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In Brazil, there are species of snakes that become involved in accidents and cause serious health problems to the inhabitants, highlighting the genus Bothrops for being responsible for approximately 90% of accidents reported annually. In the northern region of the country, this genus is responsible for the largest number of accidents, especially among rural dwellers. These populations invest in alternative treatments for with the purpose of improving the symptoms caused by snakebites. The species Mauritia flexuosa L. f., known as buriti, is traditionally used for the treatment of envenomation by snakes. Aim of the study This study aimed to evaluate the antiophidic potential of the oil of Mauritia flexuosa L. f. for Bothrops moojeni H. venom, confronting cultural and scientific knowledge. Materials and methods The physicochemical properties were determined, and the components present in the oil, extracted from fruit pulp, were analyzed by Gas Chromatography Coupled with Mass Spectrometry. The in vitro inhibitory capacity of the oil for phospholipase, metalloprotease and serine protease activities was investigated. In the in vivo studies, male Swiss mice were used to evaluate the effect of oil on lethality and toxicity, and hemorrhagic, myotoxic and edematogenic activities were assessed. Results GC‒MS analysis identification of 90.95% of the constituents of the oil, with the main components being 9-eicosenoic acid, (Z)- (34.54%), n-hexadecanoic acid (25.55%) and (E)-9-octadecenoic acid ethyl ester (12.43%). For the substrates, the outcomes indicate that the oil inhibited the activity of the main classes of toxins present in Bothrops moojeni H. venom (VBm) at the highest dose tested (0.5 μL), with inhibition of 84% for the hydrolysis of the selective substrate for serine protease and inhibition of 60% for the hydrolysis of substrates for PLA2 and metalloproteases. The antiophidic activity in vivo was evaluated with two concentrations of the oil: 1.5 mg, the dosage the population, diluted in mineral oil to a volume of 1 tablespoon and 15 mg, administered by gavage 30 min before poisoning and at time zero (concomitant to poisoning), and both concentrations administered by gavage in combination with topical use at time zero. The bleeding time in the group treated with oil at a concentration of 15 mg administered at time zero was significantly lower than that in the control group (p < 0.05). However, a greater inhibition of bleeding time was observed when local application was combined with the gavage treatment at both concentrations tested at time zero (p < 0.05). In the myotoxicity test, oil was efficient in reducing the myotoxic effects induced by the venom at the two concentrations tested, with gavage administration at time zero and gavage plus topical administration at time zero (p < 0.05). Conclusions The data obtained show that the oil is safe to use at the concentrations studied and contains fatty acids that may collaborate for cellular-level repair of the injuries caused by Bm poisoning. The in vitro and in vivo experiments showed that oil inhibits the main proteolytic enzymes present in the venom and that it has important activities to control the local effects caused by bothropic venom.
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Caterpillars of the Neotropical genus Lonomia (Lepidoptera: Saturniidae) are responsible for some fatal envenomation of humans in South America inducing hemostatic disturbances in patients upon skin contact with the caterpillars’ spines. Currently, only two species have been reported to cause hemorrhagic syndromes in humans: Lonomia achelous and Lonomia obliqua. However, species identifications have remained largely unchallenged despite improved knowledge of venom diversity and growing evidence that the taxonomy used over past decades misrepresents and underestimates species diversity. Here, we revisit the taxonomic diversity and distribution of Lonomia species using the most extensive dataset assembled to date, combining DNA barcodes, morphological comparisons, and geographical information. Considering new evidence for seven undescribed species as well as three newly proposed nomenclatural changes, our integrative approach leads to the recognition of 60 species, of which seven are known or strongly suspected to cause severe envenomation in humans. From a newly compiled synthesis of epidemiological data, we also examine the consequences of our results for understanding Lonomia envenomation risks and call for further investigations of other species’ venom activities. This is required and necessary to improve alertness in areas at risk, and to define adequate treatment strategies for envenomed patients, including performing species identification and assessing the efficacy of anti-Lonomia serums against a broader diversity of species.
