Contribution of endothelial cell and macrophage activation in the alterations induced by the venom of Micrurus tener tener in C57BL/6 mice.

An acute inflammatory response, cellular infiltrates, anemia, hemorrhage and endogenous fibrinolysis activation were previously described in C57BL/6 mice injected with M. tener tener venom (Mtt). As the endothelium and innate immunity may participate in these disturbances and due to our poor understanding of the alterations produced by these venoms when the neurotoxic component is not predominant, we evaluated the effects in an in vitro model. At 24 h, the release of pro-inflammatory mediators was detected in peritoneal macrophages. At different times, the release of pro-inflammatory (TNF-α, IL-6, NO and E-Selectin), pro-coagulant (vWF and TF) and pro-fibrinolytic (uPA) mediators were seen in liver sinusoidal endothelial cells (LSECs). These results suggest that Mtt venom activates macrophages and endothelium, thus inducing the release of mediators, such as TNF-α, that orchestrate the acute inflammatory response and the later infiltration of mononuclear cells into liver in C57BL/6 mice. In addition, endothelium activation promotes TF expression, which may in turn modulate the inflammatory and hemostatic response. These findings suggest crosstalk between inflammation and hemostasis in the alterations observed in Micrurus envenomation, where the neurotoxic manifestations do not predominate.

Mutacytin-1, a New C-Type Lectin-Like Protein from the Venezuelan Cuaima (Lachesis muta muta Linnaeus, 1766) (Serpentes: Viperidae) Snake Venom Inducing Cardiotoxicity in Developing Zebrafish (Danio rerio) Embryos.

Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 µg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.

Pro-inflammatory response and hemostatic disorder induced by venom of the coral snake Micrurus tener tener IN C57BL/6 mice.

Micrurus venoms are known to induce mainly neurotoxicity in victims. However, other manifestations, including hemorrhage, edema, myotoxicity, complement activation, and hemostatic activity have been reported. In order to develop a more complete pharmacological profile of these venoms, inflammatory responses and hemostasis were evaluated in C57BL/6 mice treated with a sub-lethal dose of M. t. tener (Mtt) venom (8 µg/mouse), inoculated intraperitoneally. The venom induced moderate bleeding into the abdominal cavity and lungs, as well as infiltration of leukocytes into the liver. After 30 min, the release of pro-inflammatory mediators (TNF-α, IL-6, and NO) were observed, being most evident at 4 h. There was a decrease in hemoglobin and hematocrit levels at 72 h, a prolongation in coagulation times (PT and aPTT), a decrease in the fibrinogen concentration and an increase in fibrinolytic activity. In this animal model, it was proposed that Mtt venom induces inflammation with the release of mediators such as TNF-α, in response to the toxins. These mediators may activate hemostatic mechanisms, producing systemic fibrinolysis and hemorrhage. These findings suggest alternative treatments in Micrurus envenomations in which neurotoxic manifestations do not predominate.

Intraspecies geographical variability in the South American tigra mariposa (Bothrops venezuelensis Sandner 1952) snake venom activities.

