Search Results (40)

Venoms of the Palearctic vipers in the Macrovipera genus cause severe procoagulant clinical effects, yet the precise molecular targets remain incompletely defined. To fill this toxicological knowledge gap, we tested five Macrovipera venoms—M. lebetina cernovi, M. l. obtusa, M. l. turanica (Turkmenistan and Uzbekistan localities), and M. schweizeri—using plasma clotting assays, Factors VII, X, XI, and XII and prothrombin zymogen activation assays, and SDS-PAGE to visualise Factor V (FV) cleavage. All venoms induced extremely rapid clot formation (10.5–12.5 s) compared with the negative control (spontaneous clotting) of 334.6 ± 3.6 s) and the positive control (kaolin trigger) of 55.8 ± 1.9 s. Activation of FVII or FXI was negligible, whereas consistent FX activation and species-variable FXII activation, both moderate, were observed. Prothrombin remained inert in the absence of cofactors, but the presence of FV or FVa elicited potent thrombin generation. SDS-PAGE confirmed proteolytic conversion of the 330 kDa FV zymogen into the ~105 kDa heavy and ~80 kDa light chains of FVa by the venoms of all species. This data demonstrates that Macrovipera venoms rely on a dual enzyme strategy: (i) activation of FV to FVa by serine proteases and (ii) FVa-dependent prothrombin activation by metalloproteases. These results reveal that prothrombin activation is the dominant procoagulant pathway and overshadows the historically emphasised FX activation. This mechanism mirrors, yet is evolutionarily independent from, the FXa:FVa prothrombinase formation seen in Australian elapid venoms, highlighting convergent evolution of cofactor-hijacking strategies among snakes. The discovery of potent FVa-mediated prothrombin activation in Macrovipera challenges existing paradigms of viperid venom action, prompts re-evaluation of related genera (e.g., Daboia), and underpins the design of targeted antivenom and therapeutic interventions. Full article

Plant compounds that inhibit snake venom activities are relevant and can provide active molecules to counteract snake venom effects. Numerous studies on snake viperid venoms found that metalloproteinases play a significant role in the pathophysiology of hemorrhage that occurs on envenomation. Preclinical studies using vitro and in vivo protocols investigated natural compounds and viperid snake venoms, evaluating the enzymatic, procoagulant, hemorrhagic, edematogenic, myotoxic, and lethal activities. Many studies focused on Bothrops venoms and ascribed that angiorrhexis and hemorrhage resulted from the metalloproteinase action on collagen in the basal lamina. This effect resulted in a combined action with phospholipase A2 and hyaluronidase, inducing hemorrhage, edema, and necrosis. Due to the lack of efficient antivenoms in remote areas, traditional native plant treatments remain common, especially in the Amazon. Our group studied plant extracts, isolated compounds, and lapachol synthetic derivative analogs with selective inhibition for Bothrops venom proteolytic and hemorrhagic activity and devoid of phospholipase activity. We highlight those new synthetic naphthoquinones which inhibit snake venom metalloproteinases and that are devoid of other venom enzyme inhibition. This review shows the potential use of snake venom effects, mainly Bothrops venom metalloproteinase activity, as a tool to identify and develop new active molecules against hemorrhagic effects. Full article

This study examined the pathophysiological effects of venoms from neonate and adult specimens of the viperid snake Macrovipera lebetina obtusa, focusing on their ability to activate various blood clotting factors in human plasma. All venoms exhibited strong procoagulant properties. In concentration–response tests, the clotting potency of the neonate venoms fell within the range of their parents’ maximum clotting velocities and areas under the curve. Intriguingly, females were more potent than males within each age group, but this requires a larger sample size to confirm. Antivenom neutralization efficacy was equipotent across age groups. The venoms potently activated Factor X (FX) robustly, consistent with previous knowledge of this genus. For the first time, the ability to activate Factors VII (FVII) and XII (FXII) was identified in this genus, with FXII exhibiting particularly strong activation. The study found no significant ontogenetic variation in procoagulant venom potency on human plasma, convergent with the Daboia genus, the other large-bodied lineage within the Palearctic viperid clade. However, the activation of FXII and FVII reveals previously undocumented pathways in the procoagulant activity of these venoms, contributing to the broader understanding of venom evolution and its clinical impacts. These findings have implications for venom biodiscovery and the development of antivenoms, highlighting the complexity of clotting factor activation beyond traditional investigations that have myopically focused upon FX and prothrombin pathways, thereby underscoring the importance of exploring additional clotting factors. Full article

Previous proteomic studies of viperid venom revealed that it is mainly composed of metalloproteinases (SVMPs), serine proteinases (SVSPs), phospholipase A2 (PLA2), and C-type lectins (CTLs). However, other proteins appear in minor amounts that affect prey and need to be identified. This study aimed to identify novel toxic proteins in the venom gland transcriptome of Bothrops asper and Bothrops jararaca, using data from NCBI. Bioinformatics tools were used to assemble, identify, and compare potentially novel proteins in both species, and we performed functional annotation with BLASTX against the NR database. While previous assemblies have been performed for B. jararaca, this is the first assembly of the B. asper venom gland transcriptome. Proteins with potentially novel functions were identified, including arylsulfatase and dihydroorotate dehydrogenase, among others, that could have implications for venom toxicity. These results suggest that the identified proteins may contribute to venom toxic variation and provide new opportunities for antivenom research. The study improves the understanding of the protein composition of Bothrops venom and suggests new possibilities for the development of treatments and antivenoms. Full article

