Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring.

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 µg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.

Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 μg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Hematology and biochemistry parameters of the Central American bushmaster (Lachesis stenophrys) under human care in Costa Rica.

Background: The Central American bushmaster (Lachesis stenophrys) is one of the largest pitvipers in the Americas, with relatively low abundance, suspected population declines, and continuing loss, fragmentation, and habitat degradation. Aim: Conservation actions, both in the wild and in captivity, bear the need for health parameters that allow managers and veterinarians to have a better understanding of health, especially when there are relatively few individuals in captivity to obtain robust information since there is no published information on the genus. To have hematological and biochemical reference ranges on the genus Lachesis. Methods: Blood samples were collected from 32 individuals (18 females and 14 males) under human care from 7 zoological institutions from August 2022 to January 2023 and performed hematological and biochemical analyses. Results: Reference intervals of hematological analytes included packed cell volume (17.51%-37.27%), total red blood cell count (0.36-0.92 × 1012/l), hemoglobin (61.73-145.23 g/l), white blood cell count (3.18-13.79 × 109/l), lymphocytes (2.16%-11.23%), azurophils (0.50%-4.20%), monocytes (0.00%-0.21%), heterophils (0.05%-1.12%), eosinophils (0.00%-0.19%), basophils (0.00%-2.00%), and total thrombocyte count (0.68-6.68 × 109/l), and biochemistry reference intervals included total protein (41.76-111.31 g/l), albumin (11.46-28.69 g/l), globulins (29.25-85.14 g/l), aspartate aminotransferase (1.44-68.75 U/l), creatinine kinase (52.72-625.00 U/l), uric acid (20.02-438.53 µmol/l), glucose (0.68-3.29 mml/l), cholesterol (41.74-13.25 mmol/l), calcium (1.78-6.06 mmol/l), and phosphorus (0.72-2.26 mmol/l). Conclusion: This is the first report on the genus Lachesis reporting hematological and biochemical reference ranges.

Rhomb-I, a P-I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation.

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Simultaneous identification of three clinically relevant peruvian pit vipers by multiplex loop-mediated isothermal amplification (mLAMP).

Previous knowledge about the taxonomic distribution of venomous snake species is very useful for epidemiological aspects of ophidism. Here, we sought to develop an assay for the differential identification of clinically relevant snakes in Peru: Bothrops atrox, Lachesis muta, and Crotalus durissus using a multiplex loop-mediated isothermal amplification (mLAMP) assay. For this, DNA was extracted from the shed snake skins and the mitochondrial genes Cytb, COI, and 12S rRNA were amplified and further sequenced, for the design of mLAMP reaction primers. For each snake species the forward and reverse primers, internal forward and reverse primers, and the loop primers were obtained, bearing the latter different fluorophores for product identification. Finally, the reaction was standardized in the presence of all primer sets, and an optimal amount of low molecular weight polyethyleneimine. The precipitated products were observed in a UV light transilluminator, finding a differential fluorescence according to the DNA used, with a detection limit to the naked eye in the range of 0.2-25 ng of DNA, within 30 min. This study is the first report on the use of mLAMP technology for the identification of venomous snakes.

Caracterização estrutural e funcional de uma metaloproteinase de classe P-I do veneno de Lachesis muta rhombeata

