The objective of the work presented in this thesis was to explore the role of potential alternatives to platelet transfusions and specifically to investigate whether desmopressin could be used for treatment of thrombocytopenia or platelet dysfunction. Patients with thrombocytopenia or platelet dysfunction are often treated with platelet transfusions to treat or prevent bleeding. However the evidence for the efficacy of platelet transfusion is limited and there is some evidence of harm. I have focused on thrombocytopenic patients with haematological malignancies or critically ill patients, who are amongst the groups most commonly treated with platelet transfusions. The aims of this research were to determine: 1. If levels of Von Willebrand factor (VWF) or other measures of haemostasis are predictive of bleeding in severe thrombocytopenia; 2. Whether VWF compensates for thrombocytopenia in vitro; 3. The evidence for the efficacy of desmopressin in all patients undergoing surgery or invasive procedures; 4. The evidence for desmopressin for platelet dysfunction or thrombocytopenia; 5. If it is feasible to use desmopressin to treat critically ill thrombocytopenic patients in a clinical trial. To identify derangements of haemostasis that may signify candidates for alternatives to platelet transfusions, I analysed blood samples from an observational trial of fifty patients with haematological malignancies and profound thrombocytopenia due to intensive chemotherapy. I used a panel of tests to investigate measures of primary haemostasis, thrombin generation, cross-linked fibrin formation and fibrinolysis. Using multivariable logistic regression, I found no consistent correlation between any measures of haemostasis and the risk of clinically significant bleeding. VWF antigen levels were the best predictor of clinically significant bleeding on the same day (odds ratio 0.31, 95% confidence interval 0.10 to 0.98, p=0.047) but were not predictive of severe bleeding over the 24 hours after the test (odds ratio 0.48, 95% confidence interval 0.10 to 2.34, p=0.36). In a separate set of experiments, I evaluated thrombus formation under flow in thrombocytopenia. This technique was sensitive to the platelet count . Addition of exogenous VWF to thrombocytopenic blood resulted in improvement in thrombus formation, suggesting that agents that affect or influence VWF pathways might have a role. Desmopressin can be used to increase VWF levels, so leading on from my laboratory experiments; I used systematic reviews and meta-analyses to assess whether desmopressin could be used in unselected patients to reduce bleeding peri-operatively. I identified 62 randomised controlled trials. Overall there was no evidence of benefit for administering desmopressin to unselected patients. However further analysis of eleven randomised controlled trials that focused on patients with platelet dysfunction found that desmopressin resulted in transfusion of fewer units of red cells (equivalent to a 25% reduction compared to control), less blood loss (equivalent to a 23% reduction compared to control) and a lower risk of requiring a re-operation due to bleeding (Peto odds ratio 0.39, 95% confidence interval 0.18 to 0.84). There was no evidence for an increase in thrombotic events. There was no randomised controlled trial evidence for perioperative desmopressin for patients with thrombocytopenia. These specific research gaps were addressed by designing new clinical trials. I have commenced a randomised controlled feasibility trial of desmopressin versus placebo for critically ill patients with thrombocytopenia undergoing invasive procedures. This trial is ongoing and is the first randomised trial evaluating peri-procedural desmopressin in thrombocytopenia. The programme of work arising from this research has the potential to benefit a large number of patients by preventing bleeding and reducing exposure to allogeneic blood components such as platelets. The results presented in this thesis are exploratory but are an important step on a path towards larger trials using desmopressin as an alternative, or adjunct to platelet transfusion.