Bothrops venezuelensis snake venoms, from five localities in the North-Central Venezuelan regions, showed biochemical and haemostatic differences. In this study, bioactivities of B. venezuelensis venoms from different regions (Aragua state; Waraira Repano (Capital District); Baruta, La Boyera and Lagunetica (Miranda state)) were compared using both natural and synthetic substrates. The protein contents of these venoms were Lagunetica 89%, La Boyera 79%, Baruta 71%, Waraira Repano 68% and Aragua 64%. Toxic activities effects were: Intraperitoneal LD50s: Aragua-14 mg/kg; Waraira Repano-6.4 mg/kg; Baruta: 8.3 mg/kg; La Boyera-4.4 mg/kg; Lagunetica-16.2 mg/kg. The MHD results: Aragua-21.4 µg/mouse; Waraira Repano-2.5 µg/mouse; Baruta-1.2 µg/mouse; La Boyera-1.4 µg/mouse and Lagunetica-12 µg/mouse. The hide powder azure results: Aragua-1.24 U/mg; La Boyera-2.26 U/mg; Baruta-2.83 U/mg; Lagunetica-3.28 U/mg and Waraira Repano-5.77 U/mg. Esterase specific activity on BAEE results: Waraira Repano-666.66 U/mg; La Boyera-805.5 U/mg; Baruta-900.00 U/mg; Lagunetica-922.19 U/mg and Aragua-1960.67 U/mg. Casein zymography showed digestion bands in the molecular weight above 100 and at 66.2 and 21.5 kDa. Analysis of casein degradation by SDS-PAGE showed two different degradation patterns. Fibrinolytic activity (mm2/µg) on fibrin plates results: Aragua-6.07; Lagunetica-27.6; Waraira Repano-35.7; La Boyera-44.27 and Baruta-45.63. In the fibrinogenolytic assay, the five venoms completely degraded the α chain after 1 min of incubation. None of the venoms completely degraded the ß and γ chains after 24 h incubation. The research indicated that venoms of B. venezuelensis of different geographic areas in Venezuela exhibit variances in composition and component concentrations; except the Aragua venom, all of them had high proteolytic activities.

Hemostatic disorders induced by skin contact with Lonomia obliqua (Lepidoptera, Saturniidae) caterpillars.

Patients envenomed by Lonomia sp caterpillars initially experience a mild burning pain, headache, nausea, vomiting, and skin and mucosal hemorrhages. Some patients can rapidly progress to a severe coagulopathy that presents as visceral or intracerebral hemorrhaging. We studied the hemostatic alterations that occurred in 14 patients who were envenomed by Lonomia obliqua in Southern Brazil and presented at the Hospital São Vicente de Paulo (Passo Fundo, RS), Brazil during the summers of 1993 and 1994 when Lonomia antivenom was not yet available for treatment. The patients were classified into to 4 clinical groups: 0 (two patients), I (eight patients), II (two patients), and III (two patients). The patients were admitted to the hospital between 4 hours and five days after contact with the caterpillars. In this study, the coagulation parameters of the patients were followed up for up to 172 hours after the accidents. The patients received no treatment with the exceptions of two patients who received blood transfusions and antifibrinolytic treatment. The observed abnormalities related to blood coagulation and fibrinolytic factors were similar regardless of the severity of the bleeding symptoms. These findings suggest that alterations in hemostatic parameters without thrombocytopenia are not predictors of the seriousness of such accidents. Thus, consumptive disorder and reactive fibrinolysis are not proportional to mild coagulopathy. Furthermore, these patients recovered. The hemostatic parameters of most of the patients normalized between 96 and 120 h after the accident.

Hemostatic disorders induced by skin contact with Lonomia obliqua (Lepidoptera, Saturniidae) caterpillars

Patients envenomed by Lonomia sp caterpillars initially experience a mild burning pain, headache, nausea, vomiting, and skin and mucosal hemorrhages. Some patients can rapidly progress to a severe coagulopathy that presents as visceral or intracerebral hemorrhaging. We studied the hemostatic alterations that occurred in 14 patients who were envenomed by Lonomia obliqua in Southern Brazil and presented at the Hospital Sao Vicente de Paulo (Passo Fundo, RS), Brazil during the summers of 1993 and 1994 when Lonomia antivenom was not yet available for treatment. The patients were classified into to 4 clinical groups: 0 (two patients), I (eight patients), II (two patients), and III (two patients). The patients were admitted to the hospital between 4 hours and five days after contact with the caterpillars. In this study, the coagulation parameters of the patients were followed up for up to 172 hours after the accidents. The patients received no treatment with the exceptions of two patients who received blood transfusions and antifibrinolytic treatment. The observed abnormalities related to blood coagulation and fibrinolytic factors were similar regardless of the severity of the bleeding symptoms. These findings suggest that alterations in hemostatic parameters without thrombocytopenia are not predictors of the seriousness of such accidents. Thus, consumptive disorder and reactive fibrinolysis are not proportional to mild coagulopathy. Furthermore, these patients recovered. The hemostatic parameters of most of the patients normalized between 96 and 120 h after the accident.

