Myotoxic Phospholipase A2 Research Articles

Alterations of the skeletal muscle contractile apparatus in necrosis induced by myotoxic snake venom phospholipases A2: a mini-review

Skeletal muscle necrosis is a common clinical manifestation of snakebite envenoming. The predominant myotoxic components in snake venoms are catalytically-active phospholipases A2 (PLA2) and PLA2 homologs devoid of enzymatic activity, which have been used as models to investigate various aspects of muscle degeneration. This review addresses the changes in the contractile apparatus of skeletal muscle induced by these toxins. Myotoxic components initially disrupt the integrity of sarcolemma, generating a calcium influx that causes various degenerative events, including hypercontraction of myofilaments. There is removal of specific sarcomeric proteins, owing to the hydrolytic action of muscle calpains and proteinases from invading inflammatory cells, causing an initial redistribution followed by widespread degradation of myofibrillar material. Experiments using skinned cardiomyocytes and skeletal muscle fibers show that these myotoxins do not directly affect the contractile apparatus, implying that hypercontraction is due to cytosolic calcium increase secondary to sarcolemmal damage. Such drastic hypercontraction may contribute to muscle damage by generating mechanical stress and further sarcolemmal damage.

The early inhibition of the COX-2 pathway in viperid phospholipase A2-induced skeletal muscle myotoxicity accelerates the tissue regeneration

The administration of non-steroidal anti-inflammatory drugs in the treatment of injury and muscle regeneration is still contradictory in effectiveness, especially regarding the timing of their administration. This can interfere with the production of prostaglandins originating from inflammatory isoform cyclooxygenase-2 (COX-2), which is essential to modulate tissue regeneration. The phospholipases A2 (PLA2) from viperid venoms cause myotoxicity, therefore constituting a tool for the study of supportive therapies to improve skeletal muscle regeneration. This study investigated the effect of early administration of lumiracoxib (selective inhibitor of COX-2) on the degeneration and regeneration stages of skeletal muscle after injury induced by a myotoxic PLA2. After 30 min and 48 h of intramuscular injection of PLA2, mice received lumiracoxib orally and histological, functional, and transcriptional parameters of muscle were evaluated from 6 h to 21 days. Inhibition of COX-2 in the early periods of PLA2-induced muscle degeneration reduced leukocyte influx, edema, and tissue damage. After the second administration of lumiracoxib, in regenerative stage, muscle showed increase in number of basophilic fibers, reduction in fibrosis content and advanced recovery of functionality characterized by the presence of fast type II fibers. The expression of Pax7 and myogenin were increased, indicating a great capacity for storing satellite cells and advanced mature state of tissue. Our data reveals a distinct role of COX-2-derived products during muscle degeneration and regeneration, in which early administration of lumiracoxib was a therapeutic strategy to modulate the effects of prostaglandins, providing a breakthrough in muscle tissue regeneration induced by a myotoxic PLA2.

In Vitro Efficacy of Antivenom and Varespladib in Neutralising Chinese Russell's Viper (Daboia siamensis) Venom Toxicity.

The venom of the Russell's viper (Daboia siamensis) contains neurotoxic and myotoxic phospholipase A2 toxins which can cause irreversible damage to motor nerve terminals. Due to the time delay between envenoming and antivenom administration, antivenoms may have limited efficacy against some of these venom components. Hence, there is a need for adjunct treatments to circumvent these limitations. In this study, we examined the efficacy of Chinese D. siamensis antivenom alone, and in combination with a PLA2 inhibitor, Varespladib, in reversing the in vitro neuromuscular blockade in the chick biventer cervicis nerve-muscle preparation. Pre-synaptic neurotoxicity and myotoxicity were not reversed by the addition of Chinese D. siamensis antivenom 30 or 60 min after venom (10 µg/mL). The prior addition of Varespladib prevented the neurotoxic and myotoxic activity of venom (10 µg/mL) and was also able to prevent further reductions in neuromuscular block and muscle twitches when added 60 min after venom. The addition of the combination of Varespladib and antivenom 60 min after venom failed to produce further improvements than Varespladib alone. This demonstrates that the window of time in which antivenom remains effective is relatively short compared to Varespladib and small-molecule inhibitors may be effective in abrogating some activities of Chinese D. siamensis venom.

Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.

DM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor’s regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.

