Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

Bothrops alternatus snake venom induces apoptosis of skeletal muscle cells in a rabbit model / O veneno da serpente Bothrops alternatus induz apoptose nas células musculares de coelhos

Este estudo objetiva reportar a ocorrência de apoptose in vivo induzida pelo veneno da serpente Bothrops alternatus em células musculares esqueléticas. Cinco coelhos machos, adultos, receberam 150µg/kg de veneno no músculo vasto lateral, enquanto outros cinco animais receberam 0,1% de BSA diluído em PBS no mesmo local. Após 12 horas, os animais foram eutanasiados, e amostras do local de inoculação foram coletadas para análise histopatológica. Foram evidenciadas necrose e hemorragia nas células musculares. Além disso, a análise imuno-histoquímica para identificação de caspase-3 ativada revelou marcações granulares e agregadas no citoplasma das células musculares, compatíveis com o processo de apoptose. Este é o primeiro relato que confirma o veneno de B. alternatus como causador de apoptose in vivo em células musculares esqueléticas.(AU)

Use of EDTA in the treatment of local tissue damage caused by the Bothrops alternatus venom / Utilização do EDTA no tratamento da lesão tecidual local causado pelo veneno de Bothrops alternatus

Twelve adult rabbits were distributed in three groups and received on the femoral biceps region, via intradermal injection (ID), 25µg of Bothrops alternatus venom dissolved in NaCl 0.9% and diluted in 0.25mL of phosphate buffered saline (PBS). Thirty minutes later, the group G1 received 0.25mL of phosphate buffered saline (PBS) ID while to G2 and G3 25mg of ethylenediamine tetraacetic acid (EDTA) dissolved in 0.25mL of PBS were administered via intramuscular (IM) and intravenous (IV) injection, respectively. Evaluations included local lesion and blood profile of all animals, before (time zero) and at 1, 2, 3, 4, 5, 6, 12, 18 and 24h after venom administration. All animal treated with PBS (G1) and EDTA IV (G3) presented increase of nociceptive stimuli at the site of inoculation of the venom, followed by moderate edema that persisted for 24h. Animals treated with IM EDTA (G2) only manifested increase of nociceptive stimuli at the site of injection 1h after treatment with discrete local edema between 12 and 24h. In relation to the local hemorrhagic halo no differences were found amongst the studied groups. Blood profile revealed significant decrease of segmented neutrophils in all groups. There was also increase in triglycerides and decrease in total protein and albumin in all groups. The local lesion was not altered by the treatments.(AU)

Doze coelhos adultos, distribuídos em três grupos, receberam, na região de bíceps femoral, por via intradérmica (ID), 25µg de veneno de Bothrops alternatus, dissolvidos em NaCl 09%, diluído em 0,25mL de tampão salina fosfato (PBS). Trinta minutos após o desafio, o grupo G1 recebeu 0,25mL de (PBS) ID, e os grupos G2 e G3 receberam 25mg de ácido etilenodiamino tetra-acético (EDTA), dissolvidos em mL de PBS por via intramuscular (IM) e intravenosa (IV), respectivamente. Foram avaliados lesão local e perfil sanguíneo de todos os animais, antes - tempo zero, e à uma, às duas, três, quatro, cinco, seis, 12, 18 e 24 horas após a injeção do veneno. Tanto os animais tratados com PBS (G1) como os animais tratados com EDTA IV (G3) apresentaram aumento do estímulo nociceptivo no local da administração do veneno, seguido por moderado edema, que perdurou por 24h. Os animais tratados com EDTA IM (G2) somente manifestaram aumento do estímulo nociceptivo local uma hora após tratamento e discreto edema local entre 12 e 24 horas. Em relação ao halo hemorrágico, não houve diferença entre os três grupos estudados. No perfil hematológico, observou-se diminuição significativa dos neutrófilos segmentados nos três grupos estudados. Da mesma forma, houve aumento dos triglicerídeos e diminuição da proteína total e albumina em todos os grupos. Conclui-se que a lesão local não foi alterada pelos tratamentos.(AU)

Use of EDTA in the treatment of local tissue damage caused by the Bothrops alternatus venom / Utilização do EDTA no tratamento da lesão tecidual local causado pelo veneno de Bothrops alternatus