Hemostatic disorders induced by skin contact with Lonomia obliqua (Lepidoptera, Saturniidae) caterpillars

ABSTRACT Patients envenomed by Lonomia sp caterpillars initially experience a mild burning pain, headache, nausea, vomiting, and skin and mucosal hemorrhages. Some patients can rapidly progress to a severe coagulopathy that presents as visceral or intracerebral hemorrhaging. We studied the hemostatic alterations that occurred in 14 patients who were envenomed by Lonomia obliqua in Southern Brazil and presented at the Hospital São Vicente de Paulo (Passo Fundo, RS), Brazil during the summers of 1993 and 1994 when Lonomia antivenom was not yet available for treatment. The patients were classified into to 4 clinical groups: 0 (two patients), I (eight patients), II (two patients), and III (two patients). The patients were admitted to the hospital between 4 hours and five days after contact with the caterpillars. In this study, the coagulation parameters of the patients were followed up for up to 172 hours after the accidents. The patients received no treatment with the exceptions of two patients who received blood transfusions and antifibrinolytic treatment. The observed abnormalities related to blood coagulation and fibrinolytic factors were similar regardless of the severity of the bleeding symptoms. These findings suggest that alterations in hemostatic parameters without thrombocytopenia are not predictors of the seriousness of such accidents. Thus, consumptive disorder and reactive fibrinolysis are not proportional to mild coagulopathy. Furthermore, these patients recovered. The hemostatic parameters of most of the patients normalized between 96 and 120 h after the accident.

Role of the inflammatory response in the hemorrhagic syndrome induced by the hemolymph of the caterpillar Lonomia achelous.

INTRODUCTION: Contact with the caterpillar of Lonomia achelous causes a hemorrhagic syndrome in humans prompted by two processes, an initial mild DIC that is later masked by overwhelming fibrinolytic activity. Although the venom affects both the hemostatic and inflammatory systems separately, it is not clear whether the hematological and hemostatic disturbances may in part be due to an indirect effect via inflammatory mediators. Here we report results on the crosstalk between these systems, particularly the effect of the pro-inflammatory cytokine TNF-α on hemostatic parameters. MATERIALS AND METHODS: the nitric oxide and TNF-α responses, as well as activation of the coagulation and fibrinolytic systems, were measured in macrophages and endothelial cells treated with Lonomia achelous hemolymph (LAH). The same responses were then determined, in a mouse model of LAH envenomation, after treatment with an anti-TNF-α antibody. RESULTS: Both macrophages and endothelial cells responded strongly to LAH in terms of pro-inflammatory mediator release and fibrinolytic activities as well as pro-coagulant activity (TF activity) in endothelial cells. Treatment with antibody against TNF-α decreased both TNF-α and NO3-/NO2- serum levels in the mice, after LAH injection. Blocking TNF-α also modified significantly the serum levels of plasminogen, fibrinogen and FXIII in mice, as well as decreased TF activity in endothelial cells. CONCLUSIONS: LAH may induce a hemostatic effect through endothelial and macrophage activation. These activated cell release hemostatic enzymes as well as pro-inflammatory mediators, principally TNF-α, that potentiate this release in an autocrine fashion, amplifying the fibrinolytic effect, which may in turn exacerbate the hemorrhagic manifestations. As far as we are aware, this is the first report of the relationship between the hemostatic system and the inflammatory responses in a hemorrhagic syndrome induce by animal secretions.

The characterization of trans-pecos copperhead (Agkistrodon contortrix pictigaster) venom and isolation of two new dimeric disintegrins.

The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead.

Biological and biochemical characterization of venom from the broad-banded copperhead (Agkistrodon contortrix laticinctus): isolation of two new dimeric disintegrins.