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+ , Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 oC. The amino acid sequence showed high homology with other SVMPs. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential BM, ECM and plasma proteins. It preferentially cleaves the α-chains of fibrin(ogen). Rhomb-I inhibited convulxin- and vWF-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low- molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors GPIbα and GPVI to release a 130-kDa and 55-kDa soluble form. Both membrane glycoproteins, GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Rhomb-I, uma metaloproteinase de 23 kDa foi isolada do veneno de L. m. rhombata. Sua ação proteolítica sobre dimetilcaseína foi inibida por α2-macroglobulina, quelantes de metais e por cátions Co2+ e Zn2+ e levemente estimulada por Ca2+ e Mg2+ . Em solução aquosa a 37 oC, rhomb-I sofreu autoproteólise gerando fragmentos de aproximadamente 20 e 11 kDa. A sequência de aminoácidos apresentou alta similaridade com outras SVMPs P-I. Rhomb-I causa hemorragia que pode ser atribuída à hidrólise de proteínas da membrana basal, da matriz extracelular e proteínas plasmáticas. Rhomb-I cliva, preferencialmente, as cadeias Aα da fibrina e do fibrinogênio. Rhomb-I inibiu a agregação induzida por convulxina e pelo fator de von Willebrand (vWF) em plaquetas humanas, sem efeito significativo na agregação estimulada por colágeno ou outros agonistas. Ela digere vWF em multímeros de massas moleculares menores bem como o domínio A1 recombinante de vWF (rvWF- A1) gerando um fragmento de aproximadamente 27 kDa, conforme evidenciado por western blot com IgG anti-rvWF-A1, produzido em camundongos. Após incubar plaquetas lavadas com rhomb-I, foi possível observar por western blot que a proteína liga e cliva as glicoproteínas de plaquetas GPIb e GPVI, liberando as formas solúveis de 130 kDa e 55 kDa, respectivamente. Ambas glicoproteínas, GPIbα que se liga ao vWF, e GPVI que se liga ao colágeno, desempenham um papel fundamental na mediação da adesão/ativação plaquetária e podem iniciar a formação fisiológica ou patológica de trombos. Conclusões: Rhomb-I é uma metaloproteinase classe P-I, hemorrágica, fibrino(geno)lítica que degrada proteínas da matriz extracelular e interfere na função plaquetária pela clivagem de vWF, GPIb e GPVI. Estes resultados indicam que rhomb-I pode estar envolvida na fisiopatologia do envenenamento por Lachesis, perturbando a hemostasia e agregação plaquetária e causando danos à vasculatura.

Unveiling the peptidome diversity of Lachesis muta snake venom: discovery of novel fragments of metalloproteinase, l-amino acid oxidase, and bradykinin potentiating peptides

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesis muta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

Rhomb-I, a P–I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Anti-oxidant Effect of High Dilutions of Arnica montana, Arsenicum Album, and Lachesis Mutus in Microglial Cells in Vitro.

Microglial cells play important roles in inflammatory responses. The level of oxidative stress is a well-known marker of inflammation. Homeopathic medicines are often used clinically to alleviate inflammation. We evaluated the anti-oxidative effect of high dilutions of Arnica montana (Arnica m.), Arsenicum album (Arsenicum a.), and Lachesis mutus (Lachesis m.) on production of reactive oxygen species (ROS) in inflamed microglial cells in vitro. Microglial cells, on exposure to lipopolysaccharide (LPS), have induced production of ROS compared with resting cells. The dilutions significantly reduced the oxidative stress by decreasing the level of ROS produced. Arnica m. 1C, 3C, 5C, 7C, 9C, and 30C dilutions had a range of ROS reduction between 15 and 42.1%; Arsenicum a. 3C, 5C, 7C, 15C, and 30C dilutions had a range of ROS reduction between 17.6 and 35.3%; and Lachesis m. 3C, 5C, 7C, 9C, 15C, and 30C dilutions had a range of ROS reduction between 25 and 41.7%. To summarize, the dilutions with the greatest effect were Arnica m. 1C (42.1%), Arsenicum a. 30C (35.3%), and Lachesis m. 7C (41.7%). Arnica m., Arsenicum a., and Lachesis m. did not have the same effect on ROS production and were not dose-dependent.

Caracterização bioquímica de uma serinoprotease com efeito ativador do plasminogênio do veneno de Lachesis muta rhombeata

Snake venom serine proteases (SVSPs) are chymotrypsin-like proteins found in some venoms of the Viperidae family. These enzymes affect physiological processes of their prey and victims, acting mainly in the hemostatic, fibrinolytic and kinin systems. SVSPs belong to the PA clan, PA (S) subclan, S1 family and A subfamily of proteolytic enzymes. This work, describes a SVSP (Lmr-PA) isolated from the venom of Lachesis muta rhombeata which activates plasminogen. The proteinase was purified by combination of gel filtration and anionic exchange chromatographies. Its homogeneity was demonstrated by SDS-PAGE, reverse-phase HPLC, and two-dimensional electrophoresis. Lmr-PA is a 30-kDa single chain glycoprotein. Its amino acid sequence (61%) was determined by mass spectrometry on nLC-MS/MS. Lmr-PA activates plasminogen to release plasmin and degrades the plasmin substrate S-2251 as well as dimethylcasein. PMSF, the specific inhibitor of serine proteases completely blocked Lmr-PA activity. The proteinase cleaves the Aα chain and partially the Bβ and γ chains of fibrinogen. In addition the protease degrades laminin, nidogen and type IV collagen from Matrigel. The enzyme digests fibrin in presence of plasminogen in vitro. Deglycosylated Lmr-PA loses approximately 26% of its activity. In addition, Lmr-PA activity is inhibited by α2-macroglobulin at a ratio of 2:1 (α2-M:E) and α2-antiplasmin inhibits plasmin generated from plasminogen. Lmr-PA does not induce aggregation of washed human platelets but, aggregates platelets in presence of exogenous fibrinogen and binds to the platelet receptors glycoproteins (GP) GPIb and GPVI. Our data indicate that Lmr-PA is a plasminogen activating serine protease, like to previously reported LV-PA from Lachesis muta muta venom. These results suggested that Lmr- PA play a role in the pathology of snake envenomation and could be a useful model to study hemostatic disorders caused by snake bites.