Orientador: Regina Kiomi Takahira / Banca: Leonardo Brandão / Banca: Marcelo Larami Santoro / Resumo: O objetivo do presente estudo foi avaliar a importância das plaquetas reticuladas nas condições trombocitopênicas associadas a hipoplasia de megacariócitos e a outras etiologias sem alteração no número de megacariócitos. Para tanto foram utilizados 30 cães sadios adultos (Grupo controle); 15 cães com trombocitopenia (< 100.000 plaquetas/mL), por causas diversas que não sejam por hipoplasia megacariocítica (Grupo A) e 15 cães com trombocitopenia (< 100.000 plaquetas/mL) e com hipoplasia megariocítica (Grupo B), constatadas por meio de citologia aspirativa de medula óssea. Foi coletado sangue e extraído o plasma rico em plaquetas para a realização da quantificação das plaquetas reticuladas em citometria de fluxo e colheita de medula óssea para a análise do número de megacariócitos por duas técnicas distintas e correlação com as plaquetas reticuladas. Não foi observada diferença estatística (p>0,05) entre as duas técnicas de quantificação de megacariócitos. Foi observada diferença estatística (p<0,05) para as plaquetas reticuladas, sendo superior nos grupos A e B, em relação ao grupo controle. Não houve correlação entre plaqueta reticulada e megacariócitos medulares. A ausência de correlação observada não nos permite estabelecer relações de contagem de PR com o aumento do número de megacariócitos na medula óssea. / Abstract: The aim of this study was to evaluate the reticulated platelet importance in evaluation of platelet production in thrombocytopenic conditions associated with megakaryocytic hypoplasia and other conditions with adequate number of bone marrow megacaryocytes. These study were obtained from 30 healthy dogs (control group), 15 thrombocytopenic dogs (< 100,000 platelets/μL) without megakaryocytic hipoplasia (group A) and 15 thrombocytopenic dogs (< 100,000 platelets /μL) with megakaryocytic hipoplasia (group B). Blood samples were collected and the platelet rich plasma (PRP) was extracted for quantification of reticulated platelets in flow cytometry, megakaryocytes were quantified in marrow particles, collected from aspiration cytology and correlated to reticulated platelets counts. No statistical significance (p>0.05) was observed between two megakaryocytes quantification techniques. For reticulated platelets was observed statistical significance difference (p<0.05), values from groups A and B were higher than control group. No correlation was verified between reticulated platelets and marrow megakaryocytes. The absence of correlation do not allow us to establish relationship of reticulated platelets with increase production of bone marrow megakaryocytes. / Mestre

Les plaquettes sont des cellules sanguines anucléées impliquées dans les phénomènes d'hémostase et de thrombose. La majorité des fonctions plaquettaires dépend de leur membrane cellulaire, qui contient de nombreux microdomaines lipidiques ordonnés appelés radeaux lipidiques. Ces microdomaines jouent un rôle central dans toutes les phases de l'hémostase médiées par les plaquettes. Les radeaux lipidiques sont essentiels pour le fonctionnement des récepteurs responsables de l'activation des plaquettes et de la transduction du signal. Le rôle des plaquettes dans la thrombose artérielle est crucial et explique l'intérêt continu de la recherche dans la thérapie antiplaquettaire. Dans ce contexte, nous avons cherché à étudier les radeaux lipidiques comme lieu d'assemblage principal des récepteurs membranaires. Nous avons également cherché à identifier des protéines impliquées dans la fonction des plaquettes, en vue de proposer de nouvelles cibles thérapeutiques. Nous avons utilisé des analyses lipidomiques et protéomiques ainsi que des analyses d'immunoblotting pour identifier les radeaux lipidiques de la membrane des plaquettes et étudier leur organisation dans les plaquettes non stimulées, stimulées et traitées par des antiagrégants plaquettaires. Des détergents, l'ultracentrifugation et les gradients de sucrase ont été utilisés principalement pour le fractionnement de la membrane et l'isolement des radeaux lipidiques. Les principaux résultats de notre travail sont: 1) Élaboration d'une méthodologie pour l'étude des radeaux lipidiques des plaquettes ; 2) Présentation d'un profil global de la composition lipidique et protéique des radeaux lipidiques ; 3) Démonstration de l'impact de l'activation plaquettaire et des antiagrégants plaquettaires sur la réorganisation des radeaux lipidiques ; Et 4) Proposition de nouvelles cibles thérapeutiques potentielles par protéomique et identification de réseau interactif de protéines autour notamment du facteur XIII (FXIII) et de la phosphoprotéine stimulée par vasodilatateur (V ASP). Nos résultats montrent que les radeaux lipidiques peuvent potentiellement être considérés comme nouvelles cibles thérapeutiques pour la découverte de nouveaux antiagrégants plaquettaires. Les études "Omics" sont importantes pour élargir nos connaissances dans ce domaine / Latelets are blood ce lis at the crossroads of both haemostasis and thrombosis. The majority of platelet functions depend on their membrane, which contains numerous, ordered lipid microdomains named lipid rafts. These microdomains play a pivotai role in all