Thesis (Ph. D.)--Duke University, 2008.

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (M.S.)--University of North Carolina Wilmington, 2009. / Title from PDF title page (February 23, 2010) Includes bibliographical references (p. 55-61)

Thesis (Ph. D.)--University of Virginia, 2002. / Includes bibliographical references (leaves 160-197). Also available online through Digital Dissertations.

Orientador: Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T23:37:17Z (GMT). No. of bitstreams: 1 BaldisseraJunior_Lineu_M.pdf: 826113 bytes, checksum: a2244710b71a5e86c7de35e81492c539 (MD5) Previous issue date: 2009 / Resumo: Há relatos de que as plaquetas desempenham função importante no desencadeamento da resposta inflamatória alérgica, como a asma brônquica. Entretanto, existem poucos estudos funcionais avaliando a interação plaqueta-asma. O objetivo deste trabalho foi investigar a função plaquetária em modelo de asma alérgica em ratos. Para tanto, ratos Wistar machos (180-200 g) foram sensibilizados à ovalbumina (OVA) e desafiados intranasalmente uma única vez, quatorze dias após a sensibilização. Para confirmar a eficácia da sensibilização à OVA, realizou-se a dosagem de IgE sérica e contagem de leucócitos no lavado broncoalveolar (LBA) após o desafio antigênico. O comportamento plaquetário foi avaliado através da contagem de plaquetas no sangue periférico entre 30 minutos a 24 horas após o desafio. As plaquetas foram isoladas em tempos variados após o desafio com OVA, a saber: 30 minutos, 2, 8 e 24 horas. Em seguida, realizou-se ensaios de adesão plaquetária em microplaca de 96 poços recoberta com fibrinogênio e ensaios de agregação plaquetária. Nossos resultados mostraram que a sensibilização e o desafio antigênico resultaram em aumento significativo dos níveis de IgE e infiltrado eosinofílico no LBA, validando o modelo alérgico adotado no estudo. Observou-se redução significativa do número de plaquetas circulantes em 30 minutos após o desafio com OVA, atingindo redução máxima em 8 horas, retornando aos valores basais 24 horas após o desafio. A adesão plaquetária ao fibrinogênio imobilizado e agregação plaquetária foram realizadas nos tempos de 30 min, 2, 8 e 24 horas após o desafio com OVA. A adesão plaquetária espontânea não foi modificada em nenhum dos tempos estudados. A adesão plaquetária induzida com ADP (5-50 µM) ou trombina (30-100 mU/mL) mostrou-se significativamente elevada em 30 minutos, e diminuída em 24 horas após o desafio com OVA. O desafio antigênico não modificou o perfil de agregação plaquetária, exceto para as plaquetas ativadas com ADP (50 µM) e/ou trombina (200 mU/mL), em 24 horas após o desafio. Com o intuito de se verificar se a via de sinalização de cálcio estaria envolvida nas mudanças observadas na adesão plaquetária, realizamos ensaios de mobilização de cálcio em plaquetas nos tempos de 30 min e 24 horas. A mobilização dos estoques internos de cálcio induzidos por ADP (20 µM) ou trombina (100 mU/mL) foram significativamente maiores em 30 minutos, e menores 24 horas após desafio antigênico à OVA. O influxo de cálcio externo foi significativamente maior em plaquetas de ratos estimuladas com ADP (20 µM) em 30 minutos após desafio com OVA. Após 24 horas, houve tendência de redução do influxo de cálcio externo em plaquetas de animais sensibilizados. Em conjunto, nossos dados mostram que o desafio intranasal com OVA acarreta, na fase imediata (30 minutos), aumento de adesão plaquetária ao fibrinogênio e elevação do transporte interno e externo de cálcio. Na fase tardia (24 horas), observamos redução da adesão plaquetária e diminuição dos estoques internos de cálcio / Abstract: Evidences show that platelets play important roles in triggering the allergic inflammatory diseases, such as bronchial asthma. However, there are few functional studies attempting to evaluate the platelet-asthma interaction. The aim of this work was to investigate platelet function in a rat model of allergic asthma. Male Wistar rats (180-200 g) were sensitized with ovalbumin (OVA) and challenged intranasally fourteen days after sensitization. In order to assess the efficacy of OVA sensitization, serum IgE levels and leukocyte counts in bronchoalveolar lavage (BAL) fluid was performed post-antigen challenge. Counts of platelets in peripheral blood were performed from 30 minutes to 24 hours post-OVA challenge. Platelets were isolated in different time-periods after OVA challenge, namely: 30 minutes, 2, 8, 24 hours; then, ex-vivo platelet adhesion to immobilized fibrinogen and aggregation assays were performed. Our results showed that antigen-sensitization and challenge resulted in increased serum levels of IgE and eosinophil infiltration in BAL fluid, thus validating the allergic model adopted in this work. A significant reduction of circulating platelet was observed at 30 minutes post OVA-challenge, reaching the maximal reduction at 8 hours, and returning to baseline at 24 hours post-challenge. Platelet adhesion to immobilized fibrinogen and aggregation were performed at 30 minutes, 2, 8 and 24 hours after OVA challenge. Spontaneous platelet adhesion remained unaffected in all studied time. ADP (5-50 µM)- and thrombin (50-100 mU/mL)-stimulated platelet adhesion were significantly increased at 30 minutes, and decreased 24 hours after OVA challenge. The antigen challenge did not modify the platelet aggregation profile, except in platelets activated with 50 µM ADP and 200 mU/mL thrombin, at 24 hours post challenge. In order to verify whether calcium signaling pathway would be involved in the changes observed in platelet adhesion, we performed Ca2+ mobilization assays at 30 minutes and 24 hours. Internal calcium stores mobilization induced by ADP (20 µM) and thrombin (100 mU/mL) were significantly enhanced at 30 minutes, and decreased 24 hours after OVA-challenge. The extracellular calcium influx was significantly increased in ADP (20 µM)-stimulated rat platelets at 30 minutes post OVA-challenge. After 24 hours, the platelet calcium influx was lesser than that in nonsensitized animals. Together, our results show that intranasal OVA challenge causes an increased platelet adhesion at an early time post OVA-challenge (30 minutes) concomitant with elevated Ca2+ internal and/or external transport. At late phase (24 hours), we observed reduced platelet adhesion and decreased Ca2+ internal storage / Mestrado / Farmacologia / Mestre em Farmacologia