As serinoproteases do veneno de serpentes (SVSPs) são proteínas semelhantes à quimotripsina presentes encontradas em alguns venenos da família Viperidae. Essas enzimas afetam os processos fisiológicos de suas presas e vítimas, atuando principalmente nos sistemas hemostático, fibrinolítico e cinina. As SVSPs pertencem ao clã PA, subclan PA (S), família S1 e subfamília A de enzimas proteolíticas. Este trabalho descreve uma SVSP (Lmr-PA) isolada do veneno de Lachesis muta rhombeata que ativa o plasminogênio. A proteinase foi purifica por combinação de cromatografias de filtração em gel e troca aniônica. Sua homogeneidade foi demonstrada por SDS-PAGE, HPLC de fase reversa e eletroforese bidimensional. Lmr-PA é uma glicoproteína de cadeia simples de 30 kDa. A sua sequência de aminoácidos (61%) foi determinada por espectrometria de massa em nLC-MS/MS. Lmr-PA ativa o plasminogênio para liberar plasmina e degrada o substrato de plasmina S-2251, bem como a dimetilcaseína. PMSF, um inibidor específico de serinoproteases bloqueou completamente a atividade da Lmr-PA. A proteinase cliva a cadeia Aα e parcialmente as cadeias Bβ e γ do fibrinogênio. Além disso, a protease degrada laminina, nidogênio e colágeno tipo IV de Matrigel. A enzima digere a fibrina na presença de plasminogênio in vitro. Lmr-PA desglicosilada perde aproximadamente 26% da sua atividade. Além disso, a atividade da Lmr-PA é inibida pela α2-macroglobulina em uma proporção de 2:1 (α2-M:E) e a α2-antiplasmina inibe a plasmina gerada a partir do plasminogênio. Lmr-PA não induz a agregação de plaquetas humanas lavadas, mas agrega plaquetas na presença de fibrinogênio exógeno e se liga às glicoproteínas dos receptores plaquetários (GP) GPIb e GPVI. Nossos dados indicam que a Lmr-PA é uma serinoprotease ativadora do plasminogênio semelhante a LV-PA relatada anteriormente do veneno de Lachesis muta muta. Esses resultados sugerem que a Lmr-PA desempenha um papel na patologia do envenenamento por serpentes e pode ser um modelo útil para estudar distúrbios hemostáticos causados por acidentes ofídicos.

When to think about a Lachesis muta envenomation in the Western Brazilian Amazon: Lessons from a case report

ABSTRACT In the Brazilian Amazon, envenomations by lancehead pit vipers prevail across the region, while bushmaster (Lachesis muta) envenomations are rarely confirmed. Here, we described a moderate snakebite, diagnosed as a lancehead pit viper envenomation upon admission and treated with four vials of Bothrops antivenom. Blood remained unclottable for 4 days of hospitalization. On day 4, after admission, the patient presented pictures of the perpetrating snake to the hospital staff, which was identified as a Lachesis muta specimen. After administering 10 vials of Lachesis antivenom, blood became clottable 12 hours after treatment. The patient was discharged without complaints.

Neutralization of hemostatic disorders induced by Lachesis muta venom using Brazilian antivenoms.