phases of platelet­mediated haemostasis. Lipid rafts are a prerequisite for the functioning of receptors in charge of platelet activation and signal transduction. The role of platelets in thrombotic diseases is crucial, and underpins the continue research interest in antiplatelet therapy. ln this context, we aimed to study the lipid rafts of platelet membranes as the principal assembly place of known receptors, and likely also other, unknown elements that participate in the thrombotic function of platelets, with a view to proposing new therapeutic targets. We used lipidomics and proteomics as well as immunoblot analysis to identify lipid rafts and investigate the organization of lipid rafts in resting, stimulated and antiplatelet-treated platelets. Detergents, ultracentrifugation and sucrose gradients were used mainly for membrane fractionatio and isolation of lipid rafts. The main findings of our work are: 1) Development of a framework or guidelines for platelet lipid raft investigation; 2) Presentation of a global profile of the lipid and protein composition of plate let lipid rafts; 3) Demonstration of the impact of activators and inhibitors on the reorganization of platelet lipid rafts; and 4) Suggestion for potential new therapeutic targets by proteomics analysis through interactive network analyzing of coagulation factor XIII (FXIII) and Vasodilator-Stimulated Phosphoprotein (VASP). Our results show that lipid rafts have potential as new therapeutic targets in pharmacological research in antiplatelets. "Omics" studies are important to expand our knowledge in this field

O período gestacional é bastante complexo e do ponto de vista vascular, ocorrem inúmeras alterações e adaptações no sistema circulatório, na funcionalidade plaquetária e no sistema endotelial com o objetivo de prevenir futuras complicações na mãe ou no concepto no momento do parto e após o nascimento. Além das alterações na circulação, nos processos vasculares e no sistema imune que são inerentes a este período, existem trabalhos demonstrando a geração de espécies reativas de oxigênio (EROs) e de nitrogênio (ERNs) as quais poderão desencadear um desequilíbrio do balanço redox. Os nucleotídeos de purina ATP, ADP, AMP, além do nucleosídeo adenosina participam da regulação vascular endotelial, de processos inflamatórios e do controle imunológico entre outras funções. O controle da produção e dos níveis intracelulares e extracelulares destes nucleotídeos e nucleosídeos são regulados por uma cascata enzimática, na qual, participam as enzimas E-NTPDases (Ecto-Nucleosídeo Trifosfato Difosfoidrolase), ENPPs (Ecto-Nucleotídeo Pirofosfatase Fosfodiesterase), 5`-Nucleotidase e adenosina desaminase. O objetivo deste trabalho foi investigar a atividade de enzimas envolvidas no processo hemostático de gestantes normais e com complicações, como a diabetes melito gestacional, a pré-eclampsia e contaminadas com o vírus HIV, além de alguns parâmetros de estresse oxidativo e a agregação plaquetária durante o terceiro trimestre gestacional. Os resultados obtidos demonstram um aumento na atividade das enzimas NTPDase e 5`-nucleotidase em plaquetas de gestantes normais e com diabetes melito gestacional e pré-eclampsia comparadas com mulheres não grávidas. Ocorreu um aumento na atividade da enzima adenosina desaminase no soro de gestantes normais e com diabetes melito gestacional, pré-eclampsia e contaminadas com o vírus HIV. Foi verificado, também, um aumento na atividade plaquetária, através da medida da agregação plaquetária, nas gestantes normais e com diabetes melito gestacional, pré-eclampsia e contaminadas com o vírus HIV. Por outro lado, foi verificado um aumento na atividade das enzimas superóxido dismutase, catalase, butirilcolinesterase e acetilcolinesterase juntamente com um aumento da peroxidação lipídica e uma redução dos níveis de ácido ascórbico e conteúdo sulfidrílico total. Além disso, não foi encontrada alteração significativa na análise de carbonilação protéica. Os resultados apresentados aqui sugerem que estas ectonucleotidases atuam na modulação de respostas vasculares, inflamatórias, imunes e circulatórias. Finalmente, foi detectado um aumento na atividade de enzimas antioxidantes e uma redução no conteúdo de moléculas antioxidantes, acompanhadas de um aumento no dano oxidativo aos lipídios de membrana o que remete à uma produção aumentada de espécies reativas e a uma tentativa do organismo em combater este desequilíbrio. / The pregnancy stage is very complex in the vascular point of view, occurring many changes and adjusts in the circulatory system, in the platelets functionality and in the endothelial system with the function of prevent future complications to the pregnant or to the newborn at the delivery and in the afterbirth. Besides the circulation changes, in the vascular processes and in the immune system that are inherent to this stage, there are studies that report the generation of reactive oxygen (ROS) and nitrogen species (RNS), which could trigger an imbalance in the redox status. The purine nucleotides ATP, ADP, AMP and the nucleoside adenosine participate in the endothelial vascular regulation, inflammatory events, immune regulation, among other functions. The control of the production, the intracellular and extracellular levels