Elevated plasma levels of hepatocyte growth factor in rats experimentally envenomated with Bothrops jararaca venom: Role of snake venom metalloproteases.

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 ±â€¯91) compared with control values (68 ±â€¯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.

Platelet participation in the pathogenesis of dermonecrosis induced by Loxosceles gaucho venom.

Loxosceles gaucho spider venom induces in vitro platelet activation and marked thrombocytopenia in rabbits. Herein, we investigated the involvement of platelets in the development of the dermonecrosis induced by L. gaucho venom, using thrombocytopenic rabbits as a model. L. gaucho venom evoked a drop in platelet and neutrophil counts 4 h after venom injection. Ecchymotic areas at the site of venom inoculation were noticed as soon as 4 h in thrombocytopenic animals but not in animals with initial normal platelet counts. After 5 days, areas of scars in thrombocytopenic animals were also larger, evidencing the marked development of lesions in the condition of thrombocytopenia. Histologically, local hemorrhage, collagen fiber disorganization, and edema were more severe in thrombocytopenic animals. Leukocyte infiltration, predominantly due to polymorphonuclears, was observed in the presence or not of thrombocytopenia. Thrombus formation was demonstrated by immunohistochemistry at the microvasculature, and it occurred even under marked thrombocytopenia. Taken together, platelets have an important role in minimizing not only the hemorrhagic phenomena but also the inflammatory and wound-healing processes, suggesting that cutaneous loxoscelism may be aggravated under thrombocytopenic conditions.

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom.

BACKGROUND: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. OBJECTIVE: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. METHODS: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. RESULTS: Platelet counts were markedly diminished in rats administered with either ARPI (± 88%) or busulfan (± 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. CONCLUSIONS: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom

Background:The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. Objective:To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. Methods:Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. Results:Platelet counts were markedly diminished in rats administered with either ARPI (±88%) or busulfan (±96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. Conclusions:Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

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.

Haematopoiesis in snakes (Ophidia) in early postnatal development.

The occurrence of haematopoiesis has been studied in various parts of the spine and in the ribs in four species of snakes (Boa constrictor L., Elaphe guttata L., Lamprophis fulaginosus Boie., Bothrops jararaca Wied.) from hatching until 150 days of postnatal development. Marrow spaces are formed by chondrolysis with various time frames depending on the studied species. Marrow cells egress to the general circulation in two ways: via migration through the endothelial cells lining the venous sinuses or by the rupture of protrusions. Erythroblasts are present in the lumen of marrow sinuses suggesting their final maturation there. Various relationships of the spleen to the pancreas have been found. No myelopoietic foci occur in the spleen, liver or kidney of any of the studied species. However, erythropoiesis (sparse islets) has been observed in Bothrops jararaca spleen.

Failure of chloramphenicol prophylaxis to reduce the frequency of abscess formation as a complication of envenoming by Bothrops snakes in Brazil: a double-blind randomized controlled trial.

Bites by many species of venomous snake may result in local necrosis at, or extending from, the site of the bite. The use of prophylactic antibiotics to prevent infection as a complication of local necrotic envenoming is controversial. A double-blind randomized controlled trial was carried out to assess whether antibiotic therapy is effective in this situation. Two hundred and fifty-one patients, with proven envenoming by snakes of the genus Bothrops, admitted to two hospitals in Brazil, between 1990 and 1996, were randomized to receive either oral chloramphenicol (500 mg every six hours for five days) or placebo. One hundred and twenty-two of these patients received chloramphenicol (group 1) and 129 were given placebo (group 2). There were no significant differences between the groups at the time of admission. Necrosis developed in seven (5.7%) patients in group 1 and in five (3.9%) patients in group 2 (P>0.05) while abscesses occurred in six patients (4.9%) in group 1 and in six (4.7%) patients in group 2 (P>0.05). In conclusion, the use of orally-administered chloramphenicol for victims of Bothrops snake bite with signs of local envenoming on admission, is not effective for the prevention of local infections.

Hematological, hemostatic and clinical chemistry disturbances induced by Crotalus durissus terrificus snake venom in dogs.

