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Master's Dissertation
DOI
https://doi.org/10.11606/D.87.2018.tde-08022018-143046
Document
Author
Full name
Daniel Akio Fukuda
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2017
Supervisor
Committee
Magalhães, Geraldo Santana (President)
Ishii, Marina
Miyaji, Eliane Namie
Oliveira, João Ezequiel de
Title in Portuguese
Identificação da ligação direta de uma Fosfolipase D de Loxosceles gaucho às plaquetas.
Keywords in Portuguese
Loxosceles gaucho
Aranhas
Citometria de fluxo
EGFP
Fosfolipase D
Ligação
Microscopia de fluorescência
Plaquetas
Proteína verde fluorescente
Toxina quimérica
Toxinas
Abstract in Portuguese
Fosfolipases D (FLD) do veneno das aranhas do gênero Loxosceles são capazes de causar entre outros efeitos, uma forte agregação plaquetária cujo mecanismo ainda não foi elucidado. Portanto, para estudar o papel das FLDs nesta atividade, uma FLD recombinante de L. gaucho (LgRec1) foi fusionada com a proteína fluorescente verde (EGFP) e utilizada como uma sonda para detectar a interação de LgRec1 com plaquetas. Essa quimera, denominada EGFP-LgRec1, manteve as principais características da LgRec1. A microscopia confocal das plaquetas mostrou que LgRec1 não requer componentes plasmáticos para se ligar às plaquetas, embora estes sejam necessários para que a LgRec1 induza agregação. Além disso, foi observado que a ação da LgRec1 leva à exposição de fosfatidilserina. Contudo, esta exposição não está relacionada à morte celular. Portanto, este trabalho mostrou que uma FLD de Loxosceles se liga a plaquetas, promovendo a exposição de fosfatidilserina, possibilitando a ligação de fatores de coagulação e resultando na agregação plaquetária.
Title in English
Identification of direct binding of a Phospholipase D from Loxosceles gaucho to platelets.
Keywords in English
Loxosceles gaucho
Binding
Chimeric toxin
EGFP
Flow cytometry
Fluorescent microscopy
Green fluorecent protein
Phospholipase D
Platelets
Spiders
Toxins
Abstract in English
Phospholipases D (PLD) from spider venom of the genus Loxosceles are capable of causing, among other effects, a strong aggregation of platelets and its mechanism has not yet been elucidated. Therefore, to study the role of PLDs in this activity, a recombinant L. gaucho PLD (LgRec1) was fused with a green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 with platelets. This chimera, named EGFP-LgRec1, remained the main activities of LgRec1. Platelet confocal microscopy has shown that LgRec1 does not require plasma components to bind to platelets, although these are required for LgRec1 to induce aggregation. In addition, it has been observed that the action of LgRec1 leads to exposures of phosphatidylserine. However, this exposure is not related to cell death. Therefore, this work showed that a Loxosceles PLD binds to platelets, promoting an exposure of phosphatidylserine, that may act as a scaffold for coagulation factors, resulting in platelet aggregation.
 
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Publishing Date
2018-02-08
 
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