Twelve adult rabbits were distributed in three groups and received on the femoral biceps region, via intradermal injection (ID), 25µg of Bothrops alternatus venom dissolved in NaCl 0.9% and diluted in 0.25mL of phosphate buffered saline (PBS). Thirty minutes later, the group G1 received 0.25mL of phosphate buffered saline (PBS) ID while to G2 and G3 25mg of ethylenediamine tetraacetic acid (EDTA) dissolved in 0.25mL of PBS were administered via intramuscular (IM) and intravenous (IV) injection, respectively. Evaluations included local lesion and blood profile of all animals, before (time zero) and at 1, 2, 3, 4, 5, 6, 12, 18 and 24h after venom administration. All animal treated with PBS (G1) and EDTA IV (G3) presented increase of nociceptive stimuli at the site of inoculation of the venom, followed by moderate edema that persisted for 24h. Animals treated with IM EDTA (G2) only manifested increase of nociceptive stimuli at the site of injection 1h after treatment with discrete local edema between 12 and 24h. In relation to the local hemorrhagic halo no differences were found amongst the studied groups. Blood profile revealed significant decrease of segmented neutrophils in all groups. There was also increase in triglycerides and decrease in total protein and albumin in all groups. The local lesion was not altered by the treatments.(AU)

Doze coelhos adultos, distribuídos em três grupos, receberam, na região de bíceps femoral, por via intradérmica (ID), 25µg de veneno de Bothrops alternatus, dissolvidos em NaCl 09%, diluído em 0,25mL de tampão salina fosfato (PBS). Trinta minutos após o desafio, o grupo G1 recebeu 0,25mL de (PBS) ID, e os grupos G2 e G3 receberam 25mg de ácido etilenodiamino tetra-acético (EDTA), dissolvidos em mL de PBS por via intramuscular (IM) e intravenosa (IV), respectivamente. Foram avaliados lesão local e perfil sanguíneo de todos os animais, antes - tempo zero, e à uma, às duas, três, quatro, cinco, seis, 12, 18 e 24 horas após a injeção do veneno. Tanto os animais tratados com PBS (G1) como os animais tratados com EDTA IV (G3) apresentaram aumento do estímulo nociceptivo no local da administração do veneno, seguido por moderado edema, que perdurou por 24h. Os animais tratados com EDTA IM (G2) somente manifestaram aumento do estímulo nociceptivo local uma hora após tratamento e discreto edema local entre 12 e 24 horas. Em relação ao halo hemorrágico, não houve diferença entre os três grupos estudados. No perfil hematológico, observou-se diminuição significativa dos neutrófilos segmentados nos três grupos estudados. Da mesma forma, houve aumento dos triglicerídeos e diminuição da proteína total e albumina em todos os grupos. Conclui-se que a lesão local não foi alterada pelos tratamentos.(AU)

Could mesenchymal stem cell therapy help in the treatment of muscle damage caused by Bothrops alternatus venom? / Podem as células tronco mesenquimais auxiliarem no tratamento da lesão muscular causada pelo veneno de Bothrops alternatus?

The aim of this study was to evaluate the use of mesenchymal stem cells (MSC) in the treatment of myonecrosis induced by Bothrops alternatus venom in rats. Seventy-five male adult Wistar rats were divided into three experimental groups. G1 and G2 were injected in the gastrocnemius muscle with 120g of B. alternatus venom, while G3 received 200L of PBS only. Three days after the venom injection, 12 rats from G1 were treated with 5.0 x 106 MSC in PBS, whereas G2 and G3 rats received PBS. Every three days, blood and muscle samples of five animals from each group were taken for serum biochemical and pathological analyses. Histological examinations showed more intense muscle lesions following MSC treatment, characterized by disorganization and loss of muscle fibers, with focal necrosis and inflammatory infiltration by mononuclear cells. In conclusion, the use of MSC for the treatment of local damage caused by inoculation of B. alternatus venom impaired muscle regeneration and interfered in the healing process.(AU)

O objetivo do presente trabalho foi avaliar a utilização das células tronco mesenquimais (MSC) no tratamento da mionecrose induzida pelo veneno de Bothrops alternatus em ratos. 75 ratos Wistar adultos foram distribuídos em três grupos experimentais. G1 e G2 receberam 120g de veneno de B. alternatus, enquanto o G3 recebeu apenas 200L de PBS. Três dias após a administração do veneno, os ratos do grupo G1 foram tratados com 5.0 x 106 MSC, enquanto G2 e G3 receberam exclusivamente PBS. A cada três dias, amostras de sangue e tecido muscular foram coletadas de cinco animais de cada grupo para avaliação bioquímica sérica e patológica, respectivamente. A análise histológica revelou lesão muscular mais intensa após a aplicação das MSC, caracterizada pela desorganização e perdas das fibras musculares, com necrose focal e infiltrado inflamatório mononuclear. É possível concluir que a utilização das MSC para o tratamento da lesão local causada pela inoculação do veneno B. alternatus deteriorou a regeneração muscular e interferiu com o processo de cicatrização.(AU)

Could mesenchymal stem cell therapy help in the treatment of muscle damage caused by Bothrops alternatus venom? / Podem as células tronco mesenquimais auxiliarem no tratamento da lesão muscular causada pelo veneno de Bothrops alternatus?

