Cupuaçu (Theobroma grandiflorum): A Multifunctional Amazonian Fruit with Extensive Benefits.

The Amazon region is known for its continental dimension, water abundance, and especially for the rich biodiversity that this biome hosts. Among the thousands of plant species in the Amazon, many represent food sources. Among these, cupuaçu (Theobroma grandiflorum (Willd. ex Spreng.) K.Schum.) stands out as an iconic fruit with an exotic flavor, appreciated for its remarkable organoleptic properties. The present review aims to provide a comprehensive description of its biology, agronomical uses, nutritional values, chemical compositions, medicinal properties, and industrial applications. The search based on scientific articles demonstrates T. grandiflorum as a valuable ingredient for the food, cosmetic and pharmaceutical sectors. Data analysis demonstrates that cupuaçu cultivation and processing contribute to the strengthening of local production chains and promotes the development of small communities, and thus the bioeconomy in the Amazon region. In this sense, since the last decade, cultivar improvement has required multidisciplinary efforts, resulting in disease-resistant plants with better productivity. Regarding its chemical composition, T. grandiflorum is a notable source of methylxanthine alkaloids, polyphenols, aroma compounds, and lipids. The presence of these compounds supports the use of cupuaçu in various products and help us to understand the potential health benefits of its consumption. Through the integration of all collected information, key gaps in basic and applied sciences were observed, highlighting the need for more research to uncover novel applications and products of T. grandiflorum. The development of new products based on biodiversity is fundamental to promoting environmental and economic sustainability, which are key steps to the survival of the Amazon rainforest. Therefore, this work summarizes the knowledge on this source and sheds light on a food source that is little known outside of the Amazon borders.

Venom Composition of Neglected Bothropoid Snakes from the Amazon Rainforest: Ecological and Toxinological Implications.

Snake venoms have evolved in several families of Caenophidae, and their toxins have been assumed to be biochemical weapons with a role as a trophic adaptation. However, it remains unclear how venom contributes to the success of venomous species for adaptation to different environments. Here we compared the venoms from Bothrocophias hyoprora, Bothrops taeniatus, Bothrops bilineatus smaragdinus, Bothrops brazili, and Bothrops atrox collected in the Amazon Rainforest, aiming to understand the ecological and toxinological consequences of venom composition. Transcriptomic and proteomic analyses indicated that the venoms presented the same toxin groups characteristic from bothropoids, but with distinct isoforms with variable qualitative and quantitative abundances, contributing to distinct enzymatic and toxic effects. Despite the particularities of each venom, commercial Bothrops antivenom recognized the venom components and neutralized the lethality of all species. No clear features could be observed between venoms from arboreal and terrestrial habitats, nor in the dispersion of the species throughout the Amazon habitats, supporting the notion that venom composition may not shape the ecological or toxinological characteristics of these snake species and that other factors influence their foraging or dispersal in different ecological niches.

Methylprednisolone therapy induces differential metabolic trajectories in severe COVID-19 patients.

IMPORTANCE: The SARS-CoV-2 virus infection in humans induces significant inflammatory and systemic reactions and complications of which corticosteroids like methylprednisolone have been recommended as treatment. Our understanding of the metabolic and metabolomic pathway dysregulations while using intravenous corticosteroids in COVID-19 is limited. This study will help enlighten the metabolic and metabolomic pathway dysregulations underlying high daily doses of intravenous methylprednisolone in COVID-19 patients compared to those receiving placebo. The information on key metabolites and pathways identified in this study together with the crosstalk with the inflammation and biochemistry components may be used, in the future, to leverage the use of methylprednisolone in any future pandemics from the coronavirus family.

Toxins from Animal Venoms as a Potential Source of Antimalarials: A Comprehensive Review.

Malaria is an infectious disease caused by Plasmodium spp. and it is mainly transmitted to humans by female mosquitoes of the genus Anopheles. Malaria is an important global public health problem due to its high rates of morbidity and mortality. At present, drug therapies and vector control with insecticides are respectively the most commonly used methods for the treatment and control of malaria. However, several studies have shown the resistance of Plasmodium to drugs that are recommended for the treatment of malaria. In view of this, it is necessary to carry out studies to discover new antimalarial molecules as lead compounds for the development of new medicines. In this sense, in the last few decades, animal venoms have attracted attention as a potential source for new antimalarial molecules. Therefore, the aim of this review was to summarize animal venom toxins with antimalarial activity found in the literature. From this research, 50 isolated substances, 4 venom fractions and 7 venom extracts from animals such as anurans, spiders, scorpions, snakes, and bees were identified. These toxins act as inhibitors at different key points in the biological cycle of Plasmodium and may be important in the context of the resistance of Plasmodium to currently available antimalarial drugs.