The aim of this study was to investigate the hematological, hemostatic and biochemical disturbances induced by the injection of Crotalus durissus terrificus venom in dogs under controlled conditions. For this purpose three groups of animals were used: an experimental group (E), which was injected i.m. with C. durissus terrificus venom (1 mg/kg); and two control groups--antivenom (AV) and control (C)--which were injected i.m. with 150 mM NaCl. Groups E and AV were treated i.v. with Crotalus antivenom 2 hours after the first injection. Serum levels of alkaline phosphatase and alanine aminotransferase were increased in groups E and AV at 24 and 48 hours after serumtherapy, respectively. The increased serum levels of myoglobin, creatine kinase and aspartate aminotransferase demonstrated that animals developed rhabdomyolysis. A persistent neutrophilic leukocytosis was already noticeable at 2 hours after envenomation and lasted even after serumtherapy. The animals of groups E and AV presented eosinopenia 24 hours after serumtherapy, and collagen-induced platelet hypoaggregation was observed without thrombocytopenia. Increased levels of fibrinogen/fibrin degradation products (FnDP/FgDP), hypofibrinogenemia, and alpha2-antiplasmin consumption were observed at 2 hours after envenomation, indicating secondary activation of fibrinolysis. Our data suggest that the biochemical and hemostatic disturbances induced by C. durissus terrificus venom in dogs are related to its myotoxic and thrombin-like activities.

Envenoming by Bothrops jararaca in Brazil: association between venom antigenaemia and severity at admission to hospital.

The association between the clinical severity of Bothrops jararaca envenoming at admission and serum venom and plasma fibrinogen concentrations before antivenom administration is reported in 137 patients admitted to Hospital Vital Brazil, Instituto Butantan, São Paulo, Brazil, between 1989 and 1990. Other variables such as age, gender, site of the bite, use of tourniquet and the time interval between the bite and start of antivenom therapy, spontaneous systemic bleeding, and the 20 minute whole blood clotting test (20WBCT) at admission showed no association with either severity or serum venom antigen concentration (SVAC). Mean SVAC in patients with mild envenoming was significantly lower than in the group with moderate envenoming (P = 0.0007). Patients with plasma fibrinogen concentrations > 1.5 g/L had a lower mean SVAC than patients with plasma fibrinogen concentrations < or = 1.5 g/L (P = 0.02). Those admitted with a tourniquet in place had significantly higher plasma fibrinogen concentrations than those without a tourniquet (P = 0.002). A multiple logistic regression model showed independent risk factors for severity: bites at sites other than legs or forearms, SVACs > or = 400 ng/mL, and the use of a tourniquet. Rapid quantification of SVAC before antivenom therapy might improve initial evaluation of severity in B. jararaca bites.

Hematopoiesis in snakes (Ophidia).

Locations of the hematopoietic tissue have been described in the following ophidian species: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe teniura teniura, Boa constrictor, and Python reticulatus. Studies were carried out on perfusion fixed vertebrae, ribs, spleen, liver, thymus, and kidney. Routine histological technique was applied using both light and electron microscopy. Hematopoietic tissue was found in the following locations of the vertebrae: neural spine, neural arch, postzygophysis processes, hypapophysis, vertebral centre. Moreover, intense hematopoiesis was found inside the ribs. In the spleen and thymus, only lymphopoiesis was found. Hematopoietic islets in the spleen were sporadically found only in young specimens. No hematopoiesis was observed in the liver and kidney. In the studied species, there were no differences in the location of hematopoietic tissue. A new model of mature and immature blood cell release to the lumen of marrow sinuses different from that known to operate in higher vertebrates is proposed.

Coagulopathy following lethal and non-lethal envenoming of humans by the South American rattlesnake (Crotalus durissus) in Brazil.

The South American tropical rattlesnake (Crotalus durissus subspp) is responsible for approximately 10% of bites from venomous snakes in Brazil. We studied 24 victims of bites by this species over 3 years, in south-eastern Brazil, particularly investigating haemostatic alterations. Thirteen patients were defined as moderately envenomed and 11 as severe. There were two deaths, which were not attributed to venom-induced haemostatic disturbances. However, envenoming by C. durissus is frequently associated with haemostatic disorders, which are probably attributable mainly to the action of the thrombin-like enzyme, with possible additional effects secondary to the powerful myotoxic activity of the venom.

Efficacy of bothropic antivenom and its IgG(T) fraction in restoring fibrinogen levels of Bothrops jararaca envenomed mice.

Bothropic antivenom and its IgG(T) fraction, administered 4 h after experimental envenoming by Bothrops jararaca in Swiss mice, were compared for their abilities to restore fibrinogen 24 or 48 h after treatment. IgG(T) was able to normalise fibrinogen levels as efficiently as conventional antivenom. As IgG(T) also neutralises most anti-toxic activities of Bothrops venom, our results suggest that IgG(T) could be a better alternative treatment for envenoming due to the reduced amount of extraneous proteins, which may facilitate the induction of early adverse reactions.

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.

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.

Snake bites by the jararacuçu (Bothrops jararacussu): clinicopathological studies of 29 proven cases in São Paulo State, Brazil.

