Introdução: A esquizofrenia é um transtorno mental grave, que com frequência leva a uma deterioração progressiva, que mais recentemente tem sido descrita como uma doença sistêmica, com alterações progressivas cerebrais e corporais, e em parte por causa disto, associada a menor expectativa de vida, em média 20 anos menos que a população geral não afetada. Apesar de não terem especificidade, já foram determinados diferentes marcadores inflamatórios e oxidativos envolvidos; em contrapartida, pouco tem sido descrito sobre marcadores sistêmicos da esquizofrenia. A experiência clínica mostra que os pacientes crônicos vão ficando com pele, musculatura, e postura diferente dos não doentes, porém não há registro de estudo buscando estudar a postura, apesar de sabermos que a mesma depende da interação de inúmeros sistemas, incluindo o estado inflamatório do sujeito. Objetivo: Definir padrões de postura dentro de parâmetros básicos (postura cifolordótica - PCL, postura relaxada ou desleixada - PRD, postura escoliótica - PE) em pacientes esquizofrênicos e correlacionar com fase de doença (inicial-tardia) e com marcadores de resposta inflamatória imediata (proteína C reativa - PCR) e tardia (fator de von Willenbrand - FvW). Método: Estudo transversal, por amostra de conveniência, aprovado pelo comitê de ética do Hospital de Clínicas de Porto Alegre (número 110083 HCPA). Foram recrutados 40 indivíduos com diagnóstico CID-10 e DSMIVTR de esquizofrenia, forma estabilizada, em tratamento ambulatorial no HCPA. Os pacientes foram subdivididos em 2 subgrupos, de acordo com o estágio da doença (estágio inicial n=15 menos de 10 anos do 1° surto, estágio tardio n=25 10 anos ou mais do 1° surto). O grupo controle (n=26) foi recrutado através de uma rede social (Facebook®). Todos os indivíduos foram submetidos à biofotogrametria pelo método SAPO® para avaliar a postura. O nível de significância adotado foi de 5% para todas as variáveis e as análises foram realizadas no programa Statistical Package for the Social Sciences (SPSS, versão 18.0). Resultados: Não houve perdas; em relação à postura o grupo estágio inicial apresentou 15 ângulos com diferenças significativas quando comparado aos valores de referência, enquanto no grupo estágio tardio apenas 7 ângulos foram significativos; já na comparação com o grupo controle, apenas 6 ângulos foram significativos num total de 19. Os marcadores inflamatórios (PCR e FvW) não foram significativos em comparação aos estágios inicial e tardio da doença, porém a PCR apresentou correlação com a gravidade da doença e o FvW com um ângulo postural da protusão da cabeça. A variável dor também apresentou correlação com 5 ângulos posturais, 2 da coluna e 3 dos membros inferiores; além disso o grupo estágio tardio teve maior prevalência de dor quando comparado ao estagio inicial. Conclusões: Existe um padrão postural comum na esquizofrenia caracterizado principalmente por protusão da cabeça, hiperlordose e escoliose, sendo que no início da doença é mais agravante e no estágio mais tardio se estabiliza. Isto pode ser explicado por dois fenômenos: aumento de peso e o enfraquecimento muscular. Adicionalmente, temos a influência da dor e dos fatores inflamatórios, onde a PCR se associou à gravidade da doença, mas não à postura; já o FvW e a dor se associaram aos ângulos posturais, demonstrando suas influências nessa doença. Isto sugere que existe uma maior atividade patológica no início da doença, não só no cérebro, mas também no organismo, incluindo músculos e tecido adiposo. A hiperlordose parece estar mais associada à obesidade e tecido adiposo, enquanto que a extensão da cabeça e escoliose mais a alterações de musculatura. Todos estes achados juntos, se confirmados em amostras maiores e mais heterogêneas, irão nos auxiliar em futuras condutas no tratamento destas alterações posturais características deste transtorno (hiperlordose lombar, anteriorização da