The aim of this study was to evaluate the use of mesenchymal stem cells (MSC) in the treatment of myonecrosis induced by Bothrops alternatus venom in rats. Seventy-five male adult Wistar rats were divided into three experimental groups. G1 and G2 were injected in the gastrocnemius muscle with 120g of B. alternatus venom, while G3 received 200L of PBS only. Three days after the venom injection, 12 rats from G1 were treated with 5.0 x 106 MSC in PBS, whereas G2 and G3 rats received PBS. Every three days, blood and muscle samples of five animals from each group were taken for serum biochemical and pathological analyses. Histological examinations showed more intense muscle lesions following MSC treatment, characterized by disorganization and loss of muscle fibers, with focal necrosis and inflammatory infiltration by mononuclear cells. In conclusion, the use of MSC for the treatment of local damage caused by inoculation of B. alternatus venom impaired muscle regeneration and interfered in the healing process.

O objetivo do presente trabalho foi avaliar a utilização das células tronco mesenquimais (MSC) no tratamento da mionecrose induzida pelo veneno de Bothrops alternatus em ratos. 75 ratos Wistar adultos foram distribuídos em três grupos experimentais. G1 e G2 receberam 120g de veneno de B. alternatus, enquanto o G3 recebeu apenas 200L de PBS. Três dias após a administração do veneno, os ratos do grupo G1 foram tratados com 5.0 x 106 MSC, enquanto G2 e G3 receberam exclusivamente PBS. A cada três dias, amostras de sangue e tecido muscular foram coletadas de cinco animais de cada grupo para avaliação bioquímica sérica e patológica, respectivamente. A análise histológica revelou lesão muscular mais intensa após a aplicação das MSC, caracterizada pela desorganização e perdas das fibras musculares, com necrose focal e infiltrado inflamatório mononuclear. É possível concluir que a utilização das MSC para o tratamento da lesão local causada pela inoculação do veneno B. alternatus deteriorou a regeneração muscular e interferiu com o processo de cicatrização.

Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...

Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...(AU)

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 groups, present in BaltDC, form hydrogen bonds with the PO 2 groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbβPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbβPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.(AU)

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes namely sbPLI, sbPLI or sbPLI depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbPLIs and sbPLIs, whereas sbPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbPLIs and sbPLIs from two Old World Gloydius brevicaudus and Malayopython reticulatus and two New World Bothrops alternatus and Crotalus durissus terrificus snake species will be emphasized.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes namely sbPLI, sbPLI or sbPLI depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbPLIs and sbPLIs, whereas sbPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbPLIs and sbPLIs from two Old World Gloydius brevicaudus and Malayopython reticulatus and two New World Bothrops alternatus and Crotalus durissus terrificus snake species will be emphasized.(AU)

Enucleação em serpente urutu (Bothrops alternatus) no tratamento de endoftalmite decorrente de traumatismo de escama corneal / Urutu (Bothrops alternatus) snake enucleation in the treatment of endophthalmitis resulting from spectacle trauma

A urutu, Bothrops alternatus, é uma espécie de serpente peçonhenta da família Viperidae e subfamília Crotalinae. Como em outras espécies, ela pode ser acometida por acidentes ofídicos em algumas situações. Relata-se o caso de uma urutu que teve a escama corneal traumatizada em decorrência do ataque de outra cobra do mesmo gênero há 30 dias. Ao exame oftálmico, notou-se a presença de crosta recobrindo exsudato caseoso localizado no espaço subespetacular. Durante a exploração cirúrgica, foi removida a escama corneal e o exsudato caseoso, notando-se o quadro de endoftalmite. Procedeu-se à enucleação do bulbo acometido e o animal se recuperou bem. Diante de alguns casos, especificamente os que envolvem animais peçonhentos, o tratamento clínico com sua manipulação várias vezes ao dia torna-se inviável, sendo recomendado um tratamento cirúrgico definitivo.

The urutu (Bothrops alternatus) belongs to the Viperidae family and to the subfamily of venomous vipers, the Crotalinae. As with all other species the urutu is also susceptible to ophidian accidents. This is a case report of an urutu snake brought to the veterinary hospital with an injured spectacle. According to the clinical history, this lesion was caused by the attack of another snake of the same genus about 30 days earlier. Ophthalmic examination revealed a crust overlaying cheesy exudates just in the subspectacle space. After removal of these from the eye, an endophthalmitis was discovered. Enucleation of the affected ocular globe was performed allowing full recovery of the animal. Poisonous animals are difficult to handle, and I are important limitation to managements that require prolonged and multiple treatments throughout the day. Hence, in such cases, the preference for radical surgical procedures might be justifiable.