Disintegrins represent a family of effective cell-cell and cell-matrix inhibitors by binding to integrin receptors. Integrins are heterodimeric, transmembrane receptors that are the bridges for these cell interactions. Disintegrins have been shown to have many therapeutic implications for the treatment of strokes, heart attacks, and cancer. Two novel heterodimeric disintegrins were isolated from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus). Crude venom separated by cation-exchange chromatography resulted in several fractions possessing hemorrhagic, fibrinolytic, gelatinase, and platelet activities. Venom fractions 2-3 and 17-19 showed fibrinolytic activity. Fractions 2-6, 8-11, and 16-21 had hemorrhagic activity. Gelatinase activity was found in fractions 3, 11, and 19. The isolation of laticinstatins 1 and 2 was accomplished by fractionating crude venom using reverse phase chromatography. Data from both SDS-PAGE and N-terminal sequencing determined that laticinstatins 1 and 2 were heterodimeric disintegrins, and both were assayed for their ability to inhibit platelet aggregation in human whole blood. Future functional evaluation of snake venom disintegrins shows considerable promise for elucidating the biochemical mechanisms of integrin-ligand interactions that will allow the development of adequate medications for hemostatic pathologies such as thrombosis, stroke, and cerebral and cardiac accidents. In this study, we are presenting the first report of the purification, and partial characterization of two new dimeric disintegrins isolated from the venom of broad-banded copperhead snakes.

Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.

Generalidades del sistema de la coagulación y pruebas para su estudio / Overwiew of the Coagulation System and laboratory tests for its study

En la década de los años sesenta, se describió la cascada de la coagulación como una secuencia de eventos enzimáticos iniciada por dos vías, la intrínseca y la extrínseca, las cuales convergían en una vía común para generar una enzima multifuncional, denominada trombina. La principal función de esta enzima consistía en transformar el fibrinógeno, en fibrina, una proteína que se polimeriza espontáneamente para formar la base estructural del coágulo. Posteriormente, se propuso el Modelo Celular según el cual la coagulación no es la consecuencia de vías de activación enzimáticas secuenciales, sino de una red de interacciones entre proteínas plasmáticas y transmembranas, así como, varios tipos celulares, que permiten la formación de complejos enzimáticos altamente eficientes con la finalidad de generar trombina. Esta revisión explica en detalle ambos enfoques, además, aborda las diferentes funciones que cumple la trombina dentro de la hemostasia y los mecanismos de inhibición que regulan la coagulación. Finalmente, se describen diferentes pruebas empleadas en la actualidad para evaluar la funcionalidad del sistema de coagulación, como: el tiempo de tromboplastina parcial activado, el tiempo de protrombina, el tiempo de trombina, el tiempo de reptilasa, el tiempo de coagulación por ecarina y el uso de sustratos cromogénicos para evaluar cada factor de la coagulación. Finalmente, dado a que la generación de trombina es clave dentro de la coagulación y a que el potencial de generar trombina puede indicar propensión a desarrollar eventos trombóticos o hemorrágicos, en este trabajo se presentan los métodos existentes para determinar la generación de trombina.

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

Caracterización bioquímica y biológica del veneno de la serpiente Neotropical Macagua (Bothrops Colombiensis) de la región de Barlovento, estado Miranda, Venezuela / Biological and biochemical characterization of the Neotropical Macagua (Bothrops colombiensis) snake venom from Barlovento region, Miranda state, Venezuela