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Contact with Lonomia caterpillars can cause severe envenomation with hemorrhagic syndrome, consumptive coagulopathy, acute renal failure, and death. In Brazil, an antivenom was produced using extracts from L. obliqua caterpillar bristles as antigen and has been used in other countries in South America to treat envenomation caused by distinct species of Lonomia. This study aimed to characterize the activities of toxins from Lonomia descimoni caterpillars found in Colombia and the neutralization of these toxins by the Brazilian Lonomia antivenom. The protein composition and coagulant, phospholipase A2, hyaluronidase, and defibrinogenating activities were evaluated and compared with the same parameters of the L. obliqua bristle extract. Immune recognition and the neutralizing ability of Lonomia antivenom were also determined. The results showed that the L. descimoni bristle extract presented marked differences in electrophoretic and mass spectrometry profiles and had coagulant, phospholipase A2, and hyaluronidase activities significantly less intense than those of the L. obliqua extract. In rats, L. descimoni extract induced coagulopathy and hemoglobinuria when injected by intravenous or intraperitoneal routes. The Lonomia antivenom recognized the toxins in the extract of L. descimoni and reversed the experimental envenomation in rats. Our results indicate that L. descimoni caterpillars possess toxins with weaker activities than those of L. obliqua but with the potential to cause envenomation. Moreover, the Lonomia antivenom recognized and neutralized the toxins in the L. descimoni bristle extract.
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In Brazil, antivenom for snakebite is currently formulated in liquid form and requires storage at 4 °C. Here, a new freeze-dried trivalent antivenom, which would enable cold-chain free storage, was determined to have efficacy in neutralizing the biological activities of Bothrops atrox venoms from Manaus (Brazil) and Leticia (Colombia), exhibiting an efficacy similar to those of currently available liquid Bothrops antivenoms. These results indicate that freeze-dried trivalent antivenom may be beneficial for applications in the Brazilian and Colombian Amazon regions.
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Bleeding is a common hemostatic disorder that occurs in Bothrops envenomations. We evaluated the changes in coagulation, fibrinolysis components, and platelets in Bothrops atrox envenomations with bleeding. This is an observational study with B. atrox snakebite patients (n = 100) treated in Manaus, Brazilian Amazon. Bleeding was recorded on admission and during hospitalization. We found that the platelet count in our patients presented a weak correlation to tissue factor, factor II, and plasminogen. Tissue factor presented weak correlation to factor V, II, D-dimer, plasminogen, alpha 2-antiplasmin, and moderate correlation to fibrinogen and fibrin/fibrinogen degradation product (FDP). Patients with systemic bleeding (n = 20) presented low levels of factor V, II, fibrinogen, plasminogen, and alpha 2-antiplasmin, and high levels of tissue factor and FDP compared to those without bleeding. Patients with only local bleeding (n = 41) and without bleeding showed similar levels of hemostatic factors. Thrombocytopenia was observed mainly in patients with systemic bleeding and increased levels of serum venom. No association was found between venom levels and systemic bleeding, or between venom levels and clinical severity of envenomation. This is the first report that shows the participation of the extrinsic coagulation pathway in the consumption coagulopathy of B. atrox envenomations with systemic bleeding due to tissue factor release.
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Introduction: Bothrops atrox snakebites are a major public health problem in the Amazon region and also cause hemostatic disorders. In this study, we assessed the recovery from hemostatic disorders in Bothrops snakebite patients after being given antivenom therapy. Methods: This is a prospective study of Bothrops snakebite patients (n=100) treated at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazilian Amazon, between January 2016 and December 2017. Blood samples were taken for the measurement of venom concentrations, platelets, clotting time and factors of patients on admission, 12, 24 and 48h after antivenom therapy, and taken again on discharge. The presence of systemic bleeding was recorded during the follow-up. Results: On admission, systemic bleeding was observed in 14% of the patients. Thrombocytopenia was noted in 10% of the patients. A total of 54% of the patients presented unclottable blood with low levels of fibrinogen and alpha 2-antiplasmin, and high levels of fibrin/fibrinogen degradation product (FDP) and D-dimers. Unclottable blood and systemic bleeding were overcome in most patients 12h after the antivenom therapy. Three patients developed systemic bleeding 48h after antivenom therapy. Levels of fibrinogen and alpha 2-antiplasmin, FDP and D-dimer returned to normal around 48h after the treatment or on discharge. The frequency of thrombocytopenia with high mean platelet volume increased in the first 24h after antivenom therapy, and decreased on discharge. Bothrops venom levels in patients decreased 12h after antivenom therapy and were not correlated with coagulation and fibrinolytic parameters. There were no deaths. Conclusion: Laboratorial parameters of coagulopathy returned to normal values within 48h after the antivenom therapy until discharge. A few patients still presented bleeding signs within 48h after beginning antivenom therapy. However, the Brazilian antivenom was able to overcome the hemostatic disorders in these cases of envenomation.