cabeça, diminuição da cifose torácica), além de estimular busca de novos meios de intervenções com o enfoque nos diferentes estágios da doença. / Introduction: Schizophrenia is a severe mental disorder that leads to progressive deterioration that more recently has been described as a systemic disease with progressive changes in the brain and body, and partly because of this, associated with lower life expectancy, in average 20 years less than the not affected general population. Although not specific, it has already been determined different inflammatory and oxidative markers involved, however little has been described about systemic markers of schizophrenia. Clinical experience shows that chronic patients are presenting skin, muscles, and posture different from the non patients, but there are no records of studies seeking to study the posture, even though we know that it depends on the interaction of many systems, including the inflammatory state of the subject. Objective: To define patterns of posture within basic parameters (PCL, PRD, PE) in schizophrenic patients and correlate with illness stage and with inflammatory markers of immediate (CRP) and delayed (vWF) response. Method: Cross-sectional study of a convenience sample approved by the ethics committee of the Hospital de Clinicas de Porto Alegre (HCPA). Forty patients with stabilized schizophrenia were recruited in the clinic treatment of HCPA, and analyzed according to 2 subgroups of the stage of the illness (early stage n = 15 less than 10 years from the 1st outbreak, late stage n = 25 10 years or more from the 1st outbreak). The control group (n = 26) were recruited through a social network (Facebook ®). All subjects underwent photogrammetry by SAPO ® method to evaluate posture. The level of significance was set at 5% for all variables and the analyzes were performed using the Statistical Package for the Social Sciences (SPSS, version 18.0). Results: there was no loss, in relation to posture the initial stage group showed 15 angles with significant differences when compared with the reference values, while the late stage group only 7 angles were significant, as compared with the control group only 6angles were significant in a total of 19, the inflammatory markers (CRP and vWF) were not significant in comparison to the initial and late stages of the disease, but the CRP showed correlation with disease severity and the vWF with a postural angle of the protrusion of the head. The pain variable also showed correlation with 5 postural angles, 2 of the column and 3 of the lower limbs, besides the late stage group had a higher prevalence of pain when compared to the early stage. Conclusions: There is a common postural pattern in schizophrenia characterized primarily by protrusion of the head, hyperlordosis and scoliosis, and that early in the illness is more aggravating and in the later stage it stabilizes. And it can be explained by two phenomena: weight gain and muscle weakness. Additionally, we have the influence of pain and inflammatory factors, where the CRP was associated with disease severity but not the posture, but the vWF and the pain were associated with postural angles demonstrating their influences in this disease. This suggests that there is increased pathological activity in the onset of the disease, not only in the brain but also in the body including muscles and adipose tissue. The hyperlordosis appears to be most strongly associated with obesity and adipose tissue, whereas the length of the head and scoliosis more to changes in muscles. All together these findings, if confirmed in larger and more heterogeneous samples will help us in future conduct in treating these postural changes characteristic of this disorder (lumbar hyperlordosis, forward head posture, decreased thoracic kyphosis), besides stimulating the search for new means of interventions with the focus on different stages of the disease.