Hemodynamic impairment induced by Crotoxin using in vivo and ex vivo approach in a rat model.

Crotalus durissus snakebite represent 10 % of snakebite cases in Brazil, which cardiovascular disorders are associated with severe cases. Considering crotoxin (CTX) as the major venom component, the present study aimed to evaluate the hemodynamic alterations induced by CTX using in vivo and ex vivo approaches in a rat model. In vivo cardiac function parameters were analyzed from anesthetized rats treated with CTX or saline only (Sham), along with serum creatine kinase MB (CK-MB) and lung myeloperoxidase. From the same animals, hearts were isolated and functional parameters evaluated in Langendorff method ex vivo. CTX binding to myoblast cell line in vitro were evaluated using confocal microscopy and flow cytometry. CTX was capable of reducing arterial and diastolic blood pressure, heart rate, along with left ventricle pressure development or decay during systole (LVdP/dtmax and LVdP/dtmin) in vivo, however no differences were found in the ex vivo approach, showing that intrinsic heart function was preserved. In vitro, CTX binding to myoblast cell line was mitigated by hexamethonium, a nicotinic acetylcholine receptor antagonist. The present study has shown that CTX induce hemodynamic failure in rats, which can help improve the clinical management of cardiovascular alterations during Crotalus durissus snakebite.

Pregnancy outcomes after snakebite envenomations: A retrospective cohort in the Brazilian Amazonia.

Snakebite envenomations (SBEs) in pregnant women can result in adverse maternal or neonatal effects, such as abortion, placental abruption, preterm labor, fetal malformations, and maternal, fetal or neonatal deaths. Despite the high incidence of SBEs in the Brazilian Amazon, there is no literature on the impact of SBEs on pregnancy outcomes. The objective of this study was to describe clinical epidemiology and outcomes associated with SBEs in women of childbearing age and pregnant women in the state of Amazonas, Western Brazilian Amazon, from 2007 to 2021. Information on the population was obtained from the Reporting Information System (SINAN), Mortality Information System (SIM) and Live Birth Information System (SINASC) for the period from 2007 to 2021. A total of 36,786 SBEs were reported, of which 3,297 (9%) involved women of childbearing age, and 274 (8.3%) involved pregnant women. Severity (7.9% in pregnant versus 8.7% in non-pregnant women) (P = 0.87) and case-fatality (0.4% in pregnant versus 0.3% in non-pregnant women) rates were similar between groups (P = 0.76). Pregnant women who suffered snakebites were at higher risk for fetal death (OR: 2.17, 95%CI: 1.74-2.67) and neonatal death (OR = 2.79, 95%CI: 2.26-3.40). This study had major limitations related to the completeness of the information on the pregnancy outcomes. Although SBE incidence in pregnant women is low in the Brazilian Amazon, SBEs increased the risk of fetal and neonatal deaths.

The Amazonian kambô frog Phyllomedusa bicolor (Amphibia: Phyllomedusidae): Current knowledge on biology, phylogeography, toxinology, ethnopharmacology and medical aspects.

Phyllomedusa bicolor (Phyllomedusidae), popularly known as the kambô in Brazil, is a tree frog that is widely distributed in South American countries and is known for producing a skin secretion that is rich in bioactive peptides, which are often used in indigenous rituals. The biological effects of the skin secretion were observed in the first studies with indigenous communities. Over the last six decades, researchers have been studying the chemical composition in detail, as well as the potential pharmacological applications of its constituents. For this reason, indigenous communities and health agents fear the misuse of the kambô, or the inappropriate use of the species, which can result in health complications or even death of users. This article seeks to provide a transdisciplinary review that integrates knowledge regarding the biology of P. bicolor, ethnoknowledge about the ritual of the kambô, and the chemistry and pharmacology of the skin secretion of this species, in addition to medical aspects of the indiscriminate use of the kambô. Furthermore, this review seeks to shed light on perspectives on the future of research related to the kambô.