Introducción: el veneno de B. colombiensis no es solamente un elemento tóxico; en su composición existen múltiples componentes, que tienen un gran potencial terapéutico, principalmente en el tratamiento de patologías de la trombosis y la coagulación. Objetivos: estudiar una mezcla de venenos de Bothrops colombiensis de una ubicacion geográfica de Venezuela, a fin de hacer un barrido de sus actividades hemostáticas, que permitirá posteriormente purificar y caracterizar moléculas con actividad antitrombótica y anticoagulantes, entre otras, con potencial terapéutico. Métodos: el veneno a estudiar, es una mezcla de ellos obtenidos de serpientes provenientes de la Región de Barlovento, estado Miranda, Venezuela. Se caracterizó bioquímicamente por cromatografias de exclusión molecular, cromatografía de fase reversa C18 y por electroforesis a través de SDS­PAGE; y biológicamente por medio de actividades relacionadas con la hemostasia. Se analizaron los perfiles en relación a las actividades fibrinolítica, proteolítica sobre polvo azul y cadena ß de insulina, procoagulante, hemorrágica y letal. Resultados: la actividad hemorrágica, definida como la Dosis Hemorrágica Mínima fue de 8,7 mg/kg. La letalidad, definida como la Dosis Letal cincuenta fue 8,7 mg/kg. El veneno presentó actividad procoagulante y fibrinolítica. Las fracciones mostraron actividad fibrinolítica y proteolítica sobre polvo azul de ocultamiento y sobre la cadena ß de insulina. Conclusiones: las características biológicas de los componentes de este veneno le confieren un enorme potencial terapéutico, ya que contiene una alta actividad fibrinolítica y anticoagulante. Estos compuestos una vez purificados y caracterizados podrían explorarse como coadyuvantes en procesos trombolíticos, dado que disuelven coágulos de fibrina y degradan fibrinógeno, evitando episodios de retrombosis(AU)

Introduction: This paper is a screening of multiple toxic activities, of which some will be potentially useful for the management of coagulation pathologies. Objetives: A pool of Bothrops colombiensis venoms from a specific geographical location was studied, in order to carry out a hemostatic activities screening, allowing then to purify and characterise molecules with antithrombotic and anticoagulant activity, among others, which could have therapeutic potential. Methods: The venom was chromatographically by molecular exclusion and reverse phase C18 and SDS -PAGE characterized; its hemostatic activity was also established. Snakes were from the region of Barlovento, Miranda state, Venezuela. Profiles of fibrinolytic, proteolytic, procoagulant, hemorrhagic and lethal activities were analyzed. Hemorrhagic activity was 8.7 mg/kg. The LD50 was 8.7 mg/kg. The venom showed strongly procoagulant activity. Both, crude venom as fractions showed high fibrinolytic activity. The majority of the eluted fractions showed significant proteolytic activity in azure blue powder and on ß chain of insulin. Conclusions: The biological characteristics of the components of this venom confer enormous therapeutic potential because they contain a high fibrinolytic and anticoagulant activity. Most of these proteinases, once purified and characterized, could be explored as thrombolytic agents given that dissolves fibrin clots or prevent their formation(AU)

Overwiew of the Coagulation System and laboratory tests for its study. / Generalidades del sistema de la coagulación y pruebas para su estudio.

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

Biochemical and biological characterisation of lancehead (Bothrops venezuelensis Sandner 1952) snake venom from the Venezuelan Central Coastal range / Caracterización bioquímica y biológica del veneno de la serpiente "tigra mariposa" (Bothrops venezuelensis Sandner 1952) de la región central de la Cordillera de la Costa Venezolana

Se aislaron fracciones del veneno de Bothrops venezuelensis que demuestran ser un espectro abundante de proteínas con actividades variadas (coagulante, hemorrágica, fibrinolítica, proteolítica y de función plaquetaria), para el análisis de sus propiedades físico-químicas y biológicas, el veneno fue fraccionado por cromatografía de exclusión molecular, corrido en una electroforesis en gel y realizada una batería de ensayos biológicos. La DL50 del veneno de B. venezuelensis fue 6,39 mg/kg de peso corporal, fue determinada inyectando intraperitonealmente en ratones, diluciones seriadas de veneno de B. venezuelensis. Se colectaron doce fracciones a partir del veneno de B. venezuelensis mediante cromatografía de exclusión molecular. Las fracciones 1-5 y 7-9 tenían actividad hemorrágica. Todas las fracciones, con la excepción de las fracciones 3 y 6, tenían actividad fibrinolítica. Ninguna de las fracciones tuvo actividad de gelatinasa significativa, y sólo fracciones 4-6 demostraron actividad en polvo azul de ocultamiento. Con la excepción de las fracciones 1 y 4 , todas hidrolizaron la cadena β de la insulina. Cada fracción del veneno, así como el veneno crudo mostraron actividad procoagulante, cuando se probó en un analizador Sonoclot. Las fracciones 1, 3 , 5 y 9 inhibieron la función plaquetaria. En este estudio se señalan actividades biológicas de un veneno poco estudiado (B. venezuelensis) y sus fracciones. Al detectar actividades hemorrágicas, fibrinolíticas, procoagulantes, proteolíticas y de inhibición de la función plaquetaria. Este estudio preliminar abre el camino para la identificación de moléculas específicas que podrían tener potencial terapéutico en hemostasia y cáncer, que vienen siendo estudiados en nuestro grupo.

