RESUMO
Snake venoms are complex mixtures of proteins, which affect the vital biologic systems of prey, as well as humans. Envenomation leads to immobilization by paralysis, cardiac, and circulatory failure. These same venom proteins that cause havoc in the physiologic system could be used as therapeutic agents. Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are non-enzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction. These proteins may have potential in the treatment of strokes, heart attacks, cancers, osteoporosis, and diabetes. The present study describes the isolation and characterization of a disintegrin (colombistatin) found in the venom of the Venezuelan snake mapanare (Bothrops colombiensis). Colombistatin was purified by a two-step high-performance liquid chromatography procedure, which included reverse phase C18 and size exclusion protein Pak 60. Colombistatin inhibited ADP-induced platelet aggregation, human urinary (T24) and skin melanoma (SK-Mel-28) cancer cell adhesion to fibronectin, and cell migration. Colombistatin contained 72 amino acids with a mass of 7.778 kDa as determined by mass spectrometry. Colombistatin could be used as a therapeutic tool in the treatment of melanoma cancers and also thrombotic diseases.
Assuntos
Bothrops , Venenos de Crotalídeos/farmacologia , Desintegrinas/isolamento & purificação , Desintegrinas/farmacologia , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Venenos de Serpentes/isolamento & purificação , Venenos de Serpentes/farmacologia , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sequência Conservada , Venenos de Crotalídeos/química , Desintegrinas/química , Relação Dose-Resposta a Droga , Fibronectinas/metabolismo , Melanócitos/efeitos dos fármacos , Melanoma/metabolismo , Dados de Sequência Molecular , Peso Molecular , Inibidores da Agregação Plaquetária/química , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/metabolismo , Venenos de Serpentes/química , América do SulRESUMO
Envenomations by the southern Pacific rattlesnake (Crotalus oreganus helleri) are the most common snakebite accidents in southern California. Intraspecies venom variation may lead to unresponsiveness to antivenom therapy. Even in a known species, venom toxins are recognized as diverse in conformity with interpopulational, seasonal, ontogenetic and individual factors. Five venoms of individual C. oreganus helleri located in Riverside and San Bernardino counties of southern California were studied for their variation in their hemostatic activity. The results demonstrated that Riverside 2 and San Bernardino 1 venoms presented the highest lethal activity without hemorrhagic activity. In contrast, San Bernardino 2 and 3 venoms had the highest hemorrhagic and fibrinolytic activities with low lethal and coagulant activities. Riverside 1, Riverside 2 and San Bernardino 1 venoms presented a significant thrombin-like activity. San Bernardino 2 and 3 venoms presented an insignificant thrombin-like activity. In relation to the fibrinolytic activity, San Bernardino 3 venom was the most active on fibrin plates, which was in turn neutralized by metal chelating inhibitors. These results demonstrate the differences amongst C. oreganus helleri venoms from close localities. A metalloproteinase, hellerase, was purified by anionic and cationic exchange chromatographies from San Bernardino 3 venom. Hellerase exhibited the ability to break fibrin clots in vitro, which can be of biomedically importance in the treatment of heart attacks and strokes.
Assuntos
Venenos de Crotalídeos/farmacologia , Crotalus , Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Metaloproteases/farmacologia , Sequência de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , California , Cromatografia por Troca Iônica , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Hemólise/efeitos dos fármacos , Hemorragia/induzido quimicamente , Humanos , Dose Letal Mediana , Metaloproteases/química , Metaloproteases/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Fibrinólise/efeitos dos fármacos , Hemorragia/induzido quimicamente , Peptídeo Hidrolases/toxicidade , Dermatopatias/induzido quimicamente , Animais , Bothrops , Cromatografia em Gel , Venenos de Crotalídeos/enzimologia , Dextranos , Eletroforese em Gel de Poliacrilamida , Fator Xa/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Hemorragia/sangue , Humanos , Calicreínas/metabolismo , Dose Letal Mediana , Metaloproteases/isolamento & purificação , Metaloproteases/toxicidade , Camundongos , Peso Molecular , Peptídeo Hidrolases/isolamento & purificação , Inibidores de Proteases/farmacologia , Dermatopatias/sangue , Trombina/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , VenezuelaRESUMO
Elapid snakes throughout the world are considered very lethal, containing neurotoxic venoms that affect the nervous system. When humans are envenomated it is considered a serious medical emergency, and antivenom is the main form of treatment considered, in spite of the fact that some patients may only survive under intensive therapy treatment such as respiratory support. Coral snakes are part of the family Elapidae and envenomations by these snakes are very low (<2% of total snakebites) in most countries from southeastern United States to Argentina. In the United States, there are only two species of coral snakes of medical importance that belong to the Micrurus genera: Micrurus fulvius fulvius (Eastern coral snake) and Micrurus tener tener (Texas coral snake). In 2006, Wyeth pharmaceutical notified customers that the production of the North American coral snake antivenin (NACSA) in the US was discontinued and adequate supplies were available to meet historical needs through the end of October 2008; and therefore, it is of utmost important to consider other antivenoms as alternatives for the treatment of coral snake envenoming. One logical alternative is the coral snake antivenom, Coralmyn, produced by the Mexican company, Bioclon. In order to compare neutralization between NACSA and Coralmyn antivenoms with the North American coral snake venoms, the venom lethal doses (LD(50)) and antivenom effective doses (ED(50)) were determined in 18-20 g, female, BALB/c mice. Additionally, venom comparisons were determined through a non-reduced SDS-PAGE for M.f.fulvius, M.t.tener and the Mexican coral snake venom, Micrurus nigrocinctus nigrocinctus. Coralmyn antivenom was able to effectively neutralize three LD(50) doses of all venom from both M.t.tener and M.f.fulvius, while Wyeth antivenom only neutralized M.f.fulvius venom and was not effective in neutralizing three LD(50) doses of M.t.tener venom. Coralmyn is effective in the neutralization of both clinically important coral snake venoms in the US.