ADAMTS13 is a metalloprotease that regulates the length, and thus, the platelet-capturing capacity of von Willebrand factor. The regulation of ADAMTS13 activity remains poorly understood. Numerous circulating proteases cleave ADAMTS13 in vitro, impairing its activity, but the physiological significance of this mechanism remains unknown. Two commonly cleaved regions within ADAMTS13 were identified and mutants were developed: two with one of each region mutated (T4L and T8L mutants), one with both regions mutated (T4L/T8L or “double” mutant), and one with an additional elastase site mutated (T4L/T8L + I380G). This work characterizes the mutants’ resistance to proteolysis and compares the activity of the double mutant to wild-type ADAMTS13 (WT). Each mutant and WT was incubated with purified coagulation and neutrophil proteases, activated neutrophils, or added to plasma before initiating coagulation with or without tissue plasminogen activator. Cleavage patterns were visualized with western blot. FRETS-VWF73 and microfluidic flow assays were used to compare WT and mutant activity. Coagulation proteases cleave both predicted sites within WT, and the double mutant exhibits near complete resistance to cleavage over 3 hours. Resistance to degradation by neutrophil proteases is prolonged in the double mutant, but additional cleavage sites are present. Elastase cleavage is prevented in the T4L/T8L + I380G mutant. In plasma, WT is degraded upon initiating coagulation and subsequent fibrinolysis, which is prevented in the double mutant. WT is also degraded in the presence of activated neutrophils, and the double and T4L/T8L + I380G mutants exhibit improved but incomplete resistance. Finally, the mutants exhibit similar activity to WT using FRETS-VWF73 and the microfluidic assay. This work validates the location of two protease-sensitive regions within ADAMTS13 and confirms the resistance of the double mutant to coagulation proteases in vitro. Future work will complete the activity analysis, and compare the mutants’ therapeutic efficacy to WT in vivo. / Thesis / Master of Science (MSc) / Current drugs used to dissolve blood clots can cause major bleeding. Therefore, safer treatments need to be developed. An important step in the clotting pathway is platelet accumulation in the injured vessel. Platelets stick to string-like protein, von Willebrand Factor (VWF), and ADAMTS13 is a protein that regulates this by cutting VWF strings. ADAMTS13 shows promise as a treatment for clots without causing bleeding, but it is unclear how its activity is controlled. ADAMTS13 can be degraded by other proteins, however the importance of this process in the body is unknown. This work characterizes a degradation-resistant ADAMTS13 mutant, which may be used to study whether ADAMTS13 degradation reduces its therapeutic effectiveness. The mutant has normal VWF-cutting activity, is resistant to degradation by clotting proteins, and is partially resistant to proteins released by neutrophils, an important immune cell in clotting. Future studies will investigate its effectiveness at treating clots in animals.

Background: Chronic enteropathies (CE) predispose dogs to thromboembolic disease, but the underlying mechanisms are poorly understood. Humans with CE have decreased activity of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a von Willebrand factor (vWF) cleaving enzyme, and increased circulating vWF. The primary aim of this study is to assess plasma ADAMTS13 activity, vWF antigen (vWF:Ag) concentration, and vWF collagen binding activity (vWF:CBA) in dogs with CE. Hypotheses: Dogs with CE have reduced plasma ADAMTS13 activity, increased vWF:Ag, and increased vWF:CBA compared to healthy control dogs. Animals: Twenty privately-owned dogs with CE and 40 healthy dogs were recruited from a specialty hospital population. Methods: Prospective observational study. Dogs were confirmed to have CE using histopathology. ADAMTS13 activity was measured using a commercially available ELISA kit (DiapharmaⓇ) in 20 dogs with CE and 40 healthy control dogs. Plasma vWF:Ag was assessed in 20 dogs with CE and 20 healthy control dogs. Plasma vWF:CBA was assessed in 19 dogs with CE and 20 healthy control dogs. For statistical analysis, an unpaired t-test was used for normally distributed data and Wilcoxon rank sum was used for non-Gaussian distribution. Significance was set at P < 0.05. Results: Plasma ADAMTS13 activity and vWF:Ag were not significantly different compared to healthy dogs (P = 0.07, P = 0.16, respectively). Plasma vWF:CBA was significantly decreased compared to healthy dogs (P = 0.007). Conclusions and clinical relevance: Plasma ADAMTS13 activity was not significantly different in dogs with CE compared to healthy dogs and is unlikely to be the primary mechanism for hypercoagulability associated with CE. Forty-three percent of CE dogs with hypoalbuminemia had ADAMTS13 activity below reference interval. Further studies are warranted to evaluate ADAMTS13 activity in a subset of dogs with CE, including those with protein losing enteropathy and thromboembolism. / Master of Science / Background: Dogs with chronic gastrointestinal disease, or chronic enteropathies (CE), often have abnormal blood clotting which predisposes to thrombosis. The mechanisms leading to this abnormal blood clotting are poorly understood. Humans with CE also suffer from abnormal blood clotting and thrombosis. Decreased activity of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a von Willebrand factor (vWF) cleaving enzyme, and increased circulating vWF have been reported as contributors to this abnormal blood clotting in people. Von Willebrand factor is a protein that allows platelets to stick together and adhere to damaged tissue to facilitate blood clot formation. The concentration of vWF can be measured using vWF antigen (vWF:Ag) and the activity of vWF can be measured using vWF collagen binding activity (vWF:CBA). Hypothesis: Dogs with CE have reduced plasma ADAMTS13 activity, increased vWF:Ag, and increased vWF:CBA compared to healthy control dogs. Animals: Twenty privately-owned dogs with CE and 40 healthy dogs were recruited from a hospital population. Methods: Prospective observational study. Plasma ADAMTS13 activity, vWF:Ag and vWF:CBA were assessed in dogs with CE and compared to healthy control dogs. Results: There was no difference in plasma ADAMTS13 activity and vWF:Ag between the two groups. The mean vWF:CBA was significantly lower in CE dogs compared to healthy dogs. Conclusions and clinical relevance: Reduced ADAMTS13 activity is unlikely to be the primary mechanism for abnormal blood clotting in dogs with CE.