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The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500?µL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.
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Bothrops snakebites usually present systemic bleeding, and the clinical–epidemiological and laboratorial factors associated with the development of this manifestation are not well established. In this study, we assessed the prevalence of Bothrops snakebites with systemic bleeding reported at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, in Manaus, Amazonas State, Brazil, and the clinical–epidemiological and laboratorial factors associated with systemic bleeding. This is an observational, cross-sectional study carried out between August, 2013 and July, 2016. Patients who developed systemic bleeding on admission or during hospitalization were considered cases, and those with non-systemic bleeding were included in the control group. Systemic bleeding was observed in 63 (15.3%) of the 442 Bothrops snakebites evaluated. Bothrops snakebites mostly occurred in males (78.2%), in rural areas (89.0%) and in the age group of 11 to 30 years old (40.4%). It took most of the patients (59.8%) less than 3 h to receive medical assistance. Unclottable blood (AOR = 3.11 (95% CI = 1.53 to 6.31; p = 0.002)) and thrombocytopenia (AOR = 4.52 (95% CI = 2.03 to 10.09; p < 0.001)) on admission were independently associated with systemic bleeding during hospitalization. These hemostatic disorders on admission increase the chances of systemic bleeding during hospitalization. Prospective studies are needed to clarify the pathophysiology of systemic bleeding in Bothrops snakebites in the Amazon region.
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In South America, accidental contact with Lepidoptera larvae can produce a diversity of reactions that vary from dermatological problems to severe hemorrhagic syndromes, such as those caused by contact with caterpillars of the genus Lonomia (Saturniidae). Lonomia venom can alter the hemostatic system and lead to renal failure, internal and brain bleeding, and in severe cases, death. The only specific treatment available for these envenomations is the Lonomia Antivenom (LAV) produced by the Butantan Institute, in Brazil, using an extract of Lonomia obliqua scoli as the antigen. LAV has been used to treat exposure to other Lonomia species across South America. However, no experimental studies have been performed to test the efficacy of LAV in neutralizing the venom of species other than L. obliqua found in Southern Brazil. In this study, we tested the effectiveness of LAV in reversing the hemostatic disturbances induced by injecting Lonomia casanarensis (Lca) and Lonomia orientoandensis (Lor) scolus extracts into rats and compared the effects to the case of L. obliqua (Lob) scolus extract-induced envenomation. Lca and Lor caterpillars were collected in Colombia, and some of them were reared to adults for identification. The Minimum Defibrinating Doses (MDD) of Lca and Lor were estimated. Rats were injected (i.d.) with a dose of 3 MDD per rat of each scolus extract and treated (i.v.) with 1.5 mL of LAV or 1.5 mL of saline. Twenty-four hours after the treatment, the fibrinogen levels and platelet counts had recovered to the hemostatic levels in the groups treated with LAV. The groups treated with the saline solution had fibrinogen levels and platelet counts at non-hemostatic levels. Thromboelastometric analyses confirmed these results. In conclusion, the results showed that LAV is effective at neutralizing the envenomation induced by Lca and Lor spine extracts in rats and restoring hemostasis.
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Patients envenomed by Lonomia sp caterpillars initially experience a mild burning pain, headache, nausea, vomiting, and skin and mucosal hemorrhages. Some patients can rapidly progress to a severe coagulopathy that presents as visceral or intracerebral hemorrhaging. We studied the hemostatic alterations that occurred in 14 patients who were envenomed by Lonomia obliqua in Southern Brazil and presented at the Hospital São Vicente de Paulo (Passo Fundo, RS), Brazil during the summers of 1993 and 1994 when Lonomia antivenom was not yet available for treatment. The patients were classified into to 4 clinical groups: 0 (two patients), I (eight patients), II (two patients), and III (two patients). The patients were admitted to the hospital between 4 hours and five days after contact with the caterpillars. In this study, the coagulation parameters of the patients were followed up for up to 172 hours after the accidents. The patients received no treatment with the exceptions of two patients who received blood transfusions and antifibrinolytic treatment. The observed abnormalities related to blood coagulation and fibrinolytic factors were similar regardless of the severity of the bleeding symptoms. These findings suggest that alterations in hemostatic parameters without thrombocytopenia are not predictors of the seriousness of such accidents. Thus, consumptive disorder and reactive fibrinolysis are not proportional to mild coagulopathy. Furthermore, these patients recovered. The hemostatic parameters of most of the patients normalized between 96 and 120 h after the accident.