Assuntos
Antivenenos/uso terapêutico , Venenos Elapídicos/toxicidade , Elapidae , Animais , Antivenenos/administração & dosagem , Venenos Elapídicos/antagonistas & inibidores , Dose Letal Mediana , México , Camundongos , Mordeduras de Serpentes/tratamento farmacológico , Estados UnidosRESUMO
Crotalus durissus cumanensis snake venoms from different Venezuelan regions, showed biochemical and hemostatic variations. Fibrino(geno)lytic, hemorrhagic and procoagulant activities and gel-filtration chromatography and SDS-PAGE profiles were analyzed. Differences were observed in fibrinolytic activity: kallikrein-like amidolytic activity was highest in venoms of Santa Teresa, and Margarita. Lagunetica and Carrizales venoms showed the maximum fibrin lysis. The highest hemorrhagic activity was seen in Lagunetica venom. Margarita had the lowest LD(50) of 0.18. Lagunetica, Carrizales and Anzoátegui induced a rapid degradation of fibrinogen alpha chains and slower degradation on beta chains, which could possibly due to a higher content of alpha fibrinogenases in these venoms. This fibrinogenolytic activity is decreased by metalloprotease inhibitors. All venoms, except Carrizales, presented thrombin-like activity. Anzoátegui, Carrizales and Lagunetica, in which fibrinolytic activity was present, showed the largest concentration of high molecular mass components. These results represent a new finding, not previously described, of fibrinolytic activity in South American C. durissus venoms. Santa Teresa and Margarita had fibrinolytic activity, and lack of hemorrhagic activity, representing an important finding in Venezuelan venoms since the description of a fibrinolytic molecule without hemorrhagic activity can have valuable potential in thrombolytic therapy.
Assuntos
Venenos de Crotalídeos/toxicidade , Amidas/química , Animais , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia em Gel , Venenos de Crotalídeos/química , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/química , Fibrinólise/efeitos dos fármacos , Hemorragia/induzido quimicamente , Hemostasia/efeitos dos fármacos , Indicadores e Reagentes , Dose Letal Mediana , Masculino , Camundongos , Proteínas/química , América do Sul , VenezuelaRESUMO
The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 microg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 microg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.
Assuntos
Técnicas de Cultura de Células/economia , Venenos de Crotalídeos/toxicidade , Crotalus , Córtex Renal/citologia , Testes de Toxicidade/economia , Animais , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Cromatografia em Gel , Córtex Renal/efeitos dos fármacos , Dose Letal Mediana , Masculino , Camundongos , Testes de Toxicidade/métodos , Células VeroRESUMO
La patogénesis de la lesion renal ha sido relacionada a la miolisis, hemólisis, hipotensión y/o el efecto directo del veneno. Tanto el cultivo primario o el cultivo celular continuo proveen una alternativa in vitro para la evaluación cuantitativa de la toxicidad de venenos de serpiente. El veneno crudo de Crotalus vegrandis fue fraccionado por una cromatografía de exclusión molecular. La toxicidad del veneno crudo de C. vegrandis, sus fracciones hemorrágicas y neurotóxicas fueron evaluadas en células renales primarias de ratón y una línea continua de células Vero mantenidas in vitro. Las células fueron aisladas de la corteza renal murina y se cultivaron en placas de 96 pozos con medio Dulbecco (DMEM). Allí fueron tratadas con el veneno crudo y sus fracciones. Las células de la corteza renal murina tuvieron una morfología de células epiteliales y la mayoría se tiñeron con un anticuerpo anti-músculo actina. La citotoxicidad fue evaluada por el método colorimétrico del tetrazolium. La viabilidad de las células fue menor en las células tratadas con el veneno crudo, seguida por la fracción hemorrágica y neurotóxica, con un CT50 de 4.93, 18.41 y 50.22 µg/mL, respectivamente. Los cultivos de células Vero parecieron ser más sensibles con un CT50 de 2.9 y 1.4 µg/mL para el veneno crudo y el pico hemorrágico, respectivamente. Los resultados de este estudio muestran la potencialidad de usar sistemas de cultivo celular para evaluar la toxicidad de los venenos.