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.

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.

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.

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.

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.

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.

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.

Inflammatory Effects of Bothrops Phospholipases A2: Mechanisms Involved in Biosynthesis of Lipid Mediators and Lipid Accumulation.

Phospholipases A2s (PLA2s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA2s (svPLA2s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA2s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA2s are classified into the group IIA secreted PLA2s, which encompasses mammalian inflammatory sPLA2s. Thus, research on svPLA2s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA2s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA2s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells.

Cytotoxicity of snake venom Lys49 PLA2-like myotoxin on rat cardiomyocytes ex vivo does not involve a direct action on the contractile apparatus.

Viperid snake venoms contain a unique family of cytotoxic proteins, the Lys49 PLA2 homologs, which are devoid of enzymatic activity but disrupt the integrity of cell membranes. They are known to induce skeletal muscle damage and are therefore named 'myotoxins'. Single intact and skinned (devoid of membranes and cytoplasm but with intact sarcomeric proteins) rat cardiomyocytes were used to analyze the cytotoxic action of a myotoxin, from the venom of Bothrops asper. The toxin induced rapid hypercontraction of intact cardiomyocytes, associated with an increase in the cytosolic concentration of calcium and with cell membrane disruption. Hypercontraction of intact cardiomyocytes was abrogated by the myosin inhibitor para-aminoblebbistatin (AmBleb). No toxin-induced changes of key parameters of force development were observed in skinned cardiomyocytes. Thus, although myosin is a key effector of the observed hypercontraction, a direct effect of the toxin on the sarcomeric proteins -including the actomyosin complex- is not part of the mechanism of cytotoxicity. Owing to the sensitivity of intact cardiomyocytes to the cytotoxic action of myotoxin, this ex vivo model is a valuable tool to explore in further detail the mechanism of action of this group of snake venom toxins.

Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis.

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40-60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.

A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell's Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury.

Acute kidney injury (AKI) following Eastern Russell's viper (Daboia siamensis) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

Venom of the Annulated Sea Snake Hydrophis cyanocinctus: A Biochemically Simple but Genetically Complex Weapon.

Given that the venom system in sea snakes has a role in enhancing their secondary adaption to the marine environment, it follows that elucidating the diversity and function of venom toxins will help to understand the adaptive radiation of sea snakes. We performed proteomic and de novo NGS analyses to explore the diversity of venom toxins in the annulated sea snake (Hydrophis cyanocinctus) and estimated the adaptive molecular evolution of the toxin-coding unigenes and the toxicity of the major components. We found three-finger toxins (3-FTxs), phospholipase A2 (PLA2) and cysteine-rich secretory protein (CRISP) in the venom proteome and 59 toxin-coding unigenes belonging to 24 protein families in the venom-gland transcriptome; 3-FTx and PLA2 were the most abundant families. Nearly half of the toxin-coding unigenes had undergone positive selection. The short- (i.p. 0.09 µg/g) and long-chain neurotoxin (i.p. 0.14 µg/g) presented fairly high toxicity, whereas both basic and acidic PLA2s expressed low toxicity. The toxicity of H. cyanocinctus venom was largely determined by the 3-FTxs. Our data show the venom is used by H. cyanocinctus as a biochemically simple but genetically complex weapon and venom evolution in H. cyanocinctus is presumably driven by natural selection to deal with fast-moving prey and enemies in the marine environment.

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.

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.

Biochemical characterization of Bothrops roedingeri Mertens, 1942 snake venom and its edematogenic, hemorrhagic, and myotoxic activities / [Caracterización bioquímica del veneno de la serpiente Bothrops roedingeri Mertens, 1942, y sus actividades edematógena, hemorrágica y miotóxica].

Introduction: Snakebite envenoming is considered by the World Health Organization (WHO) as a neglected tropical disease. Currently, Bothrops snake venoms are being studied intensively, but there is little knowledge about Bothrops roedingeri venom. Objectives: To biochemically characterize B. roedingeri total venom and evaluate its myotoxic, edematogenic, and hemorrhagic activity. Materials and methods: We characterized B. roedingeri venom enzymatic activity by determining the phospholipase A2 and the proteolytic and fibrinogenolytic action using SDSPAGE electrophoresis while we characterized its venom toxicity by determining the minimum hemorrhagic dose, the minimum edema dose, and the local and systemic myotoxic effects. Results: Bothrops roedingeri venom showed a PLA2 activity of 3.45 ± 0.11 nmoles/min, proteolytic activity of 0.145 ± 0.009 nmoles/min, and a fibrinogen coagulation index of 6.67 ± 1.33 seconds. On the other hand, it produced an minimum hemorrhagic dose of 24.5 µg, an minimum edema dose of 15.6 µg, and a pronounced local myotoxic effect evidenced by the elevation of plasma creatine kinase levels after intramuscular inoculation. The venom showed no systemic myotoxicity. Conclusions: Bothrops roedingeri venom has local hemorrhagic, edematogenic, and myotoxic activity. Enzymatically, it has high PLA2 activity, which would be responsible for the myotoxic and edematogenic effects. It also has proteolytic activity, which could affect coagulation given its ability to degrade fibrinogen, and it causes bleeding through the metalloproteases.