We characterized the hemorrhagic, coagulant and defibrinogenant activities of Lachesis muta venom and evaluated the capacity of the Brazilian antivenoms in neutralizing these activities. The hemorrhagic activity of L. muta venom was similarly neutralized by Bothrops, Bothrops-Lachesis and Bothrops-Crotalus antivenoms. The coagulant and defibrinogenant activities were better neutralized by the Bothrops-Lachesis antivenom. Bothrops-Crotalus antivenom also neutralized these activities, indicating that it can be an alternative to treat Lachesis envenomations when Bothrops-Lachesis antivenom is unavailable.

A brief review on the natural history, venomics and the medical importance of bushmaster (Lachesis) pit viper snakes.

Snakes of the genus Lachesis, commonly known as bushmasters, are the largest venomous snakes in the Americas. Because these snakes have their habitats in areas of remote forests they are difficult to find, and consequently there are few studies of Lachesis taxa in their natural ecosystems. Bushmasters are distributed in tropical forest areas of South and Central America. In Brazil they can be found in the Amazon Rainforest and the Atlantic Forest. Despite the low incidence of cases, laquetic envenoming causes severe permanent sequelae due to the high amount of inoculated venom. These accidents are characterized by local pain, hemorrhage and myonecrosis that can be confused with bothropic envenomings. However, victims of Lachesis bites develop symptoms characteristic of Lachesis envenoming, known as vagal syndrome. An important message of this bibliographic synthesis exercise is that, despite having the proteomic profiles of all the taxa of the genus available, very few structure-function correlation studies have been carried out. Therefore the motivation for this review was to fill a gap in the literature on the genus Lachesis, about which there is no recent review. Here we discuss data scattered in a number of original articles published in specialized journals, spanning the evolutionary history and extant phylogeographic distribution of the bushmasters, their venom composition and diet, as well as the pathophysiology of their bites to humans and the biological activities and possible biotechnological applicability of their venom toxins.

Identification of cross-reactive human single-chain variable fragments against phospholipases A2 from Lachesis muta and Bothrops spp venoms.

Bushmasters (Lachesis spp) and lancehead vipers (Bothrops spp) are two of the most dangerous snakes found in Latin America. Victims of envenoming by these snakes require urgent administration of antivenom. Here, we report the identification of a small set of broadly neutralizing human monoclonal single-chain variable fragment (scFv) antibodies targeting key phospholipases A2 from Lachesis and Bothrops spp using phage display technology and demonstrate their in vitro efficacy using a hemolysis assay.

Disorders on cardiovascular parameters in rats and in human blood cells caused by Lachesis acrochorda snake venom.

In Colombia, Lachesis acrochorda causes 2-3% of all snake envenomations. The accidents promote a high mortality rate (90%) due to blood and cardiovascular complications. Here, the effects of the snake venom of L. acrochorda (SVLa) were analyzed on human blood cells and on cardiovascular parameters of rats. SVLa induced blood coagulation, as measured by the prothrombin time test, but did not reduce the cell viability of neutrophils and platelets evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by the lactate dehydrogenase (LDH) enzyme assay. In fact, SVLa increased the absorbance in tests made with platelets subjected to the MTT assay. SVLa induced platelet aggregation whose magnitude was comparable to that of the positive control adenosine diphosphate (ADP), and occurred earlier with increasing SVLa concentration. Acetylsalicylic acid (ASA, a cyclooxygenase inhibitor) or clopidogrel (an ADP receptor blocker) inhibited the aggregating effect of SVLa. Inhibition of SVLa-elicited platelet aggregation also resulted from the treatment with disodium ethylenediaminetetraacetate (Na2-EDTA; metalloproteinase inhibitor) and with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, serine protease inhibitor). In isolated right atrium of rats, SVLa increased slightly, but significantly, the magnitude of the spontaneous contractions and, in isolated rat aorta, SVLa relaxed KCl- or phenylephrine-induced contractions. In vivo, SVLa induced hypotension and bradycardia in rats, with detection of hemorrhage in pulmonary and renal tissues. Altogether, under experimental conditions, SVLa induced blood coagulation, platelet aggregation, hypotension and bradycardia. Part of the effects presented here may be explained by the presence of snake venom metalloproteinases (SVMPs) and snake venom serine proteases (SVSPs), constituents of SVLa.