Changes in basement membrane components in an experimental model of skeletal muscle degeneration and regeneration induced by snake venom and myotoxic phospholipase A2

Skeletal muscle regeneration is impaired after myonecrosis induced by viperid snake venoms, but the mechanisms behind such poor regenerative outcome are not fully understood. This study compared the changes in basement membrane (BM) components in mouse skeletal muscle in two different scenarios of muscle injury: (a) injection of Bothrops asper venom, as a model of poor regeneration, and (b) injection of a myotoxic fraction (Mtx) isolated from this venom, as a model of successful regeneration. The degradation and reposition of laminin, type IV collagen and fibronectin were assessed over time by a combination of immunohistochemistry, Western blot, and real time polymerase chain reaction. Both treatments induced degradation of laminin and type IV collagen in areas of muscle necrosis since day one, however, there were differences in the pattern of degradation and reposition of these proteins along time. Overall, Mtx induced a higher synthesis of fibronectin and higher degradation of laminin at intermediate time points, together with higher levels of transcripts for the chains of the three proteins. Instead, venom induced a higher degradation of laminin and type IV collagen at early time intervals, followed by a reduced recovery of type IV collagen by 15 days. These differences in extracellular matrix degradation and remodeling between the two models could be associated to the poor muscle regeneration after myonecrosis induced by B. asper venom.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Effects of Two Fractions of Swietenia macrophylla and Catechin on Muscle Damage Induced by BothropsVenom and PLA₂.

Plant natural products can attenuate the myonecrosis caused by Bothrops snake venom and their phospholipases A2 (PLA2). In this study, we evaluated the effects of two fractions (F4 and F6) from Swietenia macrophylla and purified catechin on the muscle damage caused by a myotoxic PLA2 from Colombian Bothrops asper venom (BaColPLA2) in mice and by Bothrops marmoratus venom from Brazil in mouse phrenic nerve-diaphragm muscle (PND) preparations in vitro. Male mice were injected with PLA2 (50 µg) in the absence or presence of F4, F6, and catechin, in the gastrocnemius muscle and then killed 3, 7, 14, and 28 h later for histopathological analysis of myonecrosis, leukocyte infiltration, and the presence of collagen. Fractions F4 and F6 (500 µg) and catechin (90 µg) significantly reduced the extent of necrosis at all-time intervals. These two fractions and catechin also attenuated the leukocyte infiltration on day 3, as did catechin on day 14. There was medium-to-moderate collagen deposition in all groups up to day 7, but greater deposition on days 14 and 28 in the presence of F6 and catechin. Bothrops marmoratus venom (100 µg/mL) caused slight (~25%) muscle facilitation after 10 min and weak neuromuscular blockade (~64% decrease in contractile activity after a 120-min incubation). Pre-incubation of venom with F4 or F6 abolished the facilitation, whereas catechin, which was itself facilitatory, did not. All three fractions attenuated the venom-induced decrease in muscle contractions. These findings indicate that fractions and catechin from S. macrophylla can reduce the muscle damage caused by Bothrops venom and PLA2. These fractions or their components could be useful for treating venom-induced local damage.

Neutralizing properties of LY315920 toward snake venom group I and II myotoxic phospholipases A2

A need exists to develop specific and clinically useful inhibitors of toxic enzymes present in snake venoms, responsible for severe tissue damage and life-threatening effects occurring in thousands of people suffering envenomations globally. LY315920 (Varespladib, S-5920, A-001), a low molecular weight drug developed to inhibit several human secreted phospholipases A2 (PLA2s), was recently shown to also inhibit PLA2s in whole snake venoms with high potency, yet no studies have examined its direct effect on purified snake venom PLA2s. This work evaluated the ability of LY315920 to neutralize the enzymatic and toxic activities of three isolated PLA2 toxins of structural groups I (pseudexin) and II (crotoxin B and myotoxin I), and their corresponding whole venoms. In vitro, LY315920 inhibited the catalytic activity of these three enzymes upon a synthetic substrate. The drug also blocked their cytotoxic effect on cultured murine myotubes. In mice, preincubation of the toxins or venoms with LY315920, followed by their intramuscular injection, resulted in significant inhibition of muscle damage. Finally, immediate, independent injection of LY315920 at the site of toxin or venom inoculation also resulted in a large reduction of myonecrosis in the case of pseudexin and myotoxin-I, and of Pseudechis colletti and Bothrops asper whole venoms, suggesting a possible method of drug delivery in emergency situations. Present findings add evidence to suggest the possibility of using LY315920 as a field antidote in snakebites, aiming to limit the myonecrosis induced by many venom PLA2s in the clinical setting.

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.

Isolation and Functional Characterization of an Acidic Myotoxic Phospholipase A₂ from Colombian Bothrops asper Venom.

Myotoxic phospholipases A2 (PLA2) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA2 (BaCol PLA2) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA2 had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA2 showed structural homology with other acidic PLA2 isolated from Bothrops venoms, including a non-myotoxic PLA2 from Costa Rican B. asper. In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA2 had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA2 caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm.

