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

Orientador: Helena Zerlotti Wolf Grotto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T13:21:42Z (GMT). No. of bitstreams: 1 Noronha_JoseFernandodeAlmeida_D.pdf: 5060365 bytes, checksum: 9a433693a12ebc4fc3a72a0639d93c73 (MD5) Previous issue date: 2007 / Resumo: Introdução: Plaquetas reticuladas (PRs) são plaquetas jovens recentemente liberadas pela medula óssea para a circulação e que possuem alto conteúdo de RNA em seu citoplasma. As PRs são descritas como plaquetas grandes, densas e mais ativas no processo de formação do trombo hemostático. A participação das plaquetas ativadas nos fenÃ'menos vaso-oclusivos que acometem pacientes com síndrome falciformes (SF) já está documentada, mas a avaliação do papel das PRs não foi ainda descrito. Assim, avaliamos o número e a atividade das PRs, correlacionando-os com os níveis séricos de P-selectina solúvel (sCD62p), interleucinaâ?¿6 (IL-6), interleucina-3 (IL-3) e trombopoietina (TPO) em sangue periférico de pacientes em diferentes estágios clínicos das SF. Casuística e Métodos: Foram estudados 89 pacientes adultos, sendo 38 em fase â?¿estávelâ??, 27 em crise hemolítica (CH), 27 em crise vaso-oclusiva (CVO) e 30 indivíduos saudáveis (grupo controle - GC). Os parâmetros plaquetários, incluindo a quantificação de macroplaquetas foram determinados em analisador hematológico. Através da técnica da citometria de fluxo com o uso de anticorpos monoclonais e thiazole orange (TO), realizamos as identificações e quantificações das plaquetas ativadas (anti CD41a + anti CD62p), PRs (anti CD41a + TO) PRs ativadas (TO + anti CD62p). As dosagens de sCD62p, IL-6, IL-3 e TPO foram realizadas pela técnica de ELISA. Resultados: O número de macroplaquetas foi significativamente superior nas 3 fases clínicas das SF em relação ao GC. O número de plaquetas ativadas, tanto reticuladas como maduras foi superior em todas as fases clínicas quando comparadas ao GC. Os valores de PRs mostraram-se mais elevados no grupo de pacientes em CVO do que na fase â?¿estávelâ?? e na CH. Em todos os grupos as PRs apresentaram um maior grau de ativação quando comparadas às plaquetas maduras. Os pacientes em CVO apresentaram maiores níveis de sCD62p em relação ao GC. Nas diferentes fases das SF observamos níveis séricos elevados de IL-6, IL-3 e TPO, embora não tenha sido observada correlação entre essas determinações com os números absolutos de PRs, Plaquetas ativadas (CD62p+) e PRs ativadas (TO+/CD62p+). Conclusões: Os resultados sugerem a contribuição das PRs na trombogênese das Síndromes Falciformes. Níveis elevados das interleucinas provavelmente indicam a participação das mesmas no processo inflamatório que acompanha os fenÃ'menos de vaso-oclusão, mas aparentemente esses moduladores inflamatórios não exercem efeito sobre a trombopoiese em pacientes com SF / Abstract: Introduction: Reticulated Platelets (RPs) are youngest platelets released recently from bone marrow to the blood and are characterized by the high citoplasmatic RNA content. RPs are described as higher size, denser and more active in the formation of thrombus than mature platelets. The participation of activated platelets in vaso-occlusive process in sickle cell disease patients have been documented, but the evaluation of the RPs role has not been established at the moment. We evaluated the number and activity of RPs and correlated them with serum soluble P-selectin (sCD62p), Interleukin-6 (IL-6), Interleukin 3 (IL-3) and thrombopoetin (TPO) levels in patients with sickle cell diseases in different clinical expressions. Casuistic and Methods: Eighty-nine adult patients were studied: 38 in steady-state, 27 in hemolytic crisis (HC), 27 in vascular-occlusive crisis (CVO) and 30 healthy individuals (control groupâ?¿CG). Platelet parameters including the percentage of larger platelets were obtained by an automatic hematological analyzer. Monoclonal antibodies, thiazole orange (TO) dye and flow cytometric technique were used to identify and to quantify activated platelets (anti CD41a+ and anti CD62p+), RPs (anti CD41a+ and TO+) and activated RPs (TO+ and anti CD62p+). Soluble CD62p, IL-3, IL-6 and TPO determinations were measured by ELISA tests. Results: The number of macroplatelets was significantly higher in steady-state, CVO and HC groups than in CG. The number of activated mature platelets and activated RPs was higher in all stages of the disease when compared with CG. PRs values were more elevated in group of patients with CVO than in HC and steady-state. The degree of activation was higher in PRs than in mature platelets independently on sickle cell disease phase. CVO patients showed higher