Acute kidney injury induced by thrombotic microangiopathy in two cases of Bothrops envenomation

CONTEXT: Bothrops snakes are the most frequent agents of snakebites in South and Central America. Acute kidney injury (AKI) is one of its complications and has multifactorial origin. Thrombotic microangiopathy (TMA)-induced AKI in snakebites is uncommon and is not described in Bothrops envenomation. CASE DETAILS: We report two cases of patients bitten by young Bothrops jararaca who developed AKI induced by TMA. Both patients evolved with mild envenomation and received the specific antivenom within 4 h after the snakebite. None of them had hypotension or shock, bleeding or secondary infection. Patient 1 (P1) was diabetic and using oral hypoglycemic drugs, and patient 2 (P2) was hypertensive without regular use of medication. On admission, both patients had levels of fibrinogen lower than 35 mg/dL, D-dimer higher than 10,000 ng/mL. They evolved with AKI, thrombocytopenia, normal coagulation assays, anemia, lactate dehydrogenase (LDH) elevation, low haptoglobin levels, negative direct antiglobulin test, and presence of schizocytes in peripheral blood. Only P1 required renal replacement therapy, and plasmapheresis was not required. Both patients were discharged and did not require outpatient dialysis, and subsequently had normal creatinine levels. DISCUSSION: TMA may occur in Bothrops jararaca envenomation, even in mild cases that received early specific antivenom

Acute kidney injury induced by thrombotic microangiopathy in two cases of Bothrops envenomation.

CONTEXT: Bothrops snakes are the most frequent agents of snakebites in South and Central America. Acute kidney injury (AKI) is one of its complications and has multifactorial origin. Thrombotic microangiopathy (TMA)-induced AKI in snakebites is uncommon and is not described in Bothrops envenomation. CASE DETAILS: We report two cases of patients bitten by young Bothrops jararaca who developed AKI induced by TMA. Both patients evolved with mild envenomation and received the specific antivenom within 4 h after the snakebite. None of them had hypotension or shock, bleeding or secondary infection. Patient 1 (P1) was diabetic and using oral hypoglycemic drugs, and patient 2 (P2) was hypertensive without regular use of medication. On admission, both patients had levels of fibrinogen lower than 35 mg/dL, D-dimer higher than 10,000 ng/mL. They evolved with AKI, thrombocytopenia, normal coagulation assays, anemia, lactate dehydrogenase (LDH) elevation, low haptoglobin levels, negative direct antiglobulin test, and presence of schizocytes in peripheral blood. Only P1 required renal replacement therapy, and plasmapheresis was not required. Both patients were discharged and did not require outpatient dialysis, and subsequently had normal creatinine levels. DISCUSSION: TMA may occur in Bothrops jararaca envenomation, even in mild cases that received early specific antivenom.

Gene expression alterations related to mania and psychosis in peripheral blood of patients with a first episode of psychosis.

Psychotic disorders affect ~3% of the general population and are among the most severe forms of mental diseases. In early stages of psychosis, clinical aspects may be difficult to distinguish from one another. Undifferentiated psychopathology at the first-episode of psychosis (FEP) highlights the need for biomarkers that can improve and refine differential diagnosis. We investigated gene expression differences between patients with FEP-schizophrenia spectrum (SCZ; N=53) or FEP-Mania (BD; N=16) and healthy controls (N=73). We also verified whether gene expression was correlated to severity of psychotic, manic, depressive symptoms and/or functional impairment. All participants were antipsychotic-naive. After the psychiatric interview, blood samples were collected and the expression of 12 psychotic-disorder-related genes was evaluated by quantitative PCR. AKT1 and DICER1 expression levels were higher in BD patients compared with that in SCZ patients and healthy controls, suggesting that expression of these genes is associated more specifically to manic features. Furthermore, MBP and NDEL1 expression levels were higher in SCZ and BD patients than in healthy controls, indicating that these genes are psychosis related (independent of diagnosis). No correlation was found between gene expression and severity of symptoms or functional impairment. Our findings suggest that genes related to neurodevelopment are altered in psychotic disorders, and some might support the differential diagnosis between schizophrenia and bipolar disorder, with a potential impact on the treatment of these disorders.