Assuntos
Antivenenos/administração & dosagem , Venenos de Artrópodes/intoxicação , Transtornos Hemostáticos/induzido quimicamente , Lepidópteros/classificação , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Transtornos Hemostáticos/prevenção & controle , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Bothrops atrox snakes are the leading cause of snake bites in Northern Brazil. The venom of this snake is not included in the antigen pool used to obtain the Bothrops antivenom. There are discrepancies in reports on the effectiveness of this antivenom to treat victims bitten by B. atrox snakes. However, these studies were performed using a pre-incubation of the venom with the antivenom and, thus, did not simulate a true case of envenomation treatment. In addition, the local lesions induced by Bothrops venoms are not well resolved by antivenom therapy. Here, we investigated the efficacy of the Bothrops antivenom in treating the signs and symptoms caused by B. atrox venom in mice and evaluated whether the combination of dexamethasone and antivenom therapy enhanced the healing of local lesions induced by this envenomation. In animals that were administered the antivenom 10 minutes after the envenomation, we observed an important reduction of edema, dermonecrosis, and myonecrosis. When the antivenom was given 45 minutes after the envenomation, the edema and myonecrosis were reduced, and the fibrinogen levels and platelet counts were restored. The groups treated with the combination of antivenom and dexamethasone had an enhanced decrease in edema and a faster recovery of the damaged skeletal muscle. Our results show that Bothrops antivenom effectively treats the envenomation caused by Bothrops atrox and that the use of dexamethasone as an adjunct to the antivenom therapy could be useful to improve the treatment of local symptoms observed in envenomation caused by Bothrops snakes.
Assuntos
Anti-Inflamatórios/administração & dosagem , Antivenenos/administração & dosagem , Bothrops , Dexametasona/administração & dosagem , Fatores Imunológicos/administração & dosagem , Mordeduras de Serpentes/tratamento farmacológico , Mordeduras de Serpentes/patologia , Animais , Modelos Animais de Doenças , Quimioterapia Combinada/métodos , Edema/patologia , Camundongos , Necrose/patologia , Resultado do TratamentoRESUMO
ABSTRACT Patients envenomed by Lonomia sp caterpillars initially experience a mild burning pain, headache, nausea, vomiting, and skin and mucosal hemorrhages. Some patients can rapidly progress to a severe coagulopathy that presents as visceral or intracerebral hemorrhaging. We studied the hemostatic alterations that occurred in 14 patients who were envenomed by Lonomia obliqua in Southern Brazil and presented at the Hospital São Vicente de Paulo (Passo Fundo, RS), Brazil during the summers of 1993 and 1994 when Lonomia antivenom was not yet available for treatment. The patients were classified into to 4 clinical groups: 0 (two patients), I (eight patients), II (two patients), and III (two patients). The patients were admitted to the hospital between 4 hours and five days after contact with the caterpillars. In this study, the coagulation parameters of the patients were followed up for up to 172 hours after the accidents. The patients received no treatment with the exceptions of two patients who received blood transfusions and antifibrinolytic treatment. The observed abnormalities related to blood coagulation and fibrinolytic factors were similar regardless of the severity of the bleeding symptoms. These findings suggest that alterations in hemostatic parameters without thrombocytopenia are not predictors of the seriousness of such accidents. Thus, consumptive disorder and reactive fibrinolysis are not proportional to mild coagulopathy. Furthermore, these patients recovered. The hemostatic parameters of most of the patients normalized between 96 and 120 h after the accident.
Assuntos
Humanos , Animais , Masculino , Feminino , Pré-Escolar , Criança , Adulto , Pessoa de Meia-Idade , Idoso , Venenos de Artrópodes/intoxicação , Antivenenos/administração & dosagem , Transtornos Hemostáticos/induzido quimicamente , Lepidópteros/classificação , Fatores de Tempo , Índice de Gravidade de Doença , Transtornos Hemostáticos/prevenção & controleRESUMO
Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.