Venom fractions isolated from Bothrops venezuelensis were shown to contain a broad spectrum of proteins with varied activities. This study describes venom fractions with coagulant, haemorrhagic, fibrinolytic, proteolytic and antiplatelet activities, and analyses their physico-chemical properties and biological activities via molecular exclusion chromatography, gel electrophoresis and a bioassay battery. The LD50, determined by injecting intraperitoneally serial dilutions of B. venezuelensis venom into mice, was 6.39 mg/kg body weight. Twelve fractions were collected from B. venezuelensis venom using molecular exclusion chromatography. Of these, fractions 1-5 and 7-9 showed haemorrhagic activity, and all fractions except 3 and 6 showed fibrinolytic activity. However, none of the fractions had significant gelatinase activity, and only fractions 4-6 demonstrated activity on hide powder azure. With the exception of fractions 1 and 4, all fractions hydrolysed the insulin B-chain. In addition, all fractions as well as the crude venom showed strong procoagulant activity when tested using a Sonoclot Analyzer. Fractions 1, 3, 5 and 9 inhibited platelet function. In this study we have described the activities of the crude venom and its size-fractions from the scarcely studied B. venezuelensis. Haemorrhagic, fibrinolytic, procoagulant and proteolytic activities, and the inhibition of platelet function were detected. This preliminary study paves the way for the identification of specific molecules in B. venezuelensis venom that could have therapeutic potential for cancer and aberrant haemostasis treatment.

[Hemostasis alterations in sickle cell syndrome]. / Alteraciones de la hemostasia en el síndrome drepanocítico.

Sickle cell syndrome (SCS) includes a group of congenital hemolytic anemias associated to the presence of hemoglobin S, which is characterized by acute pain episodes and progressive damage of different organs. Some patients with sickle cell syndrome have shown, when compared with healthy individuals, an increased risk of presenting stroke, pulmonary hypertension, avascular necrosis of joints, acute chest syndrome and pregnancy complications, associated to a hypercoagulable state induced by alterations in different components of hemostasis, such as changes that include activation of the endothelium, platelet activity, coagulation and fibrinolytic systems. This paper compiles hemostasis disorders, associated with thrombotic manifestations, reported until now in sickle cell syndrom. These patients have an increase in activation markers of the coagulation system, such as prothrombin fragment 1.2, thrombin-antithrombin complex, etc., depletion of natural anticoagulant proteins, abnormal activation of the fibrinolytic system and increased tissue factor expression. Similarly, abnormal expression of glycoproteins and increased adhesion and platelet aggregation have been reported. All these alterations produce a hypercoagulable state, which induces, among other things, the appearance of thrombotic complications. In view of the importance of controlling the different complications that can occur in patients with sickle cell syndrome, we recommend the implementation, in diagnosis and monitoring studies, of the evaluation of the different components of the hemostatic system, identifying alterations at an early stage and applying effective treatments to prevent thrombotic complications.