Studies demonstrated ADAMTS13 possesses unique properties with a mystifying regulatory mechanism. ADAMTS13’s role is in its proteolytic function to its VWF. The disparity in the hemostatic balance between ADAMTS13 activity and the distribution of VWF multimers could result in the bleeding disorder Von Willebrand Disease (VWD) or the thrombotic disorder thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is constitutively secreted as an active protease, yet VWF retains its capacity to recruit platelets. This ability makes ADAMTS13 an enigmatic protease with an unknown regulatory mechanism. Currently, the postulated regulatory mechanism of ADAMTS13 is in its open/closed conformation, yet ADAMTS13 activity is retained in both forms. Literature showed that few proteases are capable of degrading ADAMTS13 in-vitro. We hypothesize that the partial degradation of ADAMTS13 regulates its activity, thereby stabilizing VWF and promoting thrombosis. The goals of this project were to develop and optimize in-vitro plasma BioID to identify novel interactions to ADAMTS13, validate novel interactions, identify proteases capable of degrading ADAMTS13 and their proteolytic sites, and develop protease-resistant ADAMTS13 mutants as novel therapeutics to thrombotic disorders. We optimized the BioID technique to be used in-vitro in plasma, to study novel interactions with ADAMTS13. Our results identified novel potential interactions with vitronectin or plasminogen. Validation studies disregarded vitronectin’s interaction and confirmed plasminogen’s interaction through the CUB and Kringle domains in a lysine-dependent manner. Further, the list of proteases capable of degrading ADAMTS13 was expanded to include FXIa and neutrophil-derived proteases including Cathepsin G, elastase, and hPR3. Activated neutrophils played a stronger role than coagulation proteases in degrading ADAMTS13 in vivo, while also demonstrating that elastase is a more potent regulator. Proteolytically degraded sites on ADAMTS13 were identified and proteolytic-resistant ADAMTS13 mutants were produced accordingly, which we aim to be utilized as a novel therapeutic to thrombotic disorders. / Thesis / Doctor of Philosophy (Medical Science)

This study tested the hypothesis that levothyroxine supplementation increases plasma von Willebrand factor (vWf) concentration and enhances vWf function. The effects of levothyroxine administration were evaluated in 8 euthyroid Doberman Pinschers with plasma vWf concentration <30%. Levothyroxine (0.04mg/kg PO q12hours) and placebo were administered for 30 days in a 2-period, 2-treatment, double-blinded, crossover design with a 30-day washout period between treatments. Buccal mucosal bleeding time (BMBT), vWf antigen concentration (vWf:Ag), vWf collagen binding activity (vWf:CBA), Factor VIII coagulant activity (FVIII:C), serum total thyroxine (T4), free thyroxine (fT4), 3,5,3â -triiodothyronine (T3), and thyroid stimulating hormone were measured on days 0, 2, and 30 of each treatment period. The dogs had markedly low plasma vWf:Ag concentrations (mean 8.9%; reference range 70-180%) and vWf:CBA (mean 11.1%; reference range >70%). All dogs had FVIII:C activity within reference range. The response to placebo versus active levothyroxine treatment revealed no significant differences between groups at any time for BMBT, vWf:Ag, vWf:CBA, and FVIII:C. Serum total thyroxine, fT4, and T3 were significantly higher in the levothyroxine-treated group compared to the placebo group at days 2 and 30. Thyroid stimulating hormone was significantly lower in the levothyroxine-treated group compared to the placebo group at days 2 and 30. Levothyroxine (0.04mg/kg) caused laboratory evidence of hyperthyroidism but did not affect plasma FVIII:C and vWf:Ag concentration or the vWf-dependent functional parameters of collagen binding and BMBT. The results of this study do not reveal a direct action of levothyroxine supplementation on plasma vWf concentration or activity in euthyroid Doberman Pinschers. / Master of Science