serum levels of sCD62p than CG. IL-6, IL-3 and TPO serum levels were increased in sickle cell disease, but there was not a correlation between those determinations and parameters related to platelets. Conclusions: Our results suggest that PRs contribute to the thrombogenesis process in sickle cell disease. Increased serum levels of interleukins probably indicate the participation of PRs in inflammatory process which is associated to vascular-occlusive phenomenon, but apparently those inflammatory mediators do not have an effect on thrombopoiese in sickle cell disease patients / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Orientador: Joyce Maria Annicchi-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T02:14:05Z (GMT). No. of bitstreams: 1 Flores-Nascimento_MarianeCristina_M.pdf: 1784393 bytes, checksum: 7247759965458fc5957d9f5617c9fb7e (MD5) Previous issue date: 2007 / Resumo: A Trombose Venosa Profunda (TVP) é uma doença multicausal, mas muitos fatores de risco ainda não estão definidos. Micropartículas (MPs) são pequenas vesículas liberadas da membrana celular durante ativação e apoptose. MPs podem ser um reflexo da dinâmica entre repouso, ativação e morte celular e podem contribuir com a gravidade da doença pois são procoagulantes e pro-inflamatórias. Parece haver associação entre elevado número de MPs e risco de complicações tromboembólicas, que podem ter um papel na patogênese destas doenças. Neste estudo avaliamos e caracterizamos as MPs em pacientes com TPV de membro inferior, [ao diagnóstico (5M/4H, idade média=41,1 anos), após 6 meses de tratamento (7M/3H, idade média=32,9 anos), associada à Síndrome do Anticorpo Antifosfolípide (7M/3H, idade média=33,8 anos)], em portadores assintomáticos do Fator V de Leiden (FVL) (7M, idade média=34 anos), e comparando-as a controles pareados por sexo, idade e etnia. As MPs foram isoladas de sangue periférico citratado, por centrifugação diferencial. A quantificação e caracterização foram feitas por citometria de fluxo usando os anticorpos: CD235, CD61, CD45, CD31, CD14, CD45, anti-TF e Anexina V. A atividade procoagulante plasmática foi investigada pela dosagem do fragmento 1+2 de protrombina (F1+2). A atividade procoagulante das MPs foi analisada pela dosagem de F1+2, de Dímero-D (DD2) e pelo teste de geração de trombina (TGT) em pool de indivíduos saudáveis em presença de MPs, corrigidas ou não por número na amostra (10.000 MPs). A análise estatística empregou os testes Wilcoxon ou U de Mann-Whitney, a=0.05. O número de MPs não estave diminuído em nenhum dos grupos estudados. A porcentagem de MPs estava estatisticamente aumentada nos pacientes com SAF/TVP em relação a seus controles (P=0,007). Observou-se um aumento significativo das MPs plaquetárias nos pacientes com SAF/TVP (P=0,01) e diminuição das MPs endoteliais naqueles com TVP ao diagnóstico (P=0,03), quando comparados aos seus controles. Os pacientes com SAF/TVP apresentaram diminuição significativa do F1+2 plasmático, tanto em relação aos seus controles (P=0,008) como ao CTR total (P=0,002). O F1+2 gerado pelas MPs estava significativamente diminuído em indivíduos com FVL em relação ao CTR total (0,009), e em pacientes com SAF/TVP em relação aos seus controles (P=0,001), e ao CTR total (P=0,008). O DD2 em pool de plasma, independente do número de MPs, estava significativamente aumentado na comparação entre TVP ao diagnóstico e seus controles (P=0,008) e ao CTR total (P=0,0001). O DD2 em pool com número corrigido de MPs apresentava-se aumentado significativamente nos pacientes com TVP ao diagnóstico quando comparados aos seus controles (P=0,008). Os valores do TGT com número corrigido de MPs estavam estatisticamente diminuídos em pacientes com TVP após 6 meses (P=0,01), em comparação ao CTR total. Nossos resultados demonstraram que o número de MPs está alterado em pacientes com TVP, e talvez possam ter um papel, particularmente após o evento trombótico ou em presença de anticorpos antifosfolípides. As MPs demonstraram atividade procoagulante, principalmente ao diagnóstico de TVP, podendo contribuir ou agravar o quadro clínico do paciente / Abstract: Deep Venous Thrombosis (DVT) is a multicausal disease, but many risk factors are not well defined. Microparticles (MPs) are small blebs released from cellular surfaces during activation and apoptosis. MPs may be the consequence of the dynamics between rest, activation and cellular death and can contribute to the seriousness of the illness and are therefore procoagulant and pro-inflammatory. There seems to be an association between high numbers of MPs and risk of thromboembolic complications and these may have a role in pathogenesis of these illnesses. In