Alteraciones de la hemostasia en el síndrome drepanocítico / Hemostasis alterations in sickle cell syndrome

El síndrome drepanocítico (SD) comprende un grupo de anemias hemolíticas hereditarias de tipo multisistémico asociadas a la hemoglobina S. Los pacientes que padecen este síndrome tienen un mayor riesgo, en comparación con individuos sanos, de presentar accidentes cerebrovasculares, hipertensión pulmonar, necrosis avascular de articulaciones, síndrome torácico agudo y complicaciones durante el embarazo, asociados a un estado de hipercoagulabilidad inducido por alteraciones en los diferentes componentes de la hemostasia, que incluyen la activación del endotelio y de los sistemas plaquetario, de la coagulación y de la fibrinólisis. Esta revisión resume las alteraciones en la hemostasia reportadas en los pacientes con SD, en los cuales se ha demostrado: mayor interacción de células endoteliales con leucocitos, hematíes y plaquetas; aumento de la expresión de proteínas de adhesión, como el factor von Willebrand y sus multímeros de alto peso molecular; aumento de la adhesión y la agregación plaquetaria y de la expresión de proteínas en sus membranas. En el sistema de coagulación se ha detectado aumento en la expresión del factor tisular (FT) en micropartículas derivadas de diferentes células, aumento de marcadores de activación de este sistema, entre estos los fragmentos 1.2 de la protrombina y los complejos trombina-antitrombina y una disminución de las proteínas C y S que actúan como anti-coagulantes. Adicionalmente, se han encontrado aumentados los marcadores de activación del sistema fibrinolítico como los dímeros D y los complejos plasmina/antiplasmina. Todas estas manifestaciones favorecen la aparición de complicaciones trombóticas, implicadas en el deterioro de la calidad de vida de los pacientes. Se recomienda implementar en el diagnóstico y seguimiento de esta enfermedad, la determinación de variables del sistema hemostático, con el fin de identificar alteraciones en etapas tempranas y aplicar terapias que puedan prevenir complicaciones trombóticas.

Sickle cell syndrome (SCS) includes a group of congenital hemolytic anemias associated to the presence of hemoglobin S, which is characterized by acute pain episodes and progressive damage of different organs. Some patients with sickle cell syndrome have shown, when compared with healthy individuals, an increased risk of presenting stroke, pulmonary hypertension, avascular necrosis of joints, acute chest syndrome and pregnancy complications, associated to a hypercoagulable state induced by alterations in different components of hemostasis, such as changes that include activation of the endothelium, platelet activity, coagulation and fibrinolytic systems. This paper compiles hemostasis disorders, associated with thrombotic manifestations, reported until now in sickle cell syndrom. These patients have an increase in activation markers of the coagulation system, such as prothrombin fragment 1.2, thrombin-antithrombin complex, etc., depletion of natural anticoagulant proteins, abnormal activation of the fibrinolytic system and increased tissue factor expression. Similarly, abnormal expression of glycoproteins and increased adhesion and platelet aggregation have been reported. All these alterations produce a hypercoagulable state, which induces, among other things, the appearance of thrombotic complications. In view of the importance of controlling the different complications that can occur in patients with sickle cell syndrome, we recommend the implementation, in diagnosis and monitoring studies, of the evaluation of the different components of the hemostatic system, identifying alterations at an early stage and applying effective treatments to prevent thrombotic complications.

Coral snake antivenom produced in chickens (Gallus domesticus).

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze-thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus) / Antiveneno de serpiente coral producido en gallinas (Gallus domesticus)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

La producción de antiveneno de serpiente usando sangre de grandes mamíferos se ha encontrado que es de bajo rendimiento y de trabajo arduo, en consecuencia, las inmunoglobulinas antiveneno para el tratamiento se obtienen generalmente, como suero polivalente. Hemos estandarizado una técnica poco exigente para la fabricación de inmunoglobulina purificada IgY, que consistió en generar anticuerpos policlonales contra el veneno de la serpiente coral en huevos de gallinas inmunizadas. La técnica consistió en la eliminación de lípidos de las yemas del huevo, diluidas en agua y en una secuencia de congelación-descongelación. Las inmunoglobulinas específicas estuvieron presentes en la yema de huevo hasta 180 días después de la inmunización. La unión del antígeno a las inmunoglobulinas se detectó mediante un ensayo de inmunodifusión doble. Los títulos de anticuerpos en la yema fueron estimados con un ensayo de protección (dosis efectiva media = ED50). Dado que las gallinas reproductoras son económicamente viables, la recolección de huevos es no invasiva y la purificación de anticuerpos IgY es rápida y fácil, la inmunización de la gallina es una excelente alternativa para la producción de anticuerpos policlonales. A nuestro entender, esta es el primer anti-veneno contra serpiente de coral preparado en aves.