Inhibition by essential oils of Mentha viridis and Mentha pulegium (Lamiaceae) in proteolysis, fibrinogenolysis and coagulation caused by venomous snakes

Snake venoms are widely used as laboratory tools for studies of physiological, pharmacological and toxicological mechanisms. Venoms used here are rich sources of several classes of proteases that act on factors of the coagulation cascade, fibrinogenolysis and fibrinolysis, altering the hemostatic processes, and phospholipases A2 which are involved mainly in inflammatory and clotting processes. Natural products such as essential oils are made up of active ingredients with wide application in the food, pharmaceutical and cosmetic industries. Thus, this study evaluates the essential oils from Mentha viridis and Mentha pulegium on coagulation, fibrinogenolysis and degradation of azocasein, induced by Bothrops sp and Lachesis muta muta venoms. These oils were achieved by hydrodistillation and presented, respectively, as the main constituents linalool (40.70 %), carvone (13.52 %) and α-terpinene (8.56 %); pulegone (50.01 %), menthol (31.90 %) and menthone (16.56 %). The essential oils were previously incubated with Bothrops alternatus venom, for two different times, then plasma was added and time was recorded. The M. viridis oil presented greater anticlotting potential. Meanwhile, the M. pulegium oil presented anticlotting or proclotting activity dependent on the dose tested. The incubation time also influenced the effect of the oils on the coagulation time. At azocaseinolytic assay, the oil from M. pulegium reduced the activity for all evaluated venoms. The highest inhibition was of 39.99 %; on activity induced by B. jararacussu, M. viridis reduced the activity in 57.72 %. On B. moojeni, the major inhibition observed was of 74.67 %. The fibrinogenolysis induced by B. moojeni venom was totally inhibited by both oils in the evaluated proportions. The results show the presence in oils of protease inhibitors, considering serine and metalloproteases (acting on clotting factors or with hemorrhagic activity), as well as phospholipase A2 (enzymes involved in inflammation and clotting processes) inhibitors of wide application in medical and biotechnology areas.

Los venenos de serpientes son ampliamente utilizados como herramientas de laboratorio para estudios de mecanismos fisiológicos, farmacéuticos y toxicológicos. Los venenos son recursos ricos en diferentes clases de proteasas que actúan sobre los factores de la cascada de coagulación, fibrinogenólisis y fibrinólisis, alterando los procesos homeostáticos y las fosfolipasas A2, los cuales están involucrados en procesos inflamatorios y de coagulación. Los productos naturales como los aceites esenciales se componen de ingredientes activos de amplia aplicación en las industrias alimentaria, farmacéutica y cosmética. El presente estudio evalúa los aceites esenciales de Mentha viridis y Mentha pulegium sobre la coagulación, fibrinogenólisis y degradación de azocaseína, inducida por los venenos de Bothrops sp. y Lachesis muta muta. Los aceites esenciales fueron obtenidos por hidrodestilación y sus componentes principales fueron identificados respectivamente como el linalool (40.70 %), carvona (13.52 %) y α-terpineno (8.56 %); pulegone (50.01 %), mentol (31.9 %) y mentona (15.56 %). Los aceites esenciales fueron previamente incubados con veneno de Bothrops alternatus, durante dos tiempos diferentes, luego se agregó el plasma y fue registrado el tiempo. El aceite de M. viridis presentó el más grande potencial anticoagulante. Mientras tanto, el aceite de M. pulegium presentó actividad anticoagulante o procoagulante dependiendo de la dosis evaluada. El tiempo de incubación también influyó en el efecto de los aceites sobre el tiempo de coagulación. En el ensayo azocaseínolitico, el aceite de M. pulegium redujo la actividad para todos los venenos evaluados. La inhibición más alta inducida para el veneno de B. jararacussu fue de 39.99 %, para M. viridis la actividad fue reducida en 57.72 %, mientras que para B. moojeni, la mayor inhibición observada fue de 74.67 %. La fibrinogenólisis inducida por el veneno de B. moojeni fue totalmente inhibida por ambos aceites en las proporciones evaluadas. Los resultados muestran la presencia de inhibidores de proteasas en los aceites, como las serinas y metaloproteasas (actuando sobre los factores de coagulación o sobre la actividad hemorrágica), como también fosfolipasas A2 (enzimas involucradas en los procesos de inflamación y coagulación), los cuales son inhibidores de amplia aplicación en áreas médicas y biotecnológicas.

Chemical structure and snake antivenom properties of sulfated agarans obtained from Laurencia dendroidea (Ceramiales, Rhodophyta).