of these nucleotides and nucleosides are regulated by an enzymatic cascade, in which, participate the enzymes E-NTPDases (ectonucleotise triphosphate diphosphohydrolases), E-NPPs (ectonucleotide pyrophosphatase/phosphodiesterases), 5’-nucleotidase and adenosine deaminase. The purpose of this study was to investigate the activity of enzymes involved in the hemostatic dynamics from normal pregnant and those with complications, such as the gestational diabetes mellitus, the pre-eclampsia and infected with the HIV. We also investigated some oxidative stress parameters and the platelets aggregation during the third gestational trimester. The results demonstrated an increase in the enzymes NTPDase and 5’-nucleotidase in platelets from normal pregnant and those with gestational diabetes mellitus and pre-eclampsia compared with not-pregnant women. It was also observed an increase in the enzyme adenosine deaminase activity in the serum from normal pregnant and those with gestational diabetes mellitus, pre-eclampsia and infected with the HIV. It was also verified an increase in the platelets activity through the measure of platelets aggregation in the normal pregnant, those with gestational diabetes mellitus, pre-eclampsia and infected with the HIV. Otherwise, it was verified an increase in the activity of the enzymes superoxide dismutase, catalase, butyrylcholinesterase and acetylcholinesterase together with an increase in the lipid peroxidation and a decrease in the ascorbic acid and total sulphydryl groups content. Moreover, it was not observed any significant alteration in the protein carbonilation. The results showed here suggest that these ectonucleotidases participate in the modulation of vascular, inflammatory, immune and circulatory responses. Finally, it was detected an increase in the activity of antioxidant enzymes and a decrease in the antioxidant molecules content, together with an increase in the membrane lipids oxidative damage, which brings about an increased production of reactive species and a struggle of the body to combat this unbalance.

A uremia é uma desordem sistêmica que pode estar associada à disfunção plaquetária adquirida, levando a alterações na hemostasia primária. Vários modelos de interferência entre a uremia e a falha na hemostasia já foram propostos, porém o mecanismo exato é desconhecido, e a tendência ao sangramento parece ser de origem multifatorial. O teste do tempo de sangramento da mucosa oral (TSMO) pode ser utilizado na avaliação da hemostasia primária em animais. O fator de von Willebrand (FvW:Ag) normal ou aumentado, observado na maioria dos cães urêmicos, contribui para o diagnóstico de uma alteração plaquetária adquirida. A contagem de plaquetas e os testes de coagulação normais associados a um aumento no TSMO dão suporte à suspeita de um defeito qualitativo. O objetivo deste trabalho foi investigar possíveis anormalidades na hemostasia, buscando estabelecer uma relação entre os resultados de exames laboratoriais e alterações no tempo de sangramento. A hemostasia foi avaliada em quarenta cães azotêmicos, urêmicos ou não. O aumento no TSMO foi observado em 35% dos cães azotêmicos. O teste de Spearman demonstrou haver correlação entre o TSMO e os valores de creatinina, uréia e hematócrito, porém, o ajuste pela Regressão Linear Múltipla evidenciou o hematócrito como única variável associada com o TSMO. Valores de hematócrito abaixo do intervalo de referência para a espécie foram observados em 92,86% dos pacientes que apresentaram aumento no TSMO. Esses valores reduzidos parecem contribuir para a tendência ao sangramento, embora não possam ser considerados como fator determinante preditivo, uma vez que sua ocorrência nem sempre se associou com interferências no TSMO. / Uremia is a systemic disorder that may be associated with acquired platelet dysfunction, leading to changes in primary hemostasis. Several interference models between uremia and hemostasis failure have been proposed, but the exact mechanism is unknown, and bleeding tendencies seem to have a multifactorial origin. The buccal mucosal bleeding time (BMBT) test can be used in the assessment of primary hemostasis in animals. Normal or increased von Willebrand factor (vWF:Ag), observed in most uremic dogs, contributes to the diagnosis of an acquired platelet alteration. Normal platelet counts and coagulation tests associated with BMBT raises support the suspicion of a qualitative defect. The aim of this study was to investigate possible hemostasis abnormalities, trying to establish a relation between the results of laboratory tests and changes in bleeding times. Hemostasis was evaluated in forty azotemic dogs, uremic or not. Increase in BMBTs was observed in 35% of the azotemic dogs. Spearman’s test showed a correlation between BMBT and the values of creatinine, urea and hematocrit; however, adjusting for Multiple Linear Regression showed hematocrit as the only variable associated with BMBT. Hematocrit values below reference range for the species was observed in 92,86% of the patients showed an increase in BMBT. These low values appear to contribute to the tendency to bleed, although they cannot be considered as a preditive determining factor, since their occurrence is not always associated with interference in BMBT.