Plant-Derived Toxin Inhibitors as Potential Candidates to Complement Antivenom Treatment in Snakebite Envenomations.

Snakebite envenomations (SBEs) are a neglected medical condition of global importance that mainly affect the tropical and subtropical regions. Clinical manifestations include pain, edema, hemorrhage, tissue necrosis, and neurotoxic signs, and may evolve to functional loss of the affected limb, acute renal and/or respiratory failure, and even death. The standard treatment for snake envenomations is antivenom, which is produced from the hyperimmunization of animals with snake toxins. The inhibition of the effects of SBEs using natural or synthetic compounds has been suggested as a complementary treatment particularly before admission to hospital for antivenom treatment, since these alternative molecules are also able to inhibit toxins. Biodiversity-derived molecules, namely those extracted from medicinal plants, are promising sources of toxin inhibitors that can minimize the deleterious consequences of SBEs. In this review, we systematically synthesize the literature on plant metabolites that can be used as toxin-inhibiting agents, as well as present the potential mechanisms of action of molecules derived from natural sources. These findings aim to further our understanding of the potential of natural products and provide new lead compounds as auxiliary therapies for SBEs.

Lower levels of CXCL-8 and IL-2 on admission as predictors of early adverse reactions to Bothrops antivenom in the Brazilian Amazon.

Snakebite envenomings are considered a global health problem. The specific therapy for these envenomings consists of administering animal-derived antivenoms aiming to neutralize the venom toxins. Antivenoms have been used effectively to treat snakebites for more than a century; however, their administration may result in early and/or late adverse reactions. The present study presents the prevalence of early adverse reactions (EARs) towards Bothrops antivenom therapy in a health tertiary unit in the Brazilian Amazon and explores if specific plasma cytokines and chemokines from envenomed patients could be used as predictors of EARs. A cohort of patients bitten by Bothrops atrox was followed-up at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), from 2014 to 2016. Patients were treated with the Brazilian Bothrops antivenom and CXCL-8, CCL-5, CXCL-9, CCL-2, CXCL-10, IL-6, TNF, IL-2, IL-10, IFN-y, IL-4, and IL-17A were evaluated in patients' plasma samples before and after antivenom administration. From the total of patients (n = 186), mostly were male (82.3%), inhabiting rural areas (87.1%), with an average age of 35 years. Most of the patients (83.8%) were admitted to the hospital within 6 h after the accident, 26 (14%) reported having suffered a previous snakebite, and 97 (52.1%) received between 7 and 9 antivenom vials. The frequency of antivenom-induced EARs was 11.8% (22), resulting mostly of mild reactions. Urticaria was the major EAR manifestation (46.4%). Interestingly, CXCL-8 and IL-2 showed significantly lower levels in patients who progressed to EARs, although IL-2 levels might not represent biological relevance due the small magnitude difference between groups. This study reveals that CXCL-8 and IL-2 could play a role in the onset of EARs in pit viper envenomings.

Crotalus Durissus Ruruima: Current Knowledge on Natural History, Medical Importance, and Clinical Toxinology.

Crotalus durissus ruruima is a rattlesnake subspecies mainly found in Roraima, the northernmost state of Brazil. Envenomings caused by this subspecies lead to severe clinical manifestations (e.g. respiratory muscle paralysis, rhabdomyolysis, and acute renal failure) that can lead to the victim's death. In this review, we comprehensively describe C. d. ruruima biology and the challenges this subspecies poses for human health, including morphology, distribution, epidemiology, venom cocktail, clinical envenoming, and the current and future specific treatment of envenomings by this snake. Moreover, this review presents maps of the distribution of the snake subspecies and evidence that this species is responsible for some of the most severe envenomings in the country and causes the highest lethality rates. Finally, we also discuss the efficacy of the Brazilian horse-derived antivenoms to treat C. d. ruruima envenomings in Roraima state.

Bothrops bilineatus: An Arboreal Pitviper in the Amazon and Atlantic Forest.