Human platelets respond to various macromolecules in the plasma. Uptake of specific ligands, and antibodies to various epitopes on the platelet plasma membrane, has been observed. The platelet canalicular system has been shown to be involved with this uptake. Recently, investigators have speculated on the role of endocytosis in platelets to account for the presence of plasma proteins such as fibrinogen and immunoglobulin within platelet organelles. Antibodies binding to cell-surface antigens on platelets can lead to a redistribution of these antigens. When antibodies, specific for platelet cell-surface receptors, bind to platelets they may either undergo endocytosis into intracellular vacuoles, or may merely become sequestered within the canalicular system of platelets. The present study investigated whether endocytosis occurs in platelets. Such a process would lead to the endocytic uptake of a fluid-phase marker and would involve internalization and recycling of cell surface membrane. A fluid-phase marker (FITC-dextran) was used to measure any constitutive endocytic activity. In addition, a suitable membrane marker was used to determine whether membrane internalization occurred. This involved a technique whereby radioactive galactose was covalently attached to cell-surface glycoconjugates. A monoclonal antibody to the platelet receptor, GPIIbIIIa, was used in conjunction with the membrane marker in order to determine if membrane internalization was involved during the subsequent redistribution of the receptor-antibody complex. Immunocytochemical techniques using electron-dense probes were employed to localise the sites to which this receptor-antibody complex became redistributed. In comparison with reported rates of endocytic uptake of fluid-phase marker in other cell types, no significant endocytic activity could be detected with platelets, after taking their relatively small volume into account. Similarly, membrane internalization was not detected with resting platelets. Following challenge of the platelets with anti-GPIIbIIIa antibody, no membrane internalization could be measured during redistribution of the receptor-antibody complex. The compartment to which the receptor-antibody complex was redistributed could be identified morphologically as the canalicular system. The present data provide evidence for a process of sequestration of receptor-antibody in the canalicular system of resting platelets. It remains possible that other mechanisms exist within the platelet system for uptake of extracellular material as this study dealt exclusively with the platelet response to a specific antibody. These results may have implications with respect to the interaction of platelets with anti-platelet antibodies in the normal state, as well as with clinical disorders involving elevated levels of platelet-associated IgG. As far as can be deduced from the available literature, these data represent the first use of a covalent membrane marker in conjunction with uptake of macromolecules to study endocytic events in human platelets.

Eukaryotic platelets are small cell fragments that are released into the bloodstream from megakaryocytes, and their production is initiated in the bone marrow. They are mainly involved in blood hemostasis and thrombus formation. The newly synthesized platelets are called reticulated platelets or young platelets. Zebrafish thrombocytes are equivalent to mammalian platelets and have similar characteristics and functions. Likewise, zebrafish has both young and mature thrombocytes. Only young thrombocytes as reticulated platelets are labeled with thiazole orange. Similarly, labeling zebrafish thrombocytes with a specific concentration of DiI-C18 showed two populations of thrombocytes (DiI+ and DiI-). Again, only young thrombocytes showed DiI+ labeling. The mechanism of selective labeling of young thrombocytes by is unknown. Furthermore, there is no zebrafish line where young and mature thrombocytes are differentially labeled with fluorescence proteins. Therefore, in this study, we identified and confirmed that the RFP labeled cells of Glofish were young thrombocytes. In addition, we found that myosin light chain 2 (MLC2) promoter is expressed in young thrombocytes. We also generated a transgenic zebrafish line, GloFli fish, where the young and mature thrombocytes are labeled with red and green fluorescence proteins respectively. Furthermore, this study showed a two-fold increase in glycerol-phospholipids (GP) in mature thrombocytes compared to young thrombocytes suggesting the lipid composition may be important for differential labeling. Therefore, we tested the liposomes prepared with different ratios of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and observed that the lower amounts of PE favor the DiI-C18 labeling whereas higher concentrations of PC are less efficient. Also, in both PE and PC, increased concentrations of both resulted in decreased binding. These results are consistent with our observation that mature thrombocytes have higher concentrations GP and thus DiI-C18 may not bind to them efficiently compared to young thrombocytes.

Thesis (M.S.)--University of Michigan, 1995. / Thesis date on spine.

Thesis (M.S.)--University of Michigan, 1995. / Thesis date on spine.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.