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca²⁺ ions and inhibited by an excess of Zn²⁺. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics. Full article

Viruses in the subfamily Serpentovirinae (order Nidovirales, family Tobaniviridae) can cause significant morbidity and mortality in captive snakes, but documented infections have been limited to snakes of the Boidae, Colubridae, Homalopsidae, and Pythonidae families. Infections can either be subclinical or associated with oral and/or respiratory disease. Beginning in June 2019, a population of over 150 confiscated snakes was screened for serpentovirus as part of a quarantine disease investigation. Antemortem oropharyngeal swabs or lung tissue collected postmortem were screened for serpentovirus by PCR, and 92/165 (56.0%) of snakes tested were positive for serpentovirus. Serpentoviruses were detected in fourteen species of Viperidae native to Asia, Africa, and South America and a single species of Elapidae native to Australia. When present, clinical signs included thin body condition, abnormal behavior or breathing, stomatitis, and/or mortality. Postmortem findings included variably severe inflammation, necrosis, and/or epithelial proliferation throughout the respiratory and upper gastrointestinal tracts. Genetic characterization of the detected serpentoviruses identified four unique viral clades phylogenetically distinct from recognized serpentovirus genera. Pairwise uncorrected distance analysis supported the phylogenetic analysis and indicated that the viper serpentoviruses likely represent the first members of a novel genus in the subfamily Serpentovirinae. The reported findings represent the first documentation of serpentoviruses in venomous snakes (Viperidae and Elapidae), greatly expanding the susceptible host range for these viruses and highlighting the importance of serpentovirus screening in all captive snake populations. Full article

Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (Bothrops asper, Crotalus atrox, and Daboia russelii) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms. Full article

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac^®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms. Full article

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A₂ (PLA₂s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor β (TGF-β) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings. Full article

The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, Tropidolaemus subannulatus of northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple highly diverged propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species. Full article

Snake envenomation remains an important yet neglected medical problem in many countries, with around five million people affected, and over a hundred thousand deaths annually. Plasma-derived antivenoms are the main therapeutic agent available. Monovalent antivenoms are produced via the immunization of large animals, e.g., horses, with one venom, after which the horse serum can neutralize the homologous venom, with minimal or no cross neutralization against other venoms. It is necessary, therefore, for the culprit snake to be identified, so that the appropriate specific antivenom can be selected. Polyvalent antivenoms (pAVs) are produced via immunization with a number of snake venoms, and the serum can neutralize all the venoms used in its production. Thus, pAVs can be used to treat several venoms from a country/region, and the identification of the culprit snake is not necessary. There are various parameters and processes involved in the production of pAVs, depending on the requirements and resources available. Most commercial pAVs use a mixture of both elapid and viperid venoms as immunogens, while some pAVs use either elapid or viperid venoms. Some pAVs are produced through the mixing of more than one monovalent or polyvalent antivenom. These various types of pAVs have their own characteristics, and have benefits and drawbacks. The major benefits of pAVs are the wide coverage of many medically important venoms, including many heterologous venoms. They also remove the need to identify the culprit snake, and they can be produced at a lower cost than several monovalent antivenoms. Interesting polyvalent antivenoms, termed ‘syndromic pAVs’ (s-pAVs), have recently gained attention. They are produced for use according to the syndromes manifested in snakebite patients. The venoms that produce these syndromes are used as immunogens in the production of ‘syndromic antivenoms’. For example, ‘neurotoxic polyvalent antivenom’ and ‘hematotoxic polyvalent antivenom’ are produced using the neurotoxic elapid and hematotoxic viperid venoms as immunogens, respectively. They were first marketed by the Thai Red Cross in 2012, and have since gained attention as a possible therapeutic modality to help solve the problem of snakebite envenomation globally. The merits of these s-pAVs, including their efficacy and wide paraspecificities, are discussed. Full article

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites. Full article

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A₂, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A₂. We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions. Full article

The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM–polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms. Full article

Snakebite envenoming is a neglected tropical disease prevalent in South Asia. In Pakistan, antivenoms are commonly imported from India despite the controversy over their effectiveness. To solve the problem, the locals have developed the Pakistani Viper Antivenom (PVAV), raised against Sochurek’s Saw-scaled Viper (Echis carinatus sochureki) and Russell’s Viper (Daboia russelii) of Pakistani origin. This study is set to evaluate the composition purity, immuno-specificity and neutralization efficacy of PVAV. Chromatographic and electrophoretic profiling coupled with proteomic mass spectrometry analysis showed PVAV containing high-purity immunoglobulin G with minimum impurities, notably the absence of serum albumin. PVAV is highly immuno-specific toward the venoms of the two vipers and Echis carinatus multisquamatus, which are indigenous to Pakistan. Its immunoreactivity, however, reduces toward the venoms of other Echis carinatus subspecies and D. russelii from South India as well as Sri Lanka. Meanwhile, its non-specific binding activities for the venoms of Hump-nosed Pit Vipers, Indian Cobras and kraits were extremely low. In the neutralization study, PVAV effectively mitigated the hemotoxic and lethal effects of the Pakistani viper venoms, tested in vitro and in vivo. Together, the findings suggest the potential utility of PVAV as a new domestic antivenom for the treatment of viperid envenoming in Pakistan. Full article