this study, we evaluated and characterized the MPs in patients with DVT of inferior limbs, [at diagnosis (5M/4H, medium age= 41.1 years), after 6 months of treatment (7M/3H, medium age= 32.9 years), and associated to Antibody Antiphospholipid Syndrome (7M/3H, medium age= 33.8 years)], and asymptomatic carriers of Factor V Leiden (FVL) (7M, medium age 34= years), matched to health controls by sex, age and ethnic origin. The MPs were isolated from citrated peripheral blood, by differential centrifugation. The quantification and characterization were performed by flow cytometry using the antibodies: CD235, CD61, CD45, CD31, CD14, CD45, anti-TF and Annexin V. The plasmatic procoagulantic activity was investigated by prothrombin fragment 1+2 (F1+2) dosage. The MPs procoagulant activities were analyzed by F1+2 dosage, D-dímer (DD2) and Thrombin Generation Test (TGT) in a pool of healthy individuals in the presence of MPs, corrected or not for number in the sample (10.000 MPs). Statistical analysiswas performed by Wilcoxon or the Mann-Whitney tests, a=0.05. The MPs number were not lower in any of the studied groups. The MPs percentage was statistically increased in the SAF/DVT patients compared to their matched controls (P=0.007). A significant increase in the platelet-derived MPs in SAF/DVT patients (P=0.01) and a reduction in the endothelial-derived MPs at diagnosis were observed (P=.03), when compared to their matched controls. The SAF/TVP patients show a significant reduction in plasmatic F1+2, when compared to their matched controls (P=0.008) and to the total CTR (P=0.002). The F1+2 generated by MPs were significantly lower in FVL carriers compared to the total CTR (0.009), and in SAF/DVT patients compared to their controls (P=0.001), and to the total CTR (P=0.008). The DD2 in the pool of plasma, independently of the number MPs, was significantly higher in DVT at diagnosis when compared to their matched controls (P=0.008) and the total CTR (P=0.0001). The DD2 in the pool with corrected MPs number was significantly higher in DVT at diagnosis patients when compared to their matched controls (P=0,008). The values of TGT in corrected MPs number were statistically lower in patients with DVT after 6 months (P=0.01), in comparison to the total CTR. Our results demonstrated that the number of MPs is modified in patients with DVT, and may play a role, particularly after the thrombotic event or in association with antiphospholipid antibodies. The MPs demonstrated procoagulant activity, especially at DVT diagnosis, and were able to contribute or to aggravate the patient¿s clinical situation / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Alteração da fosfolipase A2 tem sido descrita em diversas doenças neuropsiquiátricas. Esta enzima é responsável pelo metabolismo dos fosfolípides de membrana e parece estar envolvida na fisiopatologia do transtorno bipolar. Neste estudo, foram analisados os três principais subtipos da PLA2 (sPLA2, cPLA2 e iPLA2) em plaquetas de pacientes e indivíduos controles. A atividade de subtipos de PLA2 foi determinada em 20 pacientes TB sem tratamento com estabilizadores de humor e após seis semanas de tratamento com lítio; 72 pacientes medicados e 65 controles (16 pareados com os pacientes sem tratamento e 49 com os pacientes previamente medicados), pelo método radioenzimático. Os pacientes foram diagnosticados e classificados de acordo com os critérios estabelecidos no DSM-IV-TR. Foi verificado que Os pacientes no estágio inicial da doença apresentaram menor atividade de iPLA2, sPLA2 e cPLA2 quando comparadas ao grupo controle. Seis semanas de tratamento com o lítio não foram suficientes para observar alterações nos resultados obtidos. No entanto, o lítio diminuiu a sintomatologia maníaca e a depressiva, avaliadas pelas escalas de Hamilton e Young, respectivamente (p < 0,01 para as duas comparações). Os pacientes medicados não diferiram do grupo controle para os três subtipos de PLA2 avaliados. Estes resultados sugerem que no estágio inicial do TB há uma diminuição da atividade das PLA2, e que a ação de um ou mais medicamentos que são incluídos na terapêutica para este transtorno podem reverter essa diminuição. Sugere-se ainda que a diminuição da atividade da iPLA2 no estágio inicial do transtorno bipolar, além de alterar o remodelamento da membrana, possa ser um fator de risco para o desenvolvimento de demência nestes pacientes. / Changes in phospholipase A2 have been reported in several psychiatric disorders. This enzyme is responsible for the metabolism of membrane phospholipids and has been suggested to play a role in bipolar disorder physiopathology. In this study, the activity of the