Made available in DSpace on 2015-07-22T20:49:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-29 / Objetivo: Determinar se o escore MELD (Model for end-stage liver disease), classificação de Child-Turcotte-Pugh (CTP), APRI (AST to platelet ratio index), testes hepáticos e contagem de plaquetas são preditores da presença de varizes esofágicas (VE) ou de VE com necessidade de profilaxia para sangramento varicoso (VNP). Métodos: Trezentos pacientes cirróticos (193 homens; média etária de 53,10 anos; hepatite C crônica como etiologia predominante da cirrose) foram analisados prospectivamente. Considerou-se como VE de fino calibre aquelas que desapareciam com a insuflação do órgão; médio calibre aquelas que ocupavam até 1/3 da luz esofágica e de grosso calibre aquelas que ocupavam mais do que 1/3 da luz. Varizes de médio e grosso calibre foram consideradas VNP. Realizaram-se análises uni e multivariada para estabelecer a relação entre a presença de varizes (qualquer calibre e VNP) e as características destes pacientes (MELD, classificação CTP, APRI, exames de bioquímica hepática e contagem de plaquetas). Resultados: Cento e setenta e um pacientes (57%) possuíam VE, dos quais 35% (n = 105) possuíam VNP. A distribuição das VE de acordo com a classificação de CTP foi: A, 49%; B, 75,30% e C, 80% (p < 0,01). Em análise univariada os preditores independentes da presença de VE foram: MELD > 8 (p = 0,02); APRI > 1,64 (p = 0,01); contagem plaquetária inferior a 93.000/mm3 (p < 0,01); AST > 1,34xLSN (p = 0,01) e bilirrubina total > 1mg/dl (p = 0,04). Escore MELD acima de 8 teve o maior valor discriminante para a presença de VE, com sensibilidade de 80,10% e especificidade de 51,20%. Fatores independentemente associados à VNP foram: trombocitopenia (< 92.000/mm3; p < 0,01) e AST > 1,47xLSN (p = 0,03). Contagem plaquetária < 92.000/mm3 possui sensibilidade de 65,70% e especificidade de 57,90% para a presença de VNP. Conclusões: Escore MELD elevado foi o melhor preditor isolado da presença de VE de qualquer calibre e plaquetopenia < 92.000/mm3, foi o melhor preditor para varizes com necessidade de profilaxia. No entanto, nenhum destes concilia altos valores de sensibilidade e especificidade. / Purpose: The aim of this study was to determine whether MELD, Child-Turcotte-Pugh (CTP) class, APRI and laboratory tests could predict the presence of EV or varices which need prophylactic therapy (EV with medium or large size). Methods: Three hundred cirrhotic patients (193 men; mean age 53,1 years; majority with cirrhosis from chronic C hepatitis) were prospective analyzed. Uni and multivariate analysis were used to evaluate associations between the presence of EV (any size and medium or large EV) and patients’ characteristics (MELD, CTP class, APRI, platelets count and liver tests). Small varices were regarded as those which flatten with insufflation; medium varices those which protruded less than 1/3 of the lumen and large ones those which protruded more than 1/3. Results: One hundred seventy one patients (57%) had EV, of whom 35% (105) had varices which need prophylactic therapy. The distribution of EV according to the CTP class was as follows: A, 49%; B, 75,3% and C, 80%. Independent predictors of the presence of EV were: MELD > 8 (p = 0,02); APRI > 1,64 (p = 0,01); a platelet count < 93.000/mm3 (p < 0,01); AST > 1,34xUNL (p = 0,01) and total bilirubin > 1 mg/dl (p = 0,04). MELD > 8 had the highest discriminative value for presence of EV with sensitivity of 80,1% and specificity of 51,2%. Factors independently associated with EV which need prophylactic therapy were: thrombocytopenia (< 92.000/mm3; p < 0,01) and AST > 1,47xUNL (p = 0,03). A platelet count < 92.000/mm3 had sensitivity of 65,7% and specificity of 57,9% for the presence of varices which need prophylactic therapy. Conclusions: High values on MELD are associated with EV and thrombocytopenia (< 92.000/mm3), with varices which need prophylactic therapy. Considering their low sensitivity and specificity, it is suggested to maintain the recommendation of upper gastrointestinal endoscopy for all cirrhotic patients. / TEDE / BV UNIFESP: Teses e dissertações

Submitted by André Hergesel de Oliva null (holivaandre@gmail.com) on 2018-02-19T13:16:08Z No. of bitstreams: 1 Dissertação_MestradoCORRIGIDO.pdf: 2514302 bytes, checksum: 2de51633e693041838c21d79e951b406 (MD5) / Approved for entry into archive by Ana Paula Rimoli de Oliveira null (anapaula@foa.unesp.br) on 2018-02-19T14:23:57Z (GMT) No. of bitstreams: 1 oliva_ah_me_araca_int.pdf: 2514302 bytes, checksum: 2de51633e693041838c21d79e951b406 (MD5) / Made available in DSpace on 2018-02-19T14:23:57Z (GMT). No. of bitstreams: 1 oliva_ah_me_araca_int.pdf: 2514302 bytes, checksum: 2de51633e693041838c21d79e951b406 (MD5) Previous issue date: 2018-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esse estudo objetivou-se em avaliar o papel da membrana de fibrina rica em plaquetas na reparação óssea em defeito crítico de calota de ratos induzidos à hipercolesterolemia, bem como a atuação do tratamento