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.

Epidemiologic and clinical survey of victims of centipede stings admitted to Hospital Vital Brazil (São Paulo, Brazil).

We retrospectively analyzed 98 proven cases of centipede stings admitted to Hospital Vital Brazil, Butantan Institute, São Paulo, Brazil, between 1990 and 2007. Most stings occurred at the metropolitan area of São Paulo city (n=94, 95.9%), in the domiciles of patients (n=67, 68.4%), and during the warm-rainy season (n=60, 61.2%). The mean age of the victims was 32.0+/-18.8-years-old. Cryptops and Otostigmus genera were responsible for most cases. Around 86% of the patients sought medical care within 6h after the sting. Both lower (56.1 %) and upper limbs (41.8 %) were most frequently bitten, especially the feet and hands (89.8%). The most frequent local clinical manifestations were pain (94.9%), erythema (44.9%) and edema (21.4%), and the latter was mainly observed in patients bitten by Otostigmus spp. Supportive treatment was used in only 28.6% of the patients, namely administration of local anesthesia (9.2%) and systemic analgesia (13.3%). No sequels or complications were observed in patients, and the prognostic was benign.

Enzymatic and immunochemical characterization of Bothrops insularis venom and its neutralization by polyspecific Bothrops antivenom.

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A(2) activities. No hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting. The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of B. insularis venom.

Purification and characterization of patagonfibrase, a metalloproteinase showing alpha-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom.

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. In the present study, we describe the purification and characterization of a metalloproteinase (patagonfibrase), the first protein to be isolated from Philodryas patagoniensis (Colubridae) snake venom. Patagonfibrase is a single-chain protein, showing a molecular mass of 53,224 Da and an acidic isoelectric point (5.8). It hydrolyzed selectively the Aalpha-chain of fibrinogen and when incubated with fibrinogen or plasma, the thrombin clotting time was prolonged. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase. When administered into mouse gastrocnemius muscle, it induced local hemorrhage and necrosis, and systemic bleeding in lungs. Patagonfibrase showed proteolytic activity toward azocasein, which was enhanced by Ca(2+) and inhibited by Zn(2+), cysteine, dithiothreitol and Na(2)EDTA. Patagonfibrase impaired platelet aggregation induced by collagen and ADP. Thus, patagonfibrase may play a key role in the pathogenesis of disturbances that occur in P. patagoniensis envenomation, and may be used as a biological tool to explore many facets of hemostasis.

In vivo characterization of Lopap, a prothrombin activator serine protease from the Lonomia obliqua caterpillar venom.

Increasing occurrence of hemorrhagic syndrome in man, caused by contact with Lonomia obliqua caterpillars, has been reported in Southern Brazil in the past 10 years. The L. obliqua venom causes a severe consumptive coagulopathy, which can lead to hemorrhagic syndrome. L. obliqua prothrombin activator protease (Lopap) is a 69-kDa prothrombin activator serine protease isolated from L. obliqua caterpillar bristle extract, which is able to evoke thrombus formation, unclottable blood, and fibrinogen depletion when injected into the blood stream of rats. The purified protein generated thrombin from prothrombin, able to clot purified human fibrinogen and plasma. A decrease in platelet count and inhibition of collagen-induced platelet aggregation were observed, as well as leukocyte infiltration in the lungs. In addition, we observed congestion and hemorrhage in renal glomeruli and necrosis in renal distal tubules. These data support the hypothesis that Lopap contributes to the clinical syndrome caused by human contact with L. obliqua, most probably through prothrombin activation, resulting in a consumption coagulopathy.

A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2.

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.

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.

A randomized 'blinded' comparison of two doses of antivenom in the treatment of Bothrops envenoming in São Paulo, Brazil.