Venom Evolution: Gene Loss Shapes Phenotypic Adaptation

Snake venoms are variable protein mixtures with a multitude of bioactivities. New work shows, surprisingly, that it is the loss of toxin-encoding genes that strongly influences venom function in rattlesnakes, highlighting how gene loss can underpin adaptive phenotypic change.

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2

The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2μg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2.

Anticoagulant effect of myotoxic phospholipase A2 isolated from the venom of the snake Bothrops asper (Viperidae).

La fosfolipasa A 2 miotoxica del veneno de Bothrops asper (terciopelo) prolonga el tiempo de recalcificacion de plasmas de cinco especies de mamiferos. La fosfolipasa A 2 hidroliza los fosfolipidos del plasma, en tanto que su accion inhibitoria es revertida cuando se adicionan fosfolipidos plaquetarios. Estas dos observaciones sugieren que la accion anticoagulante se debe a una alteracion de los fosfolipidos plasma­ticos necesarios para la coagulacion. Tanto la actividad enzimatica como la anticoagulante se mantuvieron aun despues de calentamiento a 95°C durante 10 min. Pese a su accion anticoagulante in vitro , la fosfolipasa A 2 no prolonga el tiempo de coagulacion luego de inoculacion intravenosa en ratones.

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis.

The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.

Biochemical and functional studies of ColTx-I, a new myotoxic phospholipase A2 isolated from Crotalus oreganus lutosus (Great Basin rattlesnake) snake venom

Commonly, phospholipases A2 (PLA2s) play key roles in the pathogenesis of the local tissue damage characteristic of crotaline and viperine snake envenomations. Crotalus oreganus lutosus snake venom has not been extensively studied; therefore, the characterization of its components represents a valuable biotechnological tool for studying pathophysiological processes of envenoming and for gaining a deeper understanding of its biological effects. In this study, for the first time, a basic PLA2 myotoxin, ColTx-I, was purified from C. o. lutosus through two chromatographic steps. ColTx-I is monomeric with calculated molecular mass weight (Mw) of 14,145 Da and a primary structure closely related to basic PLA2s from viperid venoms. The pure enzyme has a specific activity of 15.87 ± 0.65 nmol/min/mg at optimal conditions (pH 8.0 and 37 °C). ColTx-I activity was found to be dependent on Ca2+, as its substitution by other ionic species as well as the addition of chelating agents significantly reduced its phospholipase activity. In vivo, ColTx-I triggered dose-dependent inflammatory responses, measured using the paw edema model, with an increase in IL-6 levels, systemic and local myotoxicity, characterized by elevated plasma creatine kinase activity. ColTx-I induced a complex series of degenerative events associated with edema, inflammatory infiltrate and skeletal muscle necrosis. These biochemical and functional results suggest that ColTx-I, a myotoxic and inflammatory mediator, plays a relevant role in C. o. lutosus envenomation. Thus, detailed studies on its mechanism of action, such as evaluating the synergism between ColTx-I and other venom components may reveal targets for the development of more specific and effective therapies.

PhTX-II a basic myotoxic phospholipase A₂ from Porthidium hyoprora snake venom, pharmacological characterization and amino acid sequence by mass spectrometry.

A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.

Two color morphs of the pelagic yellow-bellied sea snake, Pelamis platura, from different locations of Costa Rica: Snake venomics, toxicity, and neutralization by antivenom

