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Caracterização de enzimas do veneno de Physalia physalis: estudo bioquímico e ação na coagulação ex vivo
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Abstract(s)
A Physalia physalis, comumente conhecida como Caravela Portuguesa, é um hidrozoário colonial flutuante encontrado em ampla distribuição pelo Oceano Atlântico. A sua ocorrência é frequentemente observada no arquipélago dos Açores, ocorrendo sazonalmente em grandes aglomerados de maio a agosto. O contato com suas células urticantes resulta no envenenamento agudo que pode provocar inflamação e perturbações na hemostasia, afetando a agregação plaquetária e a coagulação. Embora em outras águas-vivas esses sintomas tenham sido associados à presença de enzimas proteolíticas, pouco se sabe sobre o veneno da P. physalis. Este estudo tem como objetivo analisar a ação do veneno da P. physalis na hemostasia, centrando-se na investigação das enzimas presentes no veneno e o seu mecanismo de ação sobre a coagulação.
O veneno da P. physalis foi obtido por meio de cinco métodos distintos. A extração através da homogeneização dos tentáculos com “ultraturrax” (Vh) e a indução das células urticantes (nematocistos) por electroestimulação (VT), foram os métodos que resultaram num extrato de veneno com maior atividade enzimática. Os venenos obtidos por esses dois métodos apresentaram diferentes padrões proteicos, pelo que foram ambos utilizados no estudo das enzimas e suas ações na coagulação.
A análise com substratos e inibidores específicos revelou pela primeira vez que o veneno de P. physalis exibe atividade proteolítica, do tipo serino protéase, especificamente quimotripsina, e serino metaloproteases. O veneno obtido pelos métodos Vh e VT exibiu atividades distintas sobre o fibrinogénio e o coágulo de fibrina, com impacto reduzido na cascata de coagulação. O veneno obtido por indução dos nematocistos (VT) demonstrou elevada eficiência na digestão do coágulo de fibrina, apresentando simultaneamente atividade fibrinolítica e fibrinogenolítica. Por outro lado, o veneno obtido por homogeneização dos tentáculos (Vh) não apresentou atividade fibrinolítica, mas exibiu uma hidrólise altamente específica do fibrinogénio, gerando péptidos de fibrina com aproximadamente 23 kDa, semelhantes aos formados pela ação da trombina. Estes péptidos de fibrina demonstraram a capacidade de formar um coágulo instantâneo, sugerindo um efeito pró-coagulante. A atividade pró-coagulante da amostra Vh e a ação trombolítica da amostra VT foram confirmadas nos ensaios de lise do coágulo de euglobulina em plasma humano. Nenhuma das amostras testadas apresentou atividade hemolítica.
O fracionamento da amostra Vh, por FPLC, resultou em quatro frações com padrões proteicos e enzimáticos distintos. Duas dessas frações (#2 e #3) apresentaram ação pró-coagulante, enquanto as outras duas frações (#1 e #4) apresentaram ação fibrinogenolítica e trombolítica. A caracterização da atividade enzimática das frações obtidas por cromatografia permitiu determinar que a atividade trombolítica é promovida por metaloproteases, enquanto o efeito pró-coagulante é mediado por serino protéases. Este estudo permitiu identificar, pela primeira vez, proteínas do veneno que podem estar envolvidas nos distúrbios hemostáticos resultantes do envenenamento agudo por P. physalis e que por este motivo podem constituir importantes fatores de toxicidade. O seu estudo pode contribuir para o desenvolvimento futuro de antídotos ou para o desenvolvimento de novas moléculas para terapias anti trombóticas.