three main subtypes of phospholipase A2 (PLA2) were analyzed (sPLA2, cPLA2 and iPLA2) in platelets of BD patients and health subjects. Subjects enrolled were: 20 drug-naïve and drug free BD patients, who were treated with lithium for six weeks; 72 BD long-term treatment patients and 65 controls (16 for the drug-naïve and drug free and 49 for the long term group). PLA2 subtype activities were determined by the radio enzymatic method. Patients\' diagnostic and classification were made according to DSM-IV-TR criteria. Patients at the early stage of the disease presented lower iPLA2, sPLA2 and cPLA2 activity than the control group. Six weeks treatment with lithium did not change these activities. However, lithium reduced the maniac and depressive symptoms, evaluated with Hamilton and Young scales, respectively (p < 0.01, for both comparisons). Long-term treated patients presented similar PLA2 activities to control group. The results suggest that at the early stage of BD iPLA2 activity is decreased and that the adequate treatment of BD could reverse this reduction. We suggest that a reduction of iPLA2 activity at the early stage of BD can modify the membrane metabolism, and could be a risk for the development of dementia in BD patients.

Orientador: Nicola Amanda Conran Zorzetto, Fernando Ferreira Costa / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T19:18:22Z (GMT). No. of bitstreams: 1 Garrido_VanessaTonin_D.pdf: 6610754 bytes, checksum: 9b799d90821835bae6ac5d806b1d2f1c (MD5) Previous issue date: 2014 / Resumo: A anemia falciforme (AF) é uma hemoglobinopatia hereditária resultante de uma mutação no gene que codifica a subunidade ?-globina, levando à produção da hemoglobina S (HbS) nos eritrócitos. Com a polimerização da HbS, durante a desoxigenação, ocorre a deformação e fragilização das células vermelhas, resultando em anemia hemolítica e eventos vaso-oclusivos. As crises vaso-oclusivas são a principal causa de morbidade nos pacientes com anemia falciforme e as plaquetas parecem ter um papel importante nesse processo, pois uma vez ativadas elas secretam e expressam mediadores que induzem uma resposta inflamatória tanto em leucócitos como em células endoteliais. A proposta deste trabalho foi investigar a produção e expressão dos mediadores inflamatórios derivados de plaquetas, LIGHT e CD40L, em controles (indivíduos saudáveis; CON), pacientes com anemia falciforme (AF) e pacientes com anemia falciforme em terapia com hidroxiureia (AFHU). Também avaliamos o envolvimento das plaquetas e seus mediadores na ativação de leucócitos e células endoteliais. Os níveis plasmáticos de ambas citocinas foram significativamente maiores em indivíduos AF e AFHU do que nos indivíduos controle e, curiosamente, apesar da hidroxiureia ser capaz de diminuir a concentração plasmática de algumas citocinas inflamatórias, a terapia com essa droga não foi associada com qualquer alteração nos níveis de LIGHT ou CD40L. Foi observada uma correlação expressiva da concentração de LIGHT com níveis plasmáticos de CD40L, IL-8, ICAM-1, Trombospondina-1 e TNF-?, enquanto que a concentração plasmática de CD40L correlacionou-se com os níveis de TNF-? e principalmente com Trombospondina-1, indicando que tanto LIGHT como CD40L podem estar participando ou então refletindo a inflamação crônica presente na anemia falciforme. A expressão proteica de LIGHT foi significativamente maior na superfície de plaquetas de indivíduos AF e AFHU em comparação com plaquetas CON e apresentou uma correlação com marcadores de ativação plaquetária. A secreção de LIGHT pelas plaquetas foi determinada por ELISA e concentrações significativas dessa citocina puderam ser detectadas no sobrenadante de plaquetas CON e AF, sugerindo que essas células podem ser uma fonte importante de LIGHT na circulação. Apesar da expressão de CD40L não ter sido detectada na superfície das plaquetas de pacientes e controles, as plaquetas de pacientes AF secretaram uma quantidade maior de CD40L em comparação aos controles e foi observada uma correlação significativa entre a liberação de LIGHT e CD40L em plaquetas de pacientes AF, indicando que pode existir uma associação na secreção dessas duas citocinas. A expressão dos receptores de LIGHT (HVEM e LT?R) e de CD40L (CD40) foi avaliada por citometria de fluxo em plaquetas, neutrófilos, linfócitos e monócitos. Foi observado que o receptor HVEM estava mais expresso em plaquetas e linfócitos de pacientes com anemia falciforme, enquanto que a expressão do receptor CD40 estava elevada nas plaquetas, nos neutrófilos, nos linfócitos e nos monócitos de pacientes, comparando com o grupo controle. Esses dados mostram que a via de sinalização de LIGHT e CD40L pode