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol-1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l-sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d-xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

Evaluación del Tratamiento Homeopático de Mujeres en Climaterio entre los 45 y 60 años con la Menopause Rating Scale (MRS) / Treatment Evaluation Homeopathic Women in Climacteric between 45 and 60 years old with Menopause Rating Scale (MRS)

La terapia hormonal es el tratamiento convencional para contrarrestar síntomas propios del climaterio; sin embargo, algunas mujeres tienen contraindicada esta terapia debido a los efectos adversos que provoca. El objetivo de este estudio fue evaluar la utilidad del tratamiento homeopático durante el climaterio con el uso de la Menopause Rating Scale (MRS) o Escala de Calificación Menopáusica. El estudio incluyó a 31 mujeres, sin problemas cardiacos, sin terapia de reemplazo hormonal y sin antecedentes de cáncer, a quienes se les aplicó el cuestionario de la MRS para obtener un puntuaje antes del tratamiento. Para determinar el tratamiento se utilizó el repertorio electrónico Radar 10.0 para Windows, con el objetivo de repertorizar los síntomas característicos de cada paciente y determinar el medicamento similar. El medicamento fue prescrito en dinamizaciones 30CH, indicando 5 gotas sublinguales cada 8 horas. La evaluación de los pacientes se llevó a cabo cada mes durante tres meses. Se realizó una evaluación final a los tres meses por comparación de puntajes de la escala inicial y final, a través de un análisis estadístico y utilizando la prueba de Wilcoxon. Lachesis trigonocephalus y Pulsatilla nigricans fueron los medicamentos indicados con mayor frecuencia, de acuerdo con los síntomas de cada paciente. La comparación del puntaje, antes y después del tratamiento, evidenció una reducción de síntomas con diferencia estadística, lo cual reflejó una mejoría global del 35%. Esta evidencia sugiere que el tratamiento homeopático beneficia a las mujeres en el climaterio. (AU)

Hormonal therapy is the conventional treatment for counteracting climacteric symptoms; however, this treatment is contraindicated in some women due to the adverse effects that this therapy may cause. The purpose of this study was to evaluate the usefulness of homeopathic therapy during the climacteric using the Menopause Rating Scale (MRS). The study included 31 women, without cardiac problems, history of no cancer or hormone replacement therapy; a MRS scale questionnaire was applied to these women to evaluate each symptom before to treatment. The Radar 10.0 for Windows electronic repertoire was used to repertorize the characteristic symptoms of each patient and determine the similarly. Homeopathic medication was prescribed in 30CH dynamization as 5 sublingual drops every 8 hrs. Patient evaluation took place monthly for 3 months. The evaluation of the results was performed by the comparison of scale scores through a statistical analysis using the Wilcoxon test. Lachesis trigonocephalus and Pulsatilla nigricans were the most frequent treatment indications, according to the symptoms of each patient. At the end of the homeopathic treatment, symptoms reduction was observed with statistical significance in the score, which reflects an overall improvement of 35%. This evidence suggested that homeopathic treatment benefits women in climacteric. (AU)

Recurso terapeutico homeopático no tratamento da cefaleia tensional / Homeopathic therapeutic remedy in the treatment of tension headache

A cefaleia é um sintoma caracterizado por sensação de dor localizada na região anatômica da cabeça. A prevalência da cefaleia na população em geral ao longo da vida é relevante (94% dos homens e 99% das mulheres) e cerca de 70% das pessoas apresentaram ao menos um episódio no último ano. Nos consultórios clínicos, a cefaleia é a terceira queixa mais frequente (10,3%), suplantado apenas por afecções de vias aéreas e distúrbios digestivos.( SPECIALI, 2018 ) A cefaleia do tipo tensional é a mais comum das cefaleias primárias, com maior prevalência na quarta década.( SPECIALI,2018 ) A Homeopatia é uma prática que pode contribuir no tratamento dessa patologia. Deve ser considerada pelo seu princípio de terapia individual, no intuito de diminuir os riscos de toxicidade a que esses pacientes estão expostos no curso da doença, principalmente quando crônica, além de promoção de saúde. Relata-se aqui um caso clínico de uma jovem de 26 anos, com quadro típico de Cefaleia tensional, controlada exclusivamente com medicação homeopática. (AU)