Assuntos
Cabelo/enzimologia , Lepidópteros/enzimologia , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência Conservada , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Homologia de Sequência de AminoácidosRESUMO
The injury caused by the intramuscular injection of a single dose of Bothrops jararaca venom (0.24 mg/kg body weight) to mice on day 8 of pregnancy and examined on day 9 was investigated. Macroscopic and histological examination showed that the bothropic venom caused an increase in the incidence of fetal resorptions. Histologically, a characteristic involution of mature decidua was noticed in saline-treated mice; however, necrotic trophoblast giant cells and decidual cells were also present in this region of mice treated with B. jararaca venom, mainly close to the embryo. Hemorrhagic areas were also observed at maternal-fetal interface, which contained maternal erythrocytes and polymorphonuclears. Plasma fibrinogen levels were lower in envenomed group (p < or = 0.0001), but prothrombin time and activated partial thromboplastin time remained unaltered. Total and differential white blood cell counts were not statistically different between groups. Thus, B. jararaca venom causes injuries not only to the fetus, but also to decidual tissue and blood coagulation of pregnant mice. It is not clear, nonetheless, whether disturbances during the development of pregnancy are due to a direct effect of venom on uterus/fetus or to homeostatic changes in dams, such as clotting disturbances, or to both of them.
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Bothrops/fisiologia , Decídua/efeitos dos fármacos , Feto/efeitos dos fármacos , Venenos de Víboras/toxicidade , Animais , Coagulação Sanguínea/efeitos dos fármacos , Decídua/patologia , Decídua/ultraestrutura , Feminino , Contagem de Leucócitos , Camundongos , GravidezRESUMO
Human accidents caused by Loxosceles spiders may result in local dermal necrosis and, in some cases, severe systemic reactions - such as intravascular hemolysis, disseminated intravascular coagulation (DIC), renal failure and death. Since many aspects of envenomation by Loxosceles spiders remain unclear, we studied the hematological and hemostatic responses induced by the i.d. injection of 10 microg/kg Loxosceles gaucho venom in rabbits. For this purpose, total blood cell count, platelet function, coagulation tests and biochemical parameters were analysed at 3, 24, 48, 72 and 120 hours after venom administration. Thrombocytopenia and leukopenia were noted at 3 and 24 hours. Histopathological analysis of the skin lesion, performed at 24 hours after venom administration, showed a massive presence of leukocytes and platelets, hemorrhage and thrombus formation at the injection site. At 72 and 120 hours, neutrophilic leukocytosis and thrombocytosis were observed. Platelet hyperaggregation was noticeable at 48 and 72 hours. Haptoglobin and fibrinogen levels were elevated early and remained in high levels over time. Significant increases in coagulation factors V, VII, VIII, IX, X and XI were noted at 120 hours. The results showed that neither intravascular hemolysis nor DIC occurred. However, the early onset of thrombocytopenia and leukopenia are important findings that may be related to dermal necrosis formation during loxoscelism.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Leucopenia/induzido quimicamente , Diester Fosfórico Hidrolases/toxicidade , Coelhos/sangue , Serina Endopeptidases/toxicidade , Venenos de Aranha/toxicidade , Trombocitopenia/induzido quimicamente , Animais , Coagulação Sanguínea/fisiologia , Testes de Química Clínica , Testes Hematológicos , Injeções Intradérmicas , Leucopenia/sangue , Leucopenia/patologia , Masculino , Diester Fosfórico Hidrolases/administração & dosagem , Serina Endopeptidases/administração & dosagem , Pele/efeitos dos fármacos , Pele/patologia , Venenos de Aranha/administração & dosagem , Trombocitopenia/sangue , Trombocitopenia/patologiaRESUMO
Hemostatic disturbances are frequent findings in human accidents caused by Lonomia obliqua caterpillars in the southern region of Brazil. In severe envenomation, patients may present life-threatening bleedings. Such disturbances may be mimicked in rats, which also develop intravascular hemolysis. The scope of this study was to investigate the time-course and intensity of intravascular hemolysis induced by i.d. injection of 750 microg/kg crude L. obliqua bristle extract in rats. Total blood cell count, reticulocyte count, plasma hemoglobin and haptoglobin assays were performed in control and envenomed rats at 1, 6, 24 and 48 h after envenomation. Rats presented a drastic drop of haptoglobin levels at 1 and 6h with increased plasma hemoglobin levels, a decrease in packed cell volume values at 6, 24 and 48 h, and increased reticulocyte counts throughout after envenomation. Such observations indicated that intravascular hemolysis occurred as early as 1h following envenomation, and lasted for more than 6h. Intravascular hemolysis is probably induced by phospholipase A(2) and other proteins with direct hemolytic activity present in crude caterpillar bristle extract.
