Ida S. Sano-Martins

Reliability of the simple 20 minute whole blood clotting test (WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by Bothrops snakes

Reliability of the simple 20 minute whole blood clotting test (WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by Bothrops snakes. Toxicon 32, 1045-1050, 1994.--A simple whole blood clotting test (WBCT20) was assessed for its efficacy in determination of severe defibrinogenation in patients envenomed by Bothrops snakes in Brazil. There was a close relationship between the results of the WBCT20 and plasma fibrinogen levels in 69 moderately envenomed patients. The advantage of the WBCT20 over estimation of plasma fibrinogen concentrations in patients is that it is a simpler, faster and more reliable test. It is also of use in assessing the effectiveness of antivenom therapy in relation to the restoration of blood coagulability.

Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the north eastern Amazon region of Brazil

The efficacies of specific Bothrops atrox-Lachesis and standard Bothrops-Lachesis antivenoms were compared in the north eastern Amazon region of Brazil. The main aim was to investigate whether a specific antivenom raised against the venom of B. atrox, the most important Amazon snake species from a medical point of view, was necessary for the treatment of patients in this region. Seventy-four patients with local and systemic effects of envenoming by Bothrops or Lachesis snakes were randomly allocated to receive either specific (n = 38) or standard (n = 36) antivenoms. In 46 cases (24 in the standard antivenom group, 22 in the other) the snake was identified either by enzyme immunoassay or by examination of the dead snake, as B. atrox in 45, L. muta in one. Patients were similar in all clinical and epidemiological respects before treatment. Results indicated that both antivenoms were equally effective in reversing all signs of envenoming detected both clinically and in the laboratory. Venom-induced haemostatic abnormalities were resolved within 24 h after the start of antivenom therapy in most patients. The extent of local complications, such as local skin necrosis and secondary infection, was similar in both groups. There were no deaths. The incidence of early anaphylactic reactions was 18% and 19%, respectively for specific and standard antivenoms; none was life-threatening. Measurement of serum venom concentrations by enzyme immunoassay (EIA) confirmed that both antivenoms cleared venom antigenaemia effectively. EIA also revealed that one patient had been bitten by Lachesis muta, although the clinical features in this case were not distinctive.

Coagulopathy and haemorrhage in human victims of Bothrops jararaca envenoming in Brazil

Thirty-four patients envenomed by Bothrops jararaca in Brazil were studied. Of these, 20 (59%) had incoagulable blood associated with local and/or systemic bleeding and 10 of the 20 were thrombocytopenic. Among 14 patients with coagulable blood, 6 (43%) had bleeding symptoms and 3 (21%) were thrombocytopenic. High levels of von Willebrand factor (vWF), plasminogen activator inhibitor type 1 (PAI-1) and tissue type plasminogen activator (t-PA) antigens were also recorded in some patients with systemic bleeding with or without incoagulable blood. These substances may have been released from endothelial cells. Admission serum venom antigen levels were similar in both groups. The study indicated that systemic haemorrhage may occur in patients with coagulable blood and thrombocytopenia and that coagulopathy is not therefore the primary cause of haemorrhage.

Snakebite by the bushmaster (Lachesis muta) in Brazil: case report and review of the literature.

The bushmaster (Lachesis muta) of Central and South America, the worlds longest pit viper, is capable of injecting a large dose of potent venom when it bites. A 28-year-old man, bitten by a 1.82 m long L. m. muta in Brazil, developed pain and oedema at the bite site, nausea, vomiting, diarrhoea and sweating. There was peripheral neutrophil leucocytosis and evidence of fibrinogen consumption with secondary activation of the fibrinolytic system. Two hours after the bite, eight ampoules of Instituto Butantan Lachesis antivenom was administered, and haemostasis was normal 24 hr later. A review of reports of 20 cases of bites in humans reliably attributed to this snake in Costa Rica, French Guiana, Brazil, Colombia and Venezuela confirms a syndrome of nausea, vomiting, abdominal colic, diarrhoea, sweating, hypotension, bradycardia and shock, possibly autopharmacological or autonomic in origin, not seen in victims of other American crotaline snakes. These, and other symptoms of bushmaster envenoming, are explained by haemorrhagic, coagulant and neurotoxic venom activities. The therapeutic efficacy of non-specific Bothrops/Crotalus polyvalent antivenoms in these cases has been unimpressive. For the treatment of bites by a snake which potentially injects a large dose (> 300 mg dry weight) of venom with a range of life-threatening activities, there is an urgent need to develop more potent specific antivenoms and to treat the dramatic and life-threatening cardiovascular symptoms.

Comparison of Phylogeny, Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.

Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake (Crotalus durissus terrificus, C. durissus cascavella and C. durissus collilineatus)

The subspecies of the South American rattlesnake, Crotalus durissus are classified according to their external morphological features and geographical distribution. We have determined some biological activities of C. durissus cascavella, C. durissus collilineatus and C. durissus terrificus venoms. C. durissus terrificus had a significantly higher clotting activity on bovine plasma and fibrinogen, human fibrinogen and rabbit plasma. C. durissus cascavella presented a statistically higher phospholipase A2 (PLA2) activity in regard to C. durissus collilineatus. Their myotoxic and proteolytic activity, median lethal doses, or median platelet aggregating doses (on rabbit and human platelets) could not differentiate the three subspecies examined. However, the electrophoretic profile and the dose-response curve for edematogenic activity for C.d. cascavella venom were different from the others. With regard to the inorganic element content of the venoms, higher levels of Br, Cl and Mg, and a lower level of Zn, were found in C.d. cascavella venom. Crotamine-like activity could not be detected in C.d. cascavella venom. Furthermore, equine antivenom specific for C. durissus terrificus venom cross-reacted equally with the antigens of the three venom pools by ELISA and Western blotting. These results indicate that the venoms from the three studied subspecies of C. durissus were very similar, except for minor differences in paw edema-inducing activity, electrophoretic profile, phospholipase A2 activity, crotamine-like activity and inorganic element contents of C.d. cascavella venom.

Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms.

Platelet dysfunction during Bothrops jararaca snake envenomation in rabbits

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.

Platelet aggregation in patients bitten by the Brazilian snake Bothrops jararaca.

Patients bitten by the lancehead snake Bothrops jararaca usually develop systemic bleeding. Our aim was to evaluate platelet function in whole blood of 17 human patients bitten by this snake in São Paulo State, Brazil. Bleeding occurred in 71% of these patients, and thrombocytopenia in 53% of them. On admission, most of the patients presented with hypoaggregation to 50 microM ADP and 1.2 mg/ml ristocetin, and only 35% of them to 5 micrograms/ml collagen. Abnormal plasma levels of fibrinogen and fibrin(ogen) degradation products (FDP/fdp) were also observed. Twenty-four hours of finishing serumtherapy, bleeding had already ceased, fibrinogen and FDP/fdp levels returned to hemostatic levels, and values for platelet aggregation returned to the reference range of controls, except for ADP that still remained decreased. These findings evidence that disturbances of platelet function are also an important factor for the development of bleeding in Bothrops envenomation, as well as other known hemostatic disturbances that occur concomitantly.

In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes

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.