Le facteur VIII (FVIII) est une glycoprotéine de la coagulation plasmatique, co-facteur du facteur IX activé (FIXa). Son métabolisme dépend de multiples facteurs limitant ou favorisant sa survie ou sa fonction. L’objectif de ce travail était d’analyser les différents paramètres pouvant influencer le taux de FVIII circulant et sa fonction pro-coagulante. Il a compris 4 volets : 1) Nous avons montré que la rétention intra-cellulaire connue du FVIII était en grande partie liée à son agrégation et sa dégradation par les 2 voies protéasomale et lysosomale. 2) Nous avons analysé les mutations du gène F8 responsables de l’hémophilie A dans une large cohorte de patients. Leur analyse bio-informatique a permis de démontrer leur caractère délétère pour le FVIII. L’influence de ces mutations sur la survenue d’allo-anticorps anti-FVIII a été étudiée et stratifiée, en association avec l’origine ethnique et la recherche d’antécédents familiaux d’nticorps. 3) La recherche des facteurs influençant les taux de FVIII chez les conductrices d’hémophilie A a mis en évidence des déterminants majeurs : l’existence d’une maladie génétique additionnelle responsable d’un déficit en FVIII, le taux de facteur Willebrand et l’inactivation non aléatoire du chromosome X ; et des déterminants secondaires : l’âge, la sévérité de l’hémophilie, le polymorphisme D1241E du gène F8 et 5 nouveaux polymorphismes de la LRP1 localisés dans ses sites de liaison pour le FVIII. 4) Nous avons analysé 8 FVIII recombinant mutés in vitro en alanine dans la région 1808-1818 du FVIII. Les études antérieures n’analysant que la chaîne légère, ont montré que cette région était la plus affine pour le FIXa. Nous démontrons ici que la région 1808-1818 ne paraît pas si essentielle à la fonction du FVIIIcar au sein de la molécule entière, son affinité diminue et ses mutations n’altèrent que très modérément l’activité du FVIII. / The factor VIII (FVIII) is a glucoprotein of the coagulation, being the cofactor of the activated factor IX (FIXa). Its metabolism depends on various limiting factors or enhancing its survey or function. The objective of this research’s work was to analyse different parameters that could influence the plasma FVIII level and its pro-coagulant function. It included 4 parts : 1) We showed that the known intra-cellular retention of FVIII was mainly due to its aggregation and degradation following both proteasomal and lysosomal pathways. 2) We analysed FVIII gene mutations responsible for hemophilia A in a large patients cohort. The bio-informatic analysis demonstrated its deleterious consequence. The influence of these mutations on the anti-FVIII antibodies occurrence was stratified, in association with ethnicity and familial antecedent of inhibitor. 3) The research of factors influencing FVIII levels in hemophilia A carriers showed : i) major determinants such as the presence of an additional genetic disease characterised by a FVIII deficiency, the factor Willebrad’s level and the non-random inactivation of the X chromosome; and ii) minor determinants : age, severity of hemophilia, the polymorphism D1241E of FVIII gene, and 5 new polymorphisms of LRP1 located in its binding site for FVIII.4) We analysed 8 recombinant FVIII with in vitro created mutations in its 1808-1818 region. Previous studies that analysed only the FVIII light chain, have shown that this region constituted the more affine binding site of FVIII for FIXa. We demonstrated here that the 1808-1818 region is not as essential as it was reported because within the entire molecule, its affinity decreases and mutations affecting it do alter mildly the FVIII activity.