estar alterada na anemia falciforme, contribuindo com a ativação dos leucócitos. Quando avaliamos a participação das plaquetas na ativação dos leucócitos, observamos que as plaquetas de indivíduos com anemia falciforme foram eficientes em aumentar a expressão do marcador de ativação, CD69, nos linfócitos e também em induzir o fenótipo pró-inflamatório nos monócitos. Enquanto que a co-cultura de HUVECs com plaquetas demonstrou que as plaquetas de pacientes com anemia falciforme possuem uma capacidade maior de induzir a expressão de ICAM-1 em células endoteliais do que as plaquetas de indivíduos controle. Na presença de anticorpos anti-CD40L observamos uma redução drástica no aumento da expressão de ICAM-1 pelas plaquetas e apesar dessa expressão também ter sido reduzida na presença de anticorpos anti-LIGHT, esses resultados não foram estatísticamente significativos. Interessantemente, altas concentrações plasmáticas de LIGHT estavam associadas com a elevada velocidade de regurgitação tricúspide, um indicativo de hipertensão pulmonar na anemia falciforme e uma associação significativa também foi encontrada entre níveis elevados de CD40L e pacientes com histórico de Síndrome Torácica Aguda. Essas evidências sugerem que LIGHT e CD40L parecem estar contribuindo com a ativação dos leucócitos e do endotélio, exercendo um papel importante na fisiopatogenia da anemia falciforme e aparentemente nas manifestações clínicas desta doença. Os resultados encontrados neste estudo evidenciam a importância que as plaquetas e seus mediadores inflamatórios, LIGHT e CD40L, podem ter na propagação da inflamação vascular presente na anemia falciforme, se tornando possíveis alvos para novas abordagens terapêuticas / Abstract: Sickle cell disease results from a single amino acid substitution in the gene encoding the ?-globin subunit, leading to hemoglobin S production in red blood cells. Polymerization of deoxygenated sickle hemoglobin leads to decreased deformability of red blood cells, resulting in hemolytic anemia and vaso-occlusive events. Platelets appear to play an important role in the vaso-occlusive process, as following their activation they express and secrete mediators that induce an inflammatory response in endothelial cells and leukocytes. The purpose of this study was to investigate the production and expression of LIGHT and CD40L on platelets, the presence of this protein in the plasma of controls (healthy subjects; CON), sickle cell anemia patients (AF) and sickle cell anemia patients on hydroxyurea therapy (AFHU). In addition, this study evaluated the involvement of platelets and their mediators, LIGHT and CD40L, in the activation of leukocytes and endothelial cells. Plasma levels of both cytokines were significantly higher in AF and AFHU individuals than in control individuals and interestingly, HU therapy was not associated with a reduction in these levels. A significant correlation was observed between levels of LIGHT with plasma levels of CD40L, IL-8, ICAM-1, Thrombospondin-1 and TNF-?, whereas the plasma concentration of CD40L correlated with levels of TNF-? and especially with plasma Thrombospondin-1. LIGHT expression was significantly higher on the surface of platelets from AF and AFHU subjects, compared with CON individuals and this expression demonstrated a correlation with markers of platelet activation. LIGHT secretion was determined by ELISA and significant concentrations of this cytokine could be detected in the supernatant of platelets from CON and AF individuals, indicating that platelets may be an important source of LIGHT. Although CD40L expression was not detected on the platelet surface in patients or controls, sickle platelets secreted an increased amount of CD40L, compared to controls. A significant correlation was observed between CD40L and LIGHT release in sickle cell patients, indicating that the production of these two proteins may be tightly coupled. The expression of LIGHT (HVEM and LT?R) and CD40L (CD40) receptors was evaluated by flow cytometry on the surface of platelets, neutrophils, lymphocytes and monocytes. An increased HVEM receptor expression was observed on the platelets and lymphocytes of sickle cell patients, whereas the expression of the CD40 receptor was elevated on platelets, neutrophils, lymphocytes and monocytes from sickle cell patients, compared to control subjects. Evaluating the contribution of platelets to leukocyte activation, we observed that platelets from sickle cell anemia individuals increased the expression of the activation marker, CD69, on lymphocytes and also induced a pro-inflammatory phenotype on monocytes. Co-culture of HUVEC with platelets demonstrated that sickle