ABSTRACT: The Physalia physalis, commonly known as the Portuguese Man-o'-War, is a floating colonial hydrozoan found widely across the Atlantic Ocean. Its presence is often observed in the Azores archipelago, occurring seasonally in large clusters from May to August. The contact with its stinging cells results in acute poisoning that can cause inflammation and disruptions in hemostasis, affecting platelet aggregation and coagulation. While in other jellyfish these symptoms have been associated with the presence of proteolytic enzymes, little is known about the venom of P. physalis. This study aims to analyze the action of P. physalis venom on hemostasis, focusing on investigating the enzymes present in the venom and their mechanism of action on coagulation. In this study, the venom of P. physalis was obtained through five different methods (Vh, Fv, FS, VP, and VT). Extraction by homogenizing the tentacles with an “ultraturrax” (Vh) and inducing the stinging cells (nematocysts) through electrostimulation (VT) were the methods that resulted in venom extract with higher enzymatic activity. The venoms obtained by these two methods showed different protein patterns, prompting the selection of both for the study of enzymes and their actions on coagulation. Analysis with specific substrates and inhibitors revealed, for the first time, that the venom of P. physalis obtained exhibited proteolytic activity, specifically serine protease, including chymotrypsin and metalloproteases. The venom obtained from the methods Vh e VT showed different activities on the clot formation and fibrinolysis. The venom obtained by inducing nematocysts (VT) showed high efficiency in digesting the fibrin clot. The study of its mechanism of action revealed that this thrombolytic activity is promoted by metalloproteases. Additionally, it was found to hydrolyze fibrinogen into low molecular weight fibrinopeptides, inhibiting clot formation. On the other hand, the venom obtained by homogenizing the tentacles (Vh) did not digest the fibrin clot but demonstrated the ability to induce clot formation through a fibrinogen hydrolysis process mediated by serine proteases. Investigation of its mechanism of action on fibrinogen revealed a specific hydrolysis profile, generating fibrinopeptides of approximately 23 kDa, which were capable of forming a fibrin clot. The pro-coagulant effect of the Vh sample and the thrombolytic activity of the VT sample were confirmed in euglobulin clot lysis assays on human plasma. None of the venoms exhibited hemolytic activity. Fractionation of the Vh sample by FPLC chromatography resulted in four fractions with distinct protein and enzymatic patterns. Two of these fractions (#2 and #3) confirmed the pro-coagulant action, while the other two fractions (#1 and #4) demonstrated fibrinogenolytic and thrombolytic activity. The characterization of the enzymatic activity of the fractions obtained by chromatography allowed to determine that the thrombolytic activity is promoted by metalloproteases, while the pro-coagulant effect is mediated by serine proteases. This study allowed for the first-time identification of venom proteins that may be involved in the hemostatic disturbances resulting from acute P. physalis envenomation, that may constitute important toxicity factors. Their characterization could contribute to the future development of antidotes or the design of new molecules for anti-thrombotic therapies.
ABSTRACT: The Physalia physalis, commonly known as the Portuguese Man-o'-War, is a floating colonial hydrozoan found widely across the Atlantic Ocean. Its presence is often observed in the Azores archipelago, occurring seasonally in large clusters from May to August. The contact with its stinging cells results in acute poisoning that can cause inflammation and disruptions in hemostasis, affecting platelet aggregation and coagulation. While in other jellyfish these symptoms have been associated with the presence of proteolytic enzymes, little is known about the venom of P. physalis. This study aims to analyze the action of P. physalis venom on hemostasis, focusing on investigating the enzymes present in the venom and their mechanism of action on coagulation. In this study, the venom of P. physalis was obtained through five different methods (Vh, Fv, FS, VP, and VT). Extraction by homogenizing the tentacles with an “ultraturrax” (Vh) and inducing the stinging cells (nematocysts) through electrostimulation (VT) were the methods that resulted in venom extract with higher enzymatic activity. The venoms obtained by these two methods showed different protein patterns, prompting the selection of both for the study of enzymes and their actions on coagulation. Analysis with specific substrates and inhibitors revealed, for the first time, that the venom of P. physalis obtained exhibited proteolytic activity, specifically serine protease, including chymotrypsin and metalloproteases. The venom obtained from the methods Vh e VT showed different activities on the clot formation and fibrinolysis. The venom obtained by inducing nematocysts (VT) showed high efficiency in digesting the fibrin clot. The study of its mechanism of action revealed that this thrombolytic activity is promoted by metalloproteases. Additionally, it was found to hydrolyze fibrinogen into low molecular weight fibrinopeptides, inhibiting clot formation. On the other hand, the venom obtained by homogenizing the tentacles (Vh) did not digest the fibrin clot but demonstrated the ability to induce clot formation through a fibrinogen hydrolysis process mediated by serine proteases. Investigation of its mechanism of action on fibrinogen revealed a specific hydrolysis profile, generating fibrinopeptides of approximately 23 kDa, which were capable of forming a fibrin clot. The pro-coagulant effect of the Vh sample and the thrombolytic activity of the VT sample were confirmed in euglobulin clot lysis assays on human plasma. None of the venoms exhibited hemolytic activity. Fractionation of the Vh sample by FPLC chromatography resulted in four fractions with distinct protein and enzymatic patterns. Two of these fractions (#2 and #3) confirmed the pro-coagulant action, while the other two fractions (#1 and #4) demonstrated fibrinogenolytic and thrombolytic activity. The characterization of the enzymatic activity of the fractions obtained by chromatography allowed to determine that the thrombolytic activity is promoted by metalloproteases, while the pro-coagulant effect is mediated by serine proteases. This study allowed for the first-time identification of venom proteins that may be involved in the hemostatic disturbances resulting from acute P. physalis envenomation, that may constitute important toxicity factors. Their characterization could contribute to the future development of antidotes or the design of new molecules for anti-thrombotic therapies.
Description
Dissertação de Mestrado, Ciências Biomédicas, 16 de setembro de 2024, Universidade dos Açores.
Keywords
Physalia physalis Caravela Portuguesa veneno serino protease e metaloproteases coagulação e hemostasia
Pedagogical Context
Citation
Borges, Margarida Botelho. (2025). "Caracterização de enzimas do veneno de Physalia physalis: estudo bioquímico e ação na coagulação ex vivo". 49 p. (Dissertação de Mestrado em Ciências Biomédicas). Ponta Delgada: Universidade dos Açores, 2025. Disponível em http://hdl.handle.net/10400.3/8675