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Venenos de Artrópodes/toxicidade , Hemólise/efeitos dos fármacos , Mordeduras e Picadas de Insetos/sangue , Mariposas/química , Análise de Variância , Animais , Contagem de Células Sanguíneas , Brasil , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Masculino , Fragilidade Osmótica/efeitos dos fármacos , Ratos , Ratos Wistar , Reticulócitos , Fatores de TempoRESUMO
Despite being well established that snake envenomation causes blood coagulation and fibrinolysis disturbances, scant information is available about blood platelet disorders. Herein we show that experimentally Bothrops jararaca-envenomed rabbits presented thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, platelet hypoaggregation in platelet rich plasma and whole blood, normoaggregation in washed platelet suspensions, decreased platelet ATP secretion, normal plasma levels of platelet factor 4, and constant intraplatelet serotonin levels. Furthermore, by flow cytometric analyses, platelets displayed a significant decrease in the expression of a GPIIb-IIIa epitope recognized by P2 monoclonal antibody (p< 0.05) and an increased expression of a ligand-induced binding site (LIBS-1) of GPIIIa (p< 0.05), but total GPIIb-IIIa expression, evaluated with specific polyclonal antibodies, was normal. Fibrinogen and fibrin(ogen) degradation product (FfDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin were noticed on circulating platelets. The percentage of circulating reticulated platelets and the survival time of biotinylated platelets of envenomed rabbits were not statistically different from control animals. We suggest that thrombin engendered by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group demonstrates that platelets of envenomed rabbits are indeed activated in circulation, and that decreased fibrinogen or increased FfDP levels are not the primary cause of platelet dysfunction. These results imply the existence of an inhibitor in plasma that interferes with platelet aggregation in bothropic envenomation.
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Plaquetas/patologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Transtornos Plaquetários , Plaquetas/metabolismo , Bothrops , Colágeno/química , Epitopos/química , Fibrina/metabolismo , Fibrinogênio/biossíntese , Fibrinogênio/metabolismo , Citometria de Fluxo , Imuno-Histoquímica , Rim/patologia , Pulmão/patologia , Selectina-P/sangue , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Coelhos , Mordeduras de Serpentes , Venenos de Serpentes/metabolismo , Trombocitopenia/etiologia , Fatores de Tempo , Fator de von Willebrand/biossínteseRESUMO
Rabbits are frequently used as models for studying coagulation and platelet disorders. However, few reports on literature have dealt with the purification and characterization of rabbit platelet proteins. Herein a protocol for the simultaneous purification of rabbit platelet factor 4 (PF4) and platelet glycoprotein IIb-IIIa (GPIIb-IIIa, integrin alpha(IIb)beta(3)) is described. Specific antibodies were raised in laying chicken, which were used for assaying PF4 by ELISA, and GPIIb-IIIa by direct immunofluorescence and flow cytometry. Furthermore, the binding of monoclonal antibodies specific for GPIIb-IIIa complex (P2), ligand-induced binding site of GPIIIa (LIBS1) and rabbit P-selectin (12A7), as well as of polyclonal IgY specific for rabbit GPIIb-IIIa, was compared in quiescent and thrombin-activated platelets. Polyclonal anti-rabbit PF4 IgY was a specific and sensitive probe that could be used for assaying PF4 in plasma samples. GPIIb-IIIa expression was increased in thrombin-activated platelets, as evaluated by flow cytometric analysis using P2 and polyclonal antibodies raised in chickens. Rabbit GPIIb-IIIa also exhibited a conformational modification that caused the appearance of ligand-induced binding sites. Increased P-selectin expression, used as a positive control, was also noticeable in thrombin-activated platelets. These data evidence that antibodies raised in laying chickens specific to rabbit PF4 and GPIIb-IIIa, as well as certain monoclonal antibodies specific for human GPIIb-IIIa, may be used for investigating rabbit platelet physiology.
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Plaquetas/imunologia , Imunoglobulinas/química , Fator Plaquetário 4/isolamento & purificação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/isolamento & purificação , Coelhos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Galinhas , Cromatografia em Agarose , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnica Direta de Fluorescência para Anticorpo , Cobaias , Imunoglobulinas/biossíntese , Imunoglobulinas/imunologia , Masculino , Ativação Plaquetária/imunologia , Fator Plaquetário 4/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Trombina/imunologiaRESUMO
Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes. Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane.