toxins in viper venoms, and little is known about the impact of their diversity on human envenomings and neutralization by antivenoms. Here, we show the isolation of three distinct PLA2s from B. atrox venom. FA1 is a Lys-49 homologue, and FA3 and FA4 are catalytic Asp-49 PLA2s. FA1 and FA3 are basic myotoxic proteins, while FA4 is an acid non-myotoxic PLA2. FA3 was the most potent toxin, inducing higher levels of edema, inflammatory nociception, indirect hemolysis, and anticoagulant activity on human, rat, and chicken plasmas. FA4 presented lower anticoagulant activity, and FA1 had only a slight effect on human and rat plasmas. PLA2s presented differential reactivities with antivenoms, with an emphasis on FA3, which was not recognized or neutralized by the antivenoms used in this study. Our findings reveal the functional and antigenic diversity among PLA2s from B. atrox venom, highlighting the importance of assessing venom variability for understanding human envenomations and treatment with antivenoms, particularly evident here as the antivenom fails to recognize FA3, the most active multifunctional toxin described.

A Combined Bioassay and Nanofractionation Approach to Investigate the Anticoagulant Toxins of Mamba and Cobra Venoms and Their Inhibition by Varespladib.

Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A2 (PLA2) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA2s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA2 inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.