Enucleação em serpente urutu (Bothrops alternatus) no tratamento de endoftalmite decorrente de traumatismo de escama corneal / Urutu (Bothrops alternatus) snake enucleation in the treatment of endophthalmitis resulting from spectacle trauma

A urutu, Bothrops alternatus, é uma espécie de serpente peçonhenta da família Viperidae e subfamília Crotalinae. Como em outras espécies, ela pode ser acometida por acidentes ofídicos em algumas situações. Relata-se o caso de uma urutu que teve a escama corneal traumatizada em decorrência do ataque de outra cobra do mesmo gênero há 30 dias. Ao exame oftálmico, notou-se a presença de crosta recobrindo exsudato caseoso localizado no espaço subespetacular. Durante a exploração cirúrgica, foi removida a escama corneal e o exsudato caseoso, notando-se o quadro de endoftalmite. Procedeu-se à enucleação do bulbo acometido e o animal se recuperou bem. Diante de alguns casos, especificamente os que envolvem animais peçonhentos, o tratamento clínico com sua manipulação várias vezes ao dia torna-se inviável, sendo recomendado um tratamento cirúrgico definitivo.(AU)

The urutu (Bothrops alternatus) belongs to the Viperidae family and to the subfamily of venomous vipers, the Crotalinae. As with all other species the urutu is also susceptible to ophidian accidents. This is a case report of an urutu snake brought to the veterinary hospital with an injured spectacle. According to the clinical history, this lesion was caused by the attack of another snake of the same genus about 30 days earlier. Ophthalmic examination revealed a crust overlaying cheesy exudates just in the subspectacle space. After removal of these from the eye, an endophthalmitis was discovered. Enucleation of the affected ocular globe was performed allowing full recovery of the animal. Poisonous animals are difficult to handle, and I are important limitation to managements that require prolonged and multiple treatments throughout the day. Hence, in such cases, the preference for radical surgical procedures might be justifiable.(AU)

Meal size effects on the postprandial metabolic response of Bothrops alternatus (Serpentes: Viperidae)

We examined the effects of meal size on the postprandial metabolic response of the lancehead Bothrops alternatus (Duméril, Bibron & Duméril, 1894), fed mice equaling to 5, 10, 20, and 40% of the snake's body mass. The maximum O2 consumption rates measured during digestion increased with meal size, reaching levels up to 2.8-7.8-fold higher than the metabolic rate measured during fasting. Specific Dynamic Action (SDA) duration also increased with meal size, lasting from 54 to 212 hours to complete. Under our experimental conditions, 30ºC, the majority of our snakes failed to completely digest prey with a relative size of 40%. The SDA coefficient ranged from 17 to 27% of the energy content of the meal and was not affected by meal size.

Meal size effects on the postprandial metabolic response of Bothrops alternatus (Serpentes: Viperidae)

We examined the effects of meal size on the postprandial metabolic response of the lancehead Bothrops alternatus (Duméril, Bibron & Duméril, 1894), fed mice equaling to 5, 10, 20, and 40% of the snake's body mass. The maximum O2 consumption rates measured during digestion increased with meal size, reaching levels up to 2.8-7.8-fold higher than the metabolic rate measured during fasting. Specific Dynamic Action (SDA) duration also increased with meal size, lasting from 54 to 212 hours to complete. Under our experimental conditions, 30ºC, the majority of our snakes failed to completely digest prey with a relative size of 40%. The SDA coefficient ranged from 17 to 27% of the energy content of the meal and was not affected by meal size.

Meal size effects on the postprandial metabolic response of Bothrops alternatus (Serpentes: Viperidae)

We examined the effects of meal size on the postprandial metabolic response of the lancehead Bothrops alternatus (Duméril, Bibron & Duméril, 1894), fed mice equaling to 5, 10, 20, and 40% of the snake's body mass. The maximum O2 consumption rates measured during digestion increased with meal size, reaching levels up to 2.8-7.8-fold higher than the metabolic rate measured during fasting. Specific Dynamic Action (SDA) duration also increased with meal size, lasting from 54 to 212 hours to complete. Under our experimental conditions, 30ºC, the majority of our snakes failed to completely digest prey with a relative size of 40%. The SDA coefficient ranged from 17 to 27% of the energy content of the meal and was not affected by meal size.