de atorvastatina nesse processo. Quarenta defeitos críticos de 6 mm de diâmetro foram criados em calotas cranianas de ratos. Cada defeito foi aleatoriamente dividido em 8 grupos experimentais (GC, GCPRF, GCA, GCH, GHA, GPRFA, GPRFH, GPRFHA), avaliados aos 28 dias pós-operatórios com análises histológica e microtomográfica. Os resultados mostraram que houve formação óssea em todos os grupos analisados. O grupo GPRFH apresentou formação óssea mais acentuada com relação aos demais grupos. Dos grupos em que foi administrado atorvastatina, o GPRFHA apresentou maior taxa de formação óssea (p < 0,05). Com a metodologia utilizada, a fibrina rica em plaquetas, quando associada à condição de hipercolesterolemia, induziu ao aumento da formação óssea. / Objectives: The aim of this study was to evaluate the efficacy of Platelet Rich Fibrin (PRF) in the healing process of surgically created critical-size defects in rat calvarial induced to hypercholesterolemia treated or not with atorvastatin. Materials and method: 40 critical defects were created with 6 mm diameter calvaria of rats. Each defect was randomly divided into eight experimental groups (GC, GCPRF, GCA, GCH, GHA, GPRFA, GPRFH, GPRFHA), evaluated at 28 postoperative days for histomorphometry and microtomography. Results: The results showed new bone formation in all groups. The GPRFH group just did differ from all groups in the new bone formation (p < 0.05). Among the groups treated with atorvastatin, the GPRFHA group showed the highest bone formation rate. Conclusion: With the methodology used, the platelet-rich fibrin associated with the hypercholesterolemia induced to new bone formation. Clinical relevance: Based on these results, it is necessary to unveil the mechanisms of the association of platelet-rich fibrin with hypercholesterolemia and how it interferes in PRF microstructure and bone regeneration, to confer clinical predictability in patients in this condition who will undergo bone reconstruction and implant rehabilitation.

Os antígenos plaquetários humanos são estruturas imunogênicas resultantes de alterações pontuais (SNP) que levam a substituição de um aminoácido a nível proteico. O objetivo deste estudo foi determinar a frequência alélica e genotípica do sistema HPA-1 a -5 e -15 em doadores de plaquetas do Estado do RS e comparar as frequências alélicas encontradas com as observadas em outras populações. A genotipagem HPA foi realizada através do método de PCR-SSP. Um total de 201 doadores de plaquetas foram incluídos no estudo sendo 167 caucasoides e 34 não caucasoides. O alelo “a” foi o mais frequente nos sistemas HPA-1 a -5 em ambos grupos. O genótipo HPA-15AB foi predominante sobre os genótipos homozigotos para este sistema. O teste exato de Fisher revelou diferença estatisticamente significativa para o sistema HPA-5. Houve maior prevalência do alelo HPA-5B no grupo não caucasoide. Para o grupo caucasoide, o método de neighbor-joining e a PCA revelaram proximidade genética entre este grupo e as populações europeias. De um modo geral, concluímos que as frequências alélicas para os sistemas HPA-1 a -5 e -15 encontradas em nosso grupo caucasoide são similares às descritas em populações europeias. Estes dados corroboram a formação étnica da população do RS. A maior frequência do alelo HPA-5b encontrada no grupo não caucasoide de nosso estudo indica a possibilidade de alosensibilização para pacientes que recebem transfusões de plaquetas não compatibilizadas geneticamente. / Human platelet antigens are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. The present study sought to determine the allele and genotype frequencies of HPA-1 through 5 and HPA-15 in platelet donors from the state of Rio Grande do Sul, Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed via the single specific primer-polymerase chain reaction (PCR-SSP) method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele “a” was that most commonly found for HPA-1 through 5 in both groups. The HPA-15AB genotype predominated over homozygous genotypes of this system. Fisher’s exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA- 5B allele in non-Caucasians. The neighbor-joining method and principal components analysis (PCA) revealed genetic proximity between the Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 through 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of Rio Grande do Sul. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os transtornos psiquiátricos são um problema de saúde pública, ocupando cinco das dez principais causas de incapacitação no mundo. O transtorno bipolar (TB) é um transtorno de humor, segundo o DSM-IV (manual diagnóstico e estatístico de doenças mentais), o qual afeta cerca de 1% da população mundial. O TB do tipo I (TBI) é o mais frequente entre os TBs e é caracterizado pela presença de episódios maníacos ou mistos acompanhados por episódios depressivos. Assim como outros transtornos psiquiátricos, como a depressão, ansiedade e esquizofrenia, o TB representa um importante fator de risco cardiovascular, e pacientes com este transtorno apresentam mortalidade cardiovascular duas vezes maior que a população em geral. No entanto, os exatos mecanismos envolvidos nesta relação permanecem desconhecidos. Estudos sugerem o envolvimento da via L-arginina-óxido nitrico (NO) na patofisiologia do TB. O NO é responsável por diversas funções fisiológicas, incluindo a inibição da função plaquetária. A L-arginina, sua precursora, é transportada em plaquetas pelo carreador y+L, ativando a enzima NO sintase (NOS), a qual produz NO e e L-citrulina. Uma vez produzido, o NO ativa a enzima guanilato ciclase (GC), levando ao aumento dos níveis de guanosina monofosfato cíclica (GMPc). Adicionalmente, a L-arginina não é exclusivamente utilizada pela NOS, ela também pode ser metabolizada pela arginase e produzir L-ornitina e uréia. A biodisponibilidade do NO depende tanto de sua síntese como de sua degradação pelo estresse oxidativo ou pela inflamação. O objetivo deste estudo foi investigar detalhadamente a via L-arginina-NO-GMPc em plaquetas de pacientes com TBI, a expressão da arginase e outros marcadores de estresse oxidativo e inflamação. Vinte e oito pacientes com TB e dez indivíduos saudáveis foram incluídos no estudo. Nossos estudos mostraram uma redução da atividade da NOS em todos os grupos de pacientes bipolares (fases de eutimia, depressão e mania), quando comparados aos controles. Isto ocorreu na presença de concentrações normais do substrato e de seu transporte, e da expressão inalterada das isoformas eNOS e iNOS. A expressão da arginase II não diferiu entre os grupos estudados, indicando que a disponibilidade da L-arginina não está sendo desviada para o ciclo de uréia em plaquetas. A produção reduzida de GMPc foi observada mesmo com a expressão inalterada da GC. A atividade e marcadores de estresse oxidativo, avaliada através da quantificação da oxidação de proteínas e atividade da catalase, não foram modificadas em plaquetas de pacientes com TB, enquanto que a atividade da SOD estava aumentada em todas as fases. Os níveis séricos da proteína C-reativa (PCR), um marcador inflamatório, estão aumentados em pacientes maníacos, comparados aos controles. A reduzida produção de NO observada em plaquetas de pacientes bipolares pode ser um elo entre esta complexa associação entre TB e a doença cardiovascular. / Psychiatric disorders are public health problems, ocuppying five of ten major causes of incapacitation in the world. Bipolar disorder (BD) is a mood disorder, according to DMS-IV (Diagnostic and Statistic Manual of Mental Disease), which affects about 1% of world population. Bipolar I disorder (BDI) is the most frequent among the BDs and is characterized by the presence of manic or mixed episodes accompanied by depressive episodes. Similarly to other psychiatric disorders, such as depression, anxiety and schizophrenia, BD represents an important cardiovascular risk factor, and patients with this disorder have twice the cardiovascular mortality expected from general population. However, the exact mechanisms underlying this relationship remain unknown. Studies suggest the involvement of L-arginine-nitric oxide (NO) pathway on the pathogenesis of BD. NO is responsible for several physiological functions, including platelet function inhibition. L-arginine, its precursor, is transported into platelets by y+L carrier, and activates the enzyme NO synthase (NOS), which produces NO and L-citrulline. Once produced, NO activates the enzyme guanilyl cyclase (GC), increasing cyclic guanosine monophosphate (cGMP) levels. Additionally, L-arginine is not exclusively used by NOS, it can also be metabolized by arginase and produce L-ornithine and urea. NO bioavailability depends on both its synthesis and its degradation by oxidative stress and inflammation. The aim of this study was to investigate in detail L-arginine-NO-cGMP pathway in platelets of BDI patients, arginase expression and other markers that can affect NO bioavailability, such as oxidative stress and inflammation. Twenty eight patients with BD and ten healthy volunteers were included in this study. Our results showed decreased NOS activity in all groups of BDI patients (euthymic, depressive and manic episodes), when compared with controls. This occurred in the presence of normal substrate concentrations and unchanged expressions of eNOS and iNOS isoforms. Arginase II expression was not different between studied groups, indicating that L-arginine availability is not shifted to urea cycle in platelets. Decreased GMPc production was observed even in inalthered expression of GC. Activity and oxidative stress markers, evaluated through protein oxidation quantification and SOD and catalase activity, were not modified in platelets of BD patients while SOD activity was increased in all groups. Serum C-reactive protein (CRP) levels, an inflammatory marker, were higher in manic patients, compared with controls. The reduced production of NO observed in platelets of bipolar patients can be a link in this complex association between BD and cardiovascular disease.