An earlier study in São Paulo state suggested that the dose for patients with mild or moderate envenoming by Bothrops snakes (mainly Bothrops jararaca) could be effectively decreased to 4 ampoules (40 mL) of Brazilian Brothrops polyspecific antivenom. The present 'blinded' study examined the lowest dose studied in the first trial (equivalent to 4 x 10 mL ampoules) and half that dose of antivenom (equivalent to 2 x 10 mL ampoules) in 2 similar groups of 170 patients who were comparable in all respects before treatment. The majority of patients showed rapid clinical improvement after treatment with either dose regimen and rapid restoration of blood coagulability and cessation of bleeding. There was no apparent difference between the 2 groups of patients in any respect. The study confirmed that, in such patients, the dose of antivenom can be decreased from 4 ampoules to 2 ampoules without reduction of therapeutic efficacy, and it is highly likely that this reduction will result in a decrease of early anaphylactic reactions caused by the antivenom.

Decreased erythrocyte osmotic fragility during canine leptospirosis.

Erythrocyte osmotic fragility (EOF) was carried out in nineteen dogs naturally infected by Leptospira interrogans serovar icterohaemorrhagiae/copenhagi. A decreased EOF was observed, suggesting a modification of erythrocyte components secondary to disturbances that occur during canine leptospirosis, such as renal damage and hepatic disease.

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.

Randomized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers (Bothrops jararaca) in São Paulo, Brazil.

In São Paulo City, Brazil, 121 patients with moderately severe envenoming by Bothrops snakes (principally B. jararaca) were randomized for treatment with Brazilian polyspecific Bothrops antivenoms: Instituto Butantan (39 patients), Instituto Vital Brazil (41), Fundação Ezequiel Dias (FUNED) (41). The initial dose was four ampoules (40 ml) in 89 patients with less severe envenoming and eight ampoules (80 ml) in 32 patients with more severe envenoming. A second dose of four ampoules was required in 20 patients. Patients receiving the three antivenoms were comparable in all respects before treatment. There were no deaths. The majority showed rapid clinical improvement, resolution of local envenoming, cessation of bleeding and restoration of blood coagulability. No differences in the efficacy of the three antivenoms were revealed by clinical or laboratory observations, including measures of haematological, haemostatic and biochemical abnormalities. Twelve patients developed abscesses (Butantan 1, Vital Brazil 6, FUNED 5) and seven developed local necrosis (3,1,3). Of 88 patients followed up 20-30 days after the bite 33 (37.5%) still had symptoms or signs of local envenoming, especially swelling. Early (anaphylactic) reactions were unexpectedly frequent after all three antivenoms but were significantly more frequent with Butantan (87%) than with Vital Brazil (37%) or FUNED (56%) antivenoms (p < 0.001). A possible explanation was the higher total protein content and percentage immunoglobulin of Butantan antivenom. The doses of antivenom recommended in Brazil and used in this study may be unnecessarily high, resulting in an unacceptably high incidence of reactions. Results of the study should prompt a critical re-evaluation of antivenom production techniques and dosage recommendations in Brazil.

Studies on blood coagulation and fibrinolysis in patients bitten by Bothrops jararaca (jararaca).

The blood coagulation and the fibrinolytic systems of nine patients envenomed by Bothrops jararaca in São Paulo (Brazil) were studied. Five of the accidents were caused by young snakes (less than 50 cm). On admission, four patients had non-clotting and three partially-clotting blood. Fibrinogen levels were decreased due to the thrombin-like activity of the venom as expected. Consequent secondary activation of the fibrinolytic system was evident from the low levels of alpha-2-antiplasmin and the high titres of fibrin(ogen) degradation products. High titres of cross-linked fibrin fragment D (D-dimer) in seven patients together with decreased platelet counts and/or factor V, and/or factor VIII in some, suggests intrinsic thrombin formation as these factors are not consumed in the defibrinogenation induced by venom thrombin-like fractions such as Ancrod and Batroxobin. However, normal or increased levels of antithrombin III in all and normal levels of factor II in eight patients do not support this interpretation. The existence of variable concentrations of other proteins in the venom of B. jararaca such as botrocetin and thrombocytin isolated from B. jararaca and B. atrox or crotalocytin from Crotalus horridus venom should be considered. Such proteins are known to activate factors V, VIII, XIII, and platelets without affecting prothrombin (factor II) and antithrombin III. Slower recovery of the haemostatic disturbances after antivenom administration to patients bitten by young snakes suggests a more severe coagulopathy in such accidents. This is supported by clinical observations.