cell platelets have an increased ability to induce ICAM-1 expression on endothelial cells than platelets from control subjects. Furthermore, in the presence of anti-CD40L antibodies, a drastic reduction was observed in this increase. Although the expression of endothelial ICAM -1 was also reduced in the presence of anti-LIGHT antibodies, these results were not statistically significant. Interestingly, high plasma concentrations of LIGHT were associated with elevated tricuspid regurgitant velocity, indicative of pulmonary hypertension in sickle cell anemia. A significant association was also found between high levels of CD40L and patients with a lifetime history of acute chest syndrome. LIGHT and CD40L appear to contribute to leukocyte and endothelial activation, playing an important role in the pathophysiology of sickle cell anemia and apparently in the clinical manifestations of this disease. These results highlight the important role that platelets and their inflammatory mediators may play in the vascular inflammation that is known to occur in sickle cell anemia / Doutorado / Fisiopatologia Médica / Doutora em Ciências

To be able to effectively study blood platelets in different environments adevelopment of an in vitro model of a microfluidic system for plateletproduction was started. The purpose of this thesis was to fabricate systemsand then characterize them and visualize the flow. The system consists of twochannels, one in the middle and the other one enclosing it. They are connectedthrough pores where Megakaryocytes can protrude through and produce platelets.The designs were produced in PDMS. This was done by first transfer the designsas structures onto a silicon wafer through UV lithography. The wafer served asa mould for casting PDMS that later was bonded to glass. The systems were thenstudied with three different methods. Computer simulations, flow tests andultimately tests with cells. From the results new designs were made andfabricated. The new designs were then tested the same ways as the first ones.The systems can most probably produce platelets with some optimisation of thetest parameters. No definite results were gathered to prove plateletproduction. Different flow speeds were tested and the flow profile atdifferent flow rates was visualised. The full capability of the new designscould not be fully studied due to unforeseen debris of PDMS clogging thechannels. A few things need to be done to achieve better results and establishfor sure if this method of producing platelets is possible. This thesis is agood ground for future work to stand on.

Dans le cancer, l'activation de la cascade de coagulation et des plaquettes participent à la formation de thromboses, à la croissance tumorale, et les métastases. Ces thromboses représentent une complication clinique chez les patients atteints du cancer du pancréas. Cet état serait dû à expression par la tumeur et leurs microparticules (MPs) de facteur tissulaire (FT). Dans une première partie, nous avons identifié le FT et le FT pathway inhibitor exprimés par les MPs cancéreuses et la P-sélectine plaquettaire impliqués dans la progression tumorale, les métastases et la thrombose associée au cancer du pancréas .Nous avons montré le rôle des intégrines αvβ3 et αvβ et des "neutrophils extracellular traps" dans l’interaction des MPs cancéreuses avec les plaquettes dans des modèles murins de « deep vein thrombosis » et de blessure au laser. Nous avons alors évalué l’efficacité du clopidogrel qui présente une action anti tumorale et thrombotique. Cette étude a permis d’initier une étude clinique de phase III pour d’évaluer le potentiel thérapeutique du clopidogrel chez des patients atteints de cancer du pancréas. Dans une seconde partie, nous avons montré que des neutrophiles exprimant le FT agissent comme « starter » de la formation de thrombi. A l’inverse, dans un modèle d’inflammation stérile, nos travaux montrent le rôle de la P-sélectine plaquettaire dans le slow rolling, l’adhésion et la transmigration des neutrophiles a des temps précoses. L’ensemble de nos résultats suggère que les coopérations cellulaires entre l’endothélium, les plaquettes, les MPs et les neutrophiles constituent des mécanismes essentiels à la thrombose et l'inflammation. / In cancers, the blood coagulation cascade and platelets can be activated to form thrombosis. This state will mainly due by the tumor and their microparticles (MPs) expression of tissue factor (TF), key protein of the coagulation cascade. In the first part of this study, we demonstrated that TF and the TF pathway inhibitor expressed by cancer MPs and the platelet P-selectin are involved in tumor progression, metastasis