Thrombocythaemia is defined as a circulating platelet count above 450x10$^9$/L in humans. The major cause of thrombocythaemia is reactive $(secondary)$ thrombocythaemia which occurs secondary to many conditions such as infection, cancer and inflammation. However, acquired clonal mutations in mainly Janus Kinas 2 $(JAK2)$, CALR and MPL cause essential thrombocythaemia $(ET)$. ET is a rare disease that leads to an increased risk of cardiovascular thrombotic events. Current treatment of ET uses combination of low dose aspirin to decrease platelet function and cytoreductive agents to decrease thrombopoiesis. The most commonly used cytoreductive agents are hydroxyurea, anagrelide and interferon-$alpha$ and all have unwanted side effects. Cytokine receptor-like factor 3 $(CRLF3)$ is a 2.4kb gene that is ubiquitously expressed throughout the haematopoietic system. Very little is known about the function of CRLF3, with only one peer reviewed journal article in the literature which shows that CRLF3 may negatively regulate the cell cycle at the G0/G1 phase. However, nothing is known about the role of CRLF3 in platelet biology. Using a Crlf3 knockout mouse $(Crlf3-/-)$ developed by the Wellcome Trust Sanger Institute we show CRLF3’s role in platelet biogenesis and how it could be used as a novel therapeutic target to treat ET. Crlf3-/- mice have an isolated and sustained 25-40$\%$ decrease in platelet count compared to wildtype $(WT)$ controls. Platelet function is unaffected as demonstrated in a range in a range of in vitro assays. The thrombocytopenia is a consequence of abnormalities in hematopoietic cells, as shown by bone marrow transplantations. Megakaryopoiesis is upregulated in Crlf3-/- mice and proplatelet morphology is unaffected, suggesting the thrombocytopenia is due to increased platelet clearance. Indeed, splenectomised Crlf3-/- mice show normalised platelet counts within 7 days, showing rapid splenic removal of platelets is responsible for the thrombocytopenia. Abnormal large platelet structures that resemble proplatelets shafts $(preplatelets)$ are abnormally present in the circulation of elderly Crlf3/- mice. Immunohistochemistry showed increased and aberrant tubulin expression in Crlf3-/- platelets compared to WT controls, especially in the preplatelet forms. Cold induced depolymerisation of microtubules was decreased in Crlf3-/- platelets, suggestive of increased tubulin stability, however, the ratio of detyrosinated to tyrosinated tubulin was not altered. We then crossbred Crlf3-/- mice with JAK2 V617F ET mice, to determine the effect of Crlf3 ablation of thrombocythaemia. Crossbred mice showed restoration of platelet counts to WT values without grossly affecting platelet function or other blood lineages, providing the rational for CRLF3 as a novel therapeutic target for treatment of ET. Finally, we aimed to resolve the crustal structure of CRLF3 and discover its interactome. To this end, we were able to resolve the crystal structure of a C-terminal portion of the full length protein containing the predicted fibronectin type III domain. To shed light on the interactome of CRLF3, endogenous CRLF3 was tagged with a tandem affinity purification $(TAP)$ tag using CRISPR/Cas9 technology in induced pluripotent stem cells $(iPSCs)$. We have been able to produce megakaryocytes from these TAP-tagged iPSCs by forward programming. However, as yet we have not been able to generate enough MKs to have adequate material to perform immunoprecipitation assays. Therefore, the interactome of CRLF3 in MKs remains unknown. In conclusion, we identified a mechanism by which Crlf3 controls platelet biogenesis. Slowed maturation of Crlf3-/- preplatelets in the peripheral circulation potentially due to increased structural stability leads to rapid removal of these immature forms by the spleen and therefore a decrease in platelet count. The isolated effect on platelet numbers and normalisation of platelet count in ET mice deficient of Crlf3 provides the rational for further study on CRLF3 drug targeting as a novel therapeutic strategy for ET.