Biochemical and biological characterisation of lancehead (Bothrops venezuelensis Sandner 1952) snake venom from the Venezuelan Central Coastal range.

Venom fractions isolated from Bothrops venezuelensis were shown to contain a broad spectrum of proteins with varied activities. This study describes venom fractions with coagulant, haemorrhagic, fibrinolytic, proteolytic and antiplatelet activities, and analyses their physico-chemical properties and biological activities via molecular exclusion chromatography, gel electrophoresis and a bioassay battery. The LD50, determined by injecting intraperitoneally serial dilutions of B. venezuelensis venom into mice, was 6.39 mg/kg body weight. Twelve fractions were collected from B. venezuelensis venom using molecular exclusion chromatography. Of these, fractions 1-5 and 7-9 showed haemorrhagic activity, and all fractions except 3 and 6 showed fibrinolytic activity. However, none of the fractions had significant gelatinase activity, and only fractions 4-6 demonstrated activity on hide powder azure. With the exception of fractions 1 and 4, all fractions hydrolysed the insulin B-chain. In addition, all fractions as well as the crude venom showed strong procoagulant activity when tested using a Sonoclot Analyzer. Fractions 1, 3, 5 and 9 inhibited platelet function. In this study we have described the activities of the crude venom and its size-fractions from the scarcely studied B. venezuelensis. Haemorrhagic, fibrinolytic, procoagulant and proteolytic activities, and the inhibition of platelet function were detected. This preliminary study paves the way for the identification of specific molecules in B. venezuelensis venom that could have therapeutic potential for cancer and aberrant haemostasis treatment.

Se aislaron fracciones del veneno de Bothrops venezuelensis que demuestran ser un espectro abundante de proteínas con actividades variadas (coagulante, hemorrágica, fibrinolítica, proteolítica y de función plaquetaria), para el análisis de sus propiedades físico-químicas y biológicas, el veneno fue fraccionado por cromatografía de exclusión molecular, corrido en una electroforesis en gel y realizada una batería de ensayos biológicos. La DL50 del veneno de B. venezuelensis fue 6,39 mg/kg de peso corporal, fue determinada inyectando intraperitonealmente en ratones, diluciones seriadas de veneno de B. venezuelensis. Se colectaron doce fracciones a partir del veneno de B. venezuelensis mediante cromatografía de exclusión molecular. Las fracciones 1­5 y 7­9 tenían actividad hemorrágica. Todas las fracciones, con la excepción de las fracciones 3 y 6, tenían actividad fibrinolítica. Ninguna de las fracciones tuvo actividad de gelatinasa significativa, y sólo fracciones 4­6 demostraron actividad en polvo azul de ocultamiento. Con la excepción de las fracciones 1 y 4, todas hidrolizaron la cadena ß de la insulina. Cada fracción del veneno, así como el veneno crudo mostraron actividad procoagulante, cuando se probó en un analizador Sonoclot. Las fracciones 1, 3, 5 y 9 inhibieron la función plaquetaria. En este estudio se señalan actividades biológicas de un veneno poco estudiado (B. venezuelensis) y sus fracciones. Al detectar actividades hemorrágicas, fibrinolíticas, procoagulantes, proteolíticas y de inhibición de la función plaquetaria. Este estudio preliminar abre el camino para la identificación de moléculas específicas que podrían tener potencial terapéutico en hemostasia y cáncer, que vienen siendo estudiados en nuestro grupo.