The two-striped forest-pitviper (Bothrops bilineatus) is an arboreal snake that is currently represented by two subspecies (B. b. bilineatus and B. b. smaragdinus) that comprise a species complex, and its distribution is in the Amazon and the Atlantic Forest. The rarity of encounters with this snake is reflected in the low occurrence of cases of snakebites throughout its geographic distribution and the resulting low number of published clinical reports. However, in some areas, B. bilineatus proves to be more frequent and causes envenomations in a greater proportion. Herein, we review the main aspects of the species complex B. bilineatus, including its biology, ecology, taxonomy, morphology, genetic and molecular studies, geographic distribution, conservation status, venom, pathophysiology and clinical aspects, and epidemiology. In addition, the different antivenoms available for the treatment of envenomations caused by B. bilineatus are presented along with suggestions for future studies that are needed for a better understanding of the snakebites caused by this snake.

Current Knowledge on Snake Dry Bites.

Snake 'dry bites' are characterized by the absence of venom being injected into the victim during a snakebite incident. The dry bite mechanism and diagnosis are quite complex, and the lack of envenoming symptoms in these cases may be misinterpreted as a miraculous treatment or as proof that the bite from the perpetrating snake species is rather harmless. The circumstances of dry bites and their clinical diagnosis are not well-explored in the literature, which may lead to ambiguity amongst treating personnel about whether antivenom is indicated or not. Here, the epidemiology and recorded history of dry bites are reviewed, and the clinical knowledge on the dry bite phenomenon is presented and discussed. Finally, this review proposes a diagnostic and therapeutic protocol to assist medical care after snake dry bites, aiming to improve patient outcomes.

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom.

BACKGROUND: Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I. METHODS AND RESULTS: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~ 4.4 and molecular mass of 14.2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 µg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1ß and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5-160 µg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated. CONCLUSIONS: BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bß chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots.

Snake venom serine proteases (SVSPs) are commonly described as capable of affecting hemostasis by interacting with several coagulation system components. In this study, we describe the isolation and characterization of BjSP from Bothrops jararaca snake venom, a serine protease with distinctive properties. This enzyme was isolated by three consecutive chromatographic steps and showed acidic character (pI 4.4), molecular mass of 28 kDa and N-carbohydrate content around 10%. Its partial amino acid sequence presented 100% identity to a serine protease cDNA clone previously identified from B. jararaca venom gland, but not yet isolated or characterized. BjSP was significantly inhibited by specific serine protease inhibitors and showed high stability at different pH values and temperatures. The enzyme displayed no effects on washed platelets, but was able to degrade fibrin clots in vitro and also the Aα and Bß chains of fibrinogen differently from thrombin, forming additional fibrinopeptides derived from the Bß chain, which should be related to its inability to coagulate fibrinogen solutions or platelet-poor plasma. In the mapping of catalytic subsites, the protease showed high hydrolytic specificity for tyrosine, especially in subsite S1. Additionally, its amidolytic activity on different chromogenic substrates suggests possible effects on other factors of the coagulation cascade. In conclusion, BjSP is a serine protease that acts nonspecifically on fibrinogen, generating different Bß fibrinopeptides and thus not forming fibrin clots. Its distinguished properties in comparison to most SVSPs stimulate further studies in an attempt to validate its potential as a defibrinogenating agent.

CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress.

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I. Methods and Results: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~4.4 and molecular mass of 14. 2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 µg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1 ß and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5-160 µg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated. Conclusions: BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.(AU)

Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease.

Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances.

Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis.

BACKGROUND: Snake venoms are great sources of bioactive molecules, which may be used as models for new drugs. Toxins that interfere in hemostasis have received considerable attention over the years. OBJECTIVES: This study aimed at the evaluation of the antithrombotic activity of Batroxase, a P-I metalloprotease from Bothrops atrox venom, in an animal model of venous thrombosis. METHODS: The antithrombotic activity of Batroxase was tested in vivo in a model based on two factors of the Virchow's Triad: blood flow alterations (partial stenosis of the inferior vena cava), and vessel wall injury (10% ferric chloride for 5min), in comparison with sodium heparin (positive control) and saline (negative control). Bleeding/clotting time was assessed by a tail bleeding assay. The immunogenicity of Batroxase was also analyzed. RESULTS: Batroxase (12mg/kg) reduced thrombus formation in 81%, similarly to heparin (100U/kg), which reduced it in 85% in comparison with the saline group. Both Batroxase and heparin increased bleeding/clotting time in approximately 3 fold. Immunizations of rabbits with Batroxase do not result in detectable levels of antibodies against this metalloprotease. CONCLUSION: Batroxase presents antithrombotic activity in vivo. Moreover, its lack of immunogenicity increases the interest on its possible therapeutic potential over thrombogenic disorders.