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2.
Toxicon ; 56(6): 1059-65, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20674587

RESUMEN

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Bothrops/fisiología , Venenos de Crotálidos/enzimología , Metaloendopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Reacciones Cruzadas , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/toxicidad , Edema/inducido químicamente , Edema/prevención & control , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Hemorragia/inducido químicamente , Hemorragia/prevención & control , Immunoblotting , Inmunoglobulinas , Inyecciones Intraperitoneales , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/toxicidad , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización
3.
Toxicon ; 56(6): 1059-1065, Jul 17, 2010.
Artículo en Inglés | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1068259

RESUMEN

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure–function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.


Asunto(s)
Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Antivenenos/inmunología , Bothrops/clasificación , Metaloproteasas/clasificación , Metaloproteasas/toxicidad , Venenos de Serpiente/inmunología , Anticuerpos Neutralizantes , Colubridae , Elapidae , Viperidae
4.
Toxicon ; 55(6): 1093-1099, Jan 28, 2010.
Artículo en Inglés | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1068251

RESUMEN

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported thatjararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to a2b1 integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to a2b1 integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344–421), showed a significant binding to recombinant a2b1 integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding ofjararhagin to collagen, but not to recombinant a2b1 integrin nor to cell-surface-exposed a2b1 integrin (a2-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and a2b1 integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.


Asunto(s)
Animales , /análisis , /inmunología , Venenos de Serpiente/enzimología , Venenos de Serpiente/inmunología , Anticuerpos/administración & dosificación , Anticuerpos/clasificación , Colágeno , Metaloproteasas
7.
Toxicon ; 51(4): 479-87, 2008 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-18262214

RESUMEN

Local and systemic hemorrhages are major problems concerning bites by viper snakes. Therefore, accessing venom hemorrhagic activity is an important feature in order to characterize viper venom major toxicities or to assay antivenom efficacy. The methods currently used to access hemorrhagic activity involve animal experiments and according to the general ethical committees, these procedures should be substituted to in vitro assays in order to minimize animal use in research. In this work, we have developed an immunoassay to detect the content of hemorrhagic metalloproteinases in snake venoms using a neutralizing monoclonal antibody anti-jararhagin (MAJar 3). The correlation between the reactivity of this monoclonal antibody and venom-induced hemorrhage was further revealed by a study comparing the hemorrhagic activity of venom samples collected individually from 88 specimens of Bothrops jararacussu with their reactivity with MAJar 3. As a result, a significant correlation (r=0.942) was achieved between samples hemorrhagic activity and their reactivity with MAJar 3, suggesting that this assay can be used as a substitute of the conventional tests performed in vivo to estimate the hemorrhagic activity.


Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/toxicidad , Ensayo de Inmunoadsorción Enzimática/métodos , Hemorragia/inducido químicamente , Metaloproteasas/análisis , Animales , Anticuerpos Monoclonales , Ratones , Proteínas/metabolismo , Conejos , Proteínas de Reptiles/metabolismo , Proteínas de Reptiles/toxicidad
8.
Biochimie ; 90(3): 484-92, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18096518

RESUMEN

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.


Asunto(s)
Colágeno/efectos de los fármacos , Venenos de Crotálidos/toxicidad , Metaloendopeptidasas/toxicidad , Secuencia de Aminoácidos , Animales , Sitios de Unión , Colágeno/química , Colágeno/metabolismo , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Veneno de Bothrops Jararaca
9.
Toxicon ; 51(1): 54-65, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17889921

RESUMEN

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.


Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/química , Metaloendopeptidasas/farmacología , Metaloproteasas/farmacología , Secuencia de Aminoácidos , Animales , Benchmarking , Factores de Coagulación Sanguínea , Células Cultivadas , Venenos de Crotálidos/farmacología , Células Endoteliales/efectos de los fármacos , Hemorragia/inducido químicamente , Humanos , Metaloendopeptidasas/química , Metaloproteasas/química , Ratones , Datos de Secuencia Molecular , Veneno de Bothrops Jararaca
13.
Curr Pharm Des ; 13(28): 2893-905, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17979734

RESUMEN

Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and play important roles in hemostatic disorders and local tissue damage that follows snakebite. The impact of SVMPs on hemostasis has been extensively studied showing diverse effects both on soluble factors and cellular components. The action of SVMPs involves catalytic and anti-adhesive properties, as well as direct cellular activation and/or the release of endogenous bioactive components. The purpose of this review is to overview the action of SVMPs on the inhibition of platelet functions; angiogenesis, particularly inducing apoptosis of endothelial cells; and regarding the pro-inflammatory reaction that follows snakebite. We discuss the structural features of the molecules that may be involved in such activities. The versatility and availability of SVMPs make them important tools for cell biology research into the mechanisms of action of endogenous metalloproteinases, for insights into cellular-matrix interactions and for clinical investigations into the treatment of snakebites.


Asunto(s)
Plaquetas/enzimología , Células Endoteliales/enzimología , Mediadores de Inflamación/fisiología , Metaloproteasas/fisiología , Venenos de Serpiente/enzimología , Animales , Plaquetas/patología , Células Endoteliales/patología , Humanos , Mediadores de Inflamación/química , Metaloproteasas/química , Venenos de Serpiente/química
15.
Apoptosis ; 10(4): 851-61, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16133875

RESUMEN

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40microg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.


Asunto(s)
Anoicis/efectos de los fármacos , Venenos de Crotálidos/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Metaloendopeptidasas/farmacología , Metaloproteasas/farmacología , Venenos de Serpiente/enzimología , Actinas/metabolismo , Animales , Bothrops , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Veneno de Bothrops Jararaca
20.
Toxicon ; 38(5): 633-44, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10673156

RESUMEN

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by affinity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG profile and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic antivenom of Instituto Butantan being more effective.


Asunto(s)
Especificidad de Anticuerpos/inmunología , Antivenenos/inmunología , Bothrops/inmunología , Venenos de Crotálidos/inmunología , Caballos/inmunología , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/inmunología , Animales , Antivenenos/uso terapéutico , Western Blotting , Brasil , Costa Rica , Reacciones Cruzadas/inmunología , Venenos de Crotálidos/envenenamiento , Ensayo de Inmunoadsorción Enzimática , Dosificación Letal Mediana , Masculino , Ratones , Pruebas de Neutralización
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