Introducción. El envenenamiento por mordedura de serpiente es considerado por la Organización Mundial de la Salud (OMS) una enfermedad tropical desatendida. Si bien los venenos de otras serpientes Bothrops se vienen estudiado ampliamente, poco se conoce del de Bothrops roedingeri. Objetivos. Caracterizar bioquímicamente el veneno total de la serpiente B. roedingeri y evaluar su actividad miotóxica, edematógena y hemorrágica. Materiales y métodos. Se hizo la caracterización enzimática del veneno de B. roedingeri determinando la actividad de la fosfolipasa A2 (PLA2) y de las enzimas proteolíticas, así como su acción fibrinogenolítica mediante electroforesis en gel de poliacrilamida con duodecilsulfato sódico (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDSPAGE), y la caracterización tóxica del veneno estableciendo la dosis hemorrágica mínima, la dosis edematógena mínima y el efecto miotóxico local y sistémico. Resultados. La actividad de las PLA2 del veneno total de B. roedingeri fue de 3,45 ± 0,11 nmoles/minuto, la proteolítica, de 0,145 ± 0,009 nmoles/minuto, en tanto que el índice de coagulación del fibrinógeno fue de 6,67 ± 1,33 segundos. Por otro lado, el veneno produjo una dosis hemorrágica mínima de 24,5 µg, una dosis edematógena mínima de 15,6 µg y un pronunciado efecto miotóxico local evidenciado por la elevación de los niveles plasmáticos de creatina cinasa después de la inoculación por vía intramuscular. No se registró miotoxicidad sistémica. Conclusiones. El veneno de B. roedingeri tiene efectos hemorrágicos, edematógenos y miotóxicos locales, así como una elevada actividad de la PLA2, que sería responsable de los efectos miotóxico y edematógeno. También presentó actividad proteolítica, la cual podría afectar la coagulación, dada su capacidad para degradar el fibrinógeno y producir hemorragia por acción de las metaloproteasas.

Factor XII-Deficient Chicken Plasma as a Useful Target for Screening of Pro- and Anticoagulant Animal Venom Toxins.

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.

Potential of Matrix Metalloproteinase Inhibitors for the Treatment of Local Tissue Damage Induced by a Type P-I Snake Venom Metalloproteinase.

Snake bite envenoming is a public health problem that was recently included in the list of neglected tropical diseases of the World Health Organization. In the search of new therapies for the treatment of local tissue damage induced by snake venom metalloproteinases (SVMPs), we tested the inhibitory activity of peptidomimetic compounds designed as inhibitors of matrix metalloproteinases on the activities of the SVMP Batx-I, from Bothrops atrox venom. The evaluated compounds show great potential for the inhibition of Batx-I proteolytic, hemorrhagic and edema-forming activities, especially the compound CP471474, a peptidomimetic including a hydroxamate zinc binding group. Molecular dynamics simulations suggest that binding of this compound to the enzyme is mediated by the electrostatic interaction between the hydroxamate group and the zinc cofactor, as well as contacts, mainly hydrophobic, between the side chain of the compound and amino acids located in the substrate binding subsites S1 and S1 ' . These results show that CP471474 constitutes a promising compound for the development of co-adjuvants to neutralize local tissue damage induced by snake venom metalloproteinases.

Damages at the nanoscale on red blood cells promoted by fire corals.

The hydrocoral Millepora alcicornis, known as fire coral, biosynthesize protein toxins with phospholipase A2 (PLA2) activity as a main defense mechanism; proteins that rapidly catalyse the hydrolysis at the sn-2 position of phosphatidylcholine-type phospholipids of cellular membranes. This hydrolysis mechanism triggers a structural damage in the outer leaflet of the red blood cells (RBC) membrane, by generating pores in the lipid bilayer that leads to a depletion of the cellular content of the damaged cell. A secondary mechanism, tentatively caused by pore-forming proteins toxins (PFTs), has been observed. The use of atomic force microscopy (AFM) has allowed to visualize the evolution of damages produced on the surface of the cells at the nanoscale level along the time.

Isolation of two basic phospholipases A2 from Bothrops diporus snake venom: Comparative characterization and synergism between Asp49 and Lys49 variants.

Bothrops diporus, previously considered a subspecies of the B. neuwiedi complex, is a medically relevant viperid in Northeastern Argentina. The venom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, and edema. In the present study, two basic phospholipases A2 (PLA2-I and PLA2-II) were isolated from this venom, and their pathological effects upon murine skeletal muscle and myogenic cells in culture were analyzed. Partial amino acid sequencing showed that PLA2-I and PLA2-II are Asp49 and Lys49 PLA2s, respectively. In agreement with this, PLA2-I showed PLA2 activity, whereas PLA2-II did not. Functional assays revealed differences in their myotoxicity, cytotoxicity, and anti-adhesion activity, and in the ability to inhibit cell migration, all of which were greater for the Lys49 variant. Native electrophoresis showed that PLA2-I was less basic than PLA2-II. The two proteins act synergistically to affect the integrity of C2C12 myogenic cells, providing a further example of the concerted action of coexisting snake venom components. PLA2-I and PLA2-II, together with additional basic PLA2s revealed by RP-HPLC, probably play an important role in myonecrosis after envenomation by B. diporus.