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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.

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 48 h 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 12 h after the antivenom therapy. Three patients developed systemic bleeding 48 h after antivenom therapy. Levels of fibrinogen and alpha 2-antiplasmin, FDP and D-dimer returned to normal around 48 h after the treatment or on discharge. The frequency of thrombocytopenia with high mean platelet volume increased in the first 24 h after antivenom therapy, and decreased on discharge. Bothrops venom levels in patients decreased 12 h 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 48 h after the antivenom therapy until discharge. A few patients still presented bleeding signs within 48 h after beginning antivenom therapy. However, the Brazilian antivenom was able to overcome the hemostatic disorders in these cases of envenomation.

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 (Na 2 -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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2011 International Society on Thrombosis and Haemostasis.)

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., ((c) 2010 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

The effects of convulxin, isolated from the venom of Crotalus durissus terrificus, on the localization and distribution of wheat germ agglutinin (WGA) binding sites on platelet surfaces have been investigated at an ultrastructural level. A post-embedding cytochemical technique using WGA-gold complexes was used and the quantitative intensity of WGA-labeling on the surface membrane of platelets after convulxin stimulation was determined. In the presence of Ca2+, convulxin induced platelet release and aggregation, while in its absence, the platelets formed pseudopodia and showed release reaction, but without aggregation. After treatment with convulxin, WGA-labeling on the surface membrane decreased compared with intact washed (control) platelets. In the presence of Ca2+, clusters of gold label were often found on the surface membrane. However, the WGA-labeling intensity of the membrane of the open canalicular system increased significantly compared with that of platelets stimulated by convulxin in the absence of Ca2+. Direct morphological evidence demonstrates qualitative and quantitative alterations of WGA-labeling on the surface membrane of platelets, after convulxin stimulation. The possibility is considered that WGA-binding glycoproteins on the surface membrane are involved in the aggregation response after convulxin stimulation.

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.

Blood coagulation studies carried out on 78 patients up to 30 h after they were bitten by Bothrops jararaca snakes demonstrated clotting defects in 37 patients which included afibrinogenemia, reduced levels of prothrombin, of factors V and VIII, thrombocytopenia and activation of the fibrinolytic system. Factor IX and X levels were within normal range for all patients. These in vitro data suggest that the disseminated intravascular clotting observed in vivo following envenomation may be triggered by the intravascular release of patient thrombin by snake venom enzymes.