The yellow-bellied sea snake, Pelamis platura, is the most broadly distributed snake species. Despite being endowed with a highly lethal venom, a proteomic analysis of its toxin composition was unavailable. The venoms of specimens collected in Golfo de Papagayo and Golfo Dulce (Costa Rica), where two distinctive color morphs occur, were chromatographically compared. The latter inhabits a fjord-like gulf where the transit of oceanic sea snakes into and from the basin is restricted, thus possibly affecting gene flow. RP-HPLC evidenced a conserved venom protein profile in both populations, despite their divergent color phenotypes. Following a trend observed in other sea snakes, P. platura venom is relatively simple, being composed of proteins of the three-finger toxin (3FTx), phospholipase A2 (PLA2), cysteine-rich secretory protein (CRISP), 5'-nucleotidase, and metalloproteinase families. The first three groups represent 49.9%, 32.9%, and 9.1% of total venom protein, respectively. The most abundant component (~26%) is pelamitoxin (P62388), a short-chain 3FTx, followed by a major basic PLA2 (~20%) and a group of three isoforms of CRISPs (~9%). Whereas isolated pelamitoxin was highly lethal to mice, neither the PLA2 nor the CRISP fraction caused death. However, the PLA2 rapidly increased plasma creatine kinase activity after intramuscular injection, indicating its myotoxic action. Differing from myotoxic PLA2s of viperids, this PLA2 was not cytolytic to murine myogenic cells in vitro, suggesting possible differences in its mechanism of action. The median lethal dose (LD50) estimates for P. platura crude venom in mice and in three species of fishes did not differ significantly. The sea snake antivenom manufactured by CSL Ltd. (Australia), which uses Enhydrina schistosa as immunogen, cross-recognized the three major components of P. platura venom and, accordingly, neutralized the lethal activity of crude venom and pelamitoxin, therefore being of potential usefulness in the treatment of envenomations by this species. Integrative analyses of animal venoms that combine the power of proteomics (venomics) with the characterization of their functional and immunological properties are significantly expanding knowledge on these remarkable bioweapons, both from a basic and a medical perspective. Costa Rica harbors a unique population of the yellow-bellied sea snake, Pelamis platura, that is restricted to a fjord-like gulf (Golfo Dulce). This population differs markedly from oceanic populations found elsewhere along the Pacific coast of this country, by presenting a patternless bright yellow coloration, instead of the typical bicolored or tricolored pattern of this species. It has been suggested that the dominance of this yellow-morph in Golfo Dulce might reflect gene flow restrictions, caused by the oceanographic conditions at this location. The present study demonstrates that the remarkable phenotypic variation between the two color morphs inhabiting Golfo Dulce and Golfo de Papagayo, respectively, is not associated with differences in the expression of venom components, as shown by their conserved RP-HPLC profiles. Proteomic analysis revealed the relatively simple toxin composition of P. platura venom, which contains three predominant types of proteins: three-finger toxins (protein abundance: 49.9%), phospholipases A2 (32.9%), and cysteine-rich secretory proteins (9.1%), together with few minor components. Further, the involvement of these most abundant proteins in the toxic effects of the venom, and their cross-recognition and neutralization by a sea snake antivenom produced against the venom of Enhydrina schistosa, were analyzed.

Activation of J77A.1 Macrophages by Three Phospholipases A2Isolated fromBothrops atroxSnake Venom

In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

Snake Venom Induced Local Toxicities: Plant Secondary Metabolites as an Auxiliary Therapy

Snakebite is a serious medical and socio-economic problem affecting the rural and agricultural laborers of tropical and sub-tropical region across the world leading to high morbidity and mortality. In most of the snakebite incidences, victims usually end up with permanent tissue damage and sequelae with high socioeconomic and psychological impacts. Although, mortality has been reduced markedly due to anti-venom regimen, it is associated with several limitations. Snake venom metalloprotease, hyaluronidase and myotoxic phospholipase A2 are the kingpins of tissue necrosis and extracellular matrix degradation. Thus, inhibition of these enzymes is considered to be the rate limiting step in the management of snakebite. Unfortunately, tissue necrosis and extracellular matrix degradation persists even after the administration of anti-venom. At present, inhibitors from snake serum and plasma, several synthetic compounds and their analogs have been demonstrated to possess anti-snake venom activities, but the use of plant metabolites for this purpose has an added advantage of traditional knowledge and will make the treatment cheaper and more accessible to the affected population. Therefore, the clinical and research forums are highly oriented towards plant metabolites and interestingly, certain phytochemicals are implicated as the antibody elicitors against venom toxicity that can be exploited in designing effective anti-venoms. Based on these facts, we have made an effort to enlist plant based secondary metabolites with antiophidian abilities and their mechanism of action against locally acting enzymes/toxins in particular. The review also describes their functional groups responsible for therapeutic beneficial and certainly oblige in designing potent inhibitors against venom toxins.

Effect of monospecific antibodies against baltergin in myotoxicity induced by Bothrops alternatus venom from northeast of Argentina. Role of metalloproteinases in muscle damage

Myotoxicity, one of the most relevant local manifestations in envenomation by Bothrops genus, may result from a direct action of myotoxins or be due to an indirect vascular degeneration and ischemia. Baltergin, a snake venom metalloproteinase (SVMP), isolated from Bothrops alternatus venom has been used to obtain monospecific IgG, in order to determine the relative role of toxin in myotoxicity induced by whole venom. Bothrops diporus venom, another medical relevant genus of the northeastern region of Argentina, was also studied. Anti-baltergin IgG was able to neutralize completely the hemorrhagic activity of B. alternatus venom at an antibodies:venom ratio of 30:1 (w:w). However, mice injected with B. diporus venom showed a small spot remaining even at the highest ratio of IgG:venom assayed (50:1; w:w). Specific antibodies were efficient to neutralize the myotoxicity of B. alternatus venom at ratio 30:1 (w:w) but did not neutralize the same effects in B. diporus venom. Anti-baltergin polyclonal antibodies were useful tools for revealing the central role of SVMPs in the development of myotoxicity of B. alternatus venom, as well as, helping to suggest indirectly presence of potent myotoxic phospholipases A2 (PLA2s) in B. diporus venom.