The jararacuçu, one of the most dreaded snakes of Brazil, southern Bolivia, Paraguay and northeastern Argentina, is a heavily-built pit viper which may grow to a length of 2.2 m. Up to 1000 mg (dry weight) of highly-lethal venom may be milked from its venom glands on a single occasion. It has accounted for 0.8% to 10% of series of snake bites in São Paulo State, Brazil. We examined 29 cases of proven jararacuçu bites recruited over a 20-year period in two São Paulo hospitals. Severe signs of local and systemic envenoming, (local necrosis, shock, spontaneous systemic bleeding, renal failure) were seen only in patients bitten by snakes longer than 50 cm; bites by shorter specimens were more likely to cause incoagulable blood. Fourteen patients developed coagulopathy, six local necrosis (requiring amputation in one) and five local abscesses. Two became shocked and four developed renal failure. Three patients, aged 3, 11 and 65 years, died 18.75, 27.75 and 83 h after being bitten, with respiratory and circulatory failure despite large doses of specific antivenom and intensive-care-unit management. In two patients, autopsies revealed acute renal tubular necrosis, cerebral oedema, haemorrhagic rhabdomyolysis at the site of the bite and disseminated intravascular coagulation. In one survivor with chronic renal failure, renal biopsy showed bilateral cortical necrosis; the patient remains dependent on haemodialysis. Effects of polyspecific Bothrops antivenom were not impressive, and it has been suggested that anti-Bothrops and anti-Crotalus antivenoms should be given in combination.

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 world's 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.

Hematological changes induced by Bothrops jararaca venom in dogs.

The mechanism of consumption coagulopathy observed in cases of human envenomation by Bothrops jararaca is well established. However, this mechanism may vary according to the animal species studied. In order to study both the clinical and laboratory aspects of bothropic envenomation in dogs, a sublethal defibrinating dose of venom (100 micrograms/kg) was given intravenously. A coagulopathy similar to that observed in humans - including fibrinogen depletion, consumption of factors II, X, V and antithrombin III, and moderate thrombocytopenia - was observed. The presence of circulating activated platelets was also noted. Neutrophilic leukocytosis, lymphopenia, and monocytosis occurred at different times. Erythrocytic values remained normal in dogs treated with B. jararaca venom compared with those treated with saline alone. The erythrocyte sedimentation rate fell rapidly after venom administration and this fall was correlated logarithmically with fibrinogen concentration. Since the effects of envenomation in dogs is similar to that in humans, it was concluded that the dog can be used as a good animal model for studying human venom-induced coagulopathy.

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. Butantan Institute Antivenom Study Group.

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.

In Vivo Platelet Activation Induced by Bothrops jararaca Venom in Rabbits.

Disturbances of platelet morphology, coagulation and fibrinolysis were studied 3, 6, and 24 h following administration of Bothrops jararaca snake venom to rabbits (80 µg/kg, i.v.). The activation of coagulation and fibrinolytic systems was demonstrated by a significant decrease in fibrinogen concentration, and an increase in fibrin(ogen) degradation product concentration, respectively. However, the prothrombin activity remained within normal limits throughout. Significant thrombocytopenia was observed 3 h following venom administration. A decrease in platelet dense body numbers was observed until 24 h. 'Exhausted' platelets and evidence of granular secretion were frequently observed in envenomed rabbits. The open canalicular system was only dilated in extensively degranulated platelets. The mean platelet area and boundary values were not significantly different from control group. Therefore, B. jararaca venom can stimulate platelets in vivo, inducing release of platelet granular content. The etiology of thrombocytopenia in B. jararaca envenoming seems to be a multifactorial process, causing platelet sequestration.

Different clotting mechanisms of Bothrops jararaca snake venom on human and rabbit plasmas.

Bothrops jararaca venom is approximately 3.5 times more effective at coagulating rabbit plasma than human plasma. To investigate this difference B. jararaca venom was treated with several enzymatic inhibitors and the minimum coagulant dose was determined both on plasma anticoagulated with sodium citrate or a mixture of sodium citrate and heparin, and on fibrinogen (both human and rabbit). On human plasma, the thrombin-like component of the venom accounted for c. 60% of the coagulant activity, such activity was negligible on rabbit plasma. The venom had little clotting activity on rabbit fibrinogen. The factor II- and X-activator components could be inhibited by EDTA, EGTA and 2-mercaptoethanol, whereas the thrombin-like activity was inhibited by PMSF. These differences show that (using human plasma) B. jararaca clotting activity is mainly due to the thrombin-like component, whereas the factor II- and X-activator components are more important on rabbit plasma. The delayed action of the thrombin-like enzyme on rabbit fibrinogen may be attributed to the difference between rabbit and human fibrinopeptide A. Thus, the increased coagulant activity on rabbit plasma may be due to a faster rate of activation of factor X, V or II by snake venom enzymes.