and the associated thrombosis in pancreatic cancer in mice. We showed the key role-play by αvβ3 and αvβ1 integrins and neutrophils extracellular traps in the interaction between cancer cells-derived MPs and platelets. We also evaluated the effect of clopidogrel, but not aspirin, treatment exhibits an anti-tumor action and limits thrombosis formation in preclinical models of pancreatic cancer. This study initiates a national investigation of a multicenter clinical phase III study to evaluate the therapeutic potential of clopidogrel in pancreatic cancer patients. In the second part of this study, we identified a “population of neutrophils expressing TF” that acts like a starter of the thrombus formation. At the reverse, in a sterile inflammatory model, our work showed the primordial role of platelet P-selectin in the slow rolling, the adhesion and the transmigration of neutrophils. All together our results suggest that the cooperation between the endothelium, platelets, MPs and neutrophils constitute essential mechanisms acting in the thrombosis and the inflammation.

As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated. Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond.

Diabetes mellitus has emerged as a major public health problem with pandemic growth as the International Diabetes Federation estimates that there were 415 million diabetics in 2015 with that number reaching 642 million by 2040, affecting all regions of the world. Globally we are all interconnected when we deal with problems of climate change, water shortage, HIV or Ebola. The war against type 2 diabetes and other non-communicable diseases should be no different, as effective solutions will need expanded global engagement in science to win it. The risk of cardiovascular events in type 2 diabetes remains unchanged despite good control of diabetes and other cardiovascular risk factors. A better understanding of thrombogenicity in diabetes may help to identify novel therapeutic agents and a starting point would be to identify ultrastructural changes in diabetic erythrocytes, platelets and fibrin networks. In diabetes, thrombogenicity is enhanced and is characterised by: hyperactive platelets, higher levels of clotting factors and impaired fibrinolysis. Thus, in this research study, the technique of scanning electron microscopy (SEM) was used to identify ultrastructural abnormalities in erythrocytes, platelets and fibrin networks of diabetic subjects. Distinct abnormal morphological findings were observed in the erythrocytes, platelets and fibrin fibres of diabetic subjects in comparison to the controls. Physiological parameters such as platelet markers and tissue factor levels were also assessed. Flow cytometric analysis revealed hyperactive platelets in the diabetic subjects. The measurement of tissue factor in plasma was completed by using an ELISA. Tissue factor levels in the diabetic subjects were markedly elevated when compared to controls. Biomedical research has provided evidence that has led to the hypothesis that inflammation is the culprit behind almost most chronic illnesses. Hyperglycaemia, a key feature of diabetes, is known to promote a state of low-grade chronic inflammation. A natural method that can resolve acute and chronic inflammation is earthing. Earthing involves coupling your body to the Earth's surface energies by simply walking barefoot or being connected to a conductive device. When earthed, the electrons are conducted into the human body at the same electrical potential as the earth. It is also suggested that free electrons from the earth neutralize the positively charged free radicals that are the hallmark of chronic inflammation. In this study, earthing was accomplished with conductive adhesive patches placed on the sole of each foot and palm of each hand. An earthing cord was connected to the patches and led outdoors to be connected to a stainless-steel rod driven into the ground. Diabetic subjects were earthed for a session of two hours. Bloods were drawn before and just prior to the end of the two-hour session. Morphological SEM findings of the erythrocytes, platelets and fibrin networks at two-hours showed a remarkable difference when compared to findings at baseline. More importantly, the erythrocytes, platelets and fibrin findings revealed that they all almost reverted to looking like control erythrocytes, platelets and fibrin networks. It remains to be seen if earthing will reduce cardiovascular events in diabetics by improving morphology of cells involved in coagulation. / Thesis (PhD)--University of Pretoria, 2016. / Physiology / PhD / Unrestricted

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.