Le facteur Willebrand (VWF) est une proteine multimerique qui a une sensibilite unique aux forces de cisaillement et aux variations hemodynamiques du flux sanguin comme celles rencontrees lors d’utilisation de dispositifs cardiovasculaires tels qu’un remplacement valvulaire aortique transcatheter (TAVI) ou un assistance circulatoire mecanique a flux continu (ACM-FC). Des travaux anterieurs nous ont permis de mettre en evidence une secretion endotheliale declenchee par les modifications du flux liees a l’utilisation de ces dispositifs.Dans la première partie de la these, nous avons choisi d’etudier le role de la pulsatilite arterielle dans la reponse endotheliale a l’aide de plusieurs modeles animaux porcins d’ACM-FC pour isoler le role de la pulsatilite dans un environnement a forces de cisaillement elevees et constantes. Nous avons observe dans un modele dose-reponse la relation entre le niveau de pulsatilite et la multimerisation du VWF et dans un modele en cross-over le caractere dynamique du relargage endothelial en reponse a des variations aigues de pulsatilite.Ces resultats nous ont permis de conceptualiser dans la deuxième partie l'utilisation du VWF dans l’evaluation de la severite des fuites paravalvulaires (FPV) post-procedure TAVI. Deux cohortes de 183 et 201 patients ont permis de demontrer l’excellente capacite diagnostique de l’analyse multimerique du VWF avec une sensibilite, une specificite et une valeur predictive negative de respectivement 92.3%, 94.9%, et 98.7%. Le test de diagnostic rapide TO-ADP (temps d’occlusion a l’ADP) donnait des resultats equivalents pour un seuil > 180 sec.Enfin dans la troisième partie de la these nous avons concu le design d’un essai clinique permettant d’evaluer la valeur ajoutee de l’utilisation de ce test de diagnostic rapide TO-ADP en salle de catheterisme pour l’amelioration des resultats proceduraux et cliniques des procedures TAVI. / Willebrand factor (VWF) is a multimeric protein that has a unique sensitivity to shear forces and hemodynamic variations in blood flow such as those encountered when using cardiovascular devices such as transcatheter aortic valve replacement (TAVI) or continuous flow mechanical circulatory assistance (CF-CAM).

Made available in DSpace on 2015-06-17T19:34:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-29. Added 1 bitstream(s) on 2015-06-18T12:47:02Z : No. of bitstreams: 1 000831074.pdf: 615076 bytes, checksum: c97e8862ed17121a7d6c2ddd7dfb24e1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fator VIII (FVIII), o fator de von Willebrand (FvW) e o fibrinogênio são de suma importância na coagulação sanguínea, com diferentes funções fisiológicas. Por conter altas concentrações destes fatores a transfusão de crioprecipitado é uma terapia utilizada principalmente em pacientes que apresentam Doença de von Willebrand, Hemofilia A (deficiência do FVIII), ou pacientes que sofrem de hipo ou disfibrinogenemia. Este hemocomponente é um precipitado obtido após o descongelamento parcial (entre 1 e 6°C) do plasma fresco congelado, e também é conhecido como fator anti-hemofílico. Estudos têm demonstrado que o protocolo de congelamento e armazenamento do crioprecipitado afeta a qualidade do produto e a viabilidade destes fatores. Com o objetivo de avaliar a viabilidade do crioprecipitado canino em diferentes protocolos de congelamento e armazenamento foram avaliados dois grupos compostos de 10 unidades de crioprecipitado canino (n=20). Após a centrifugação das bolsas de sangue, o plasma fresco foi congelado a -80ºC (grupo I) e a -20ºC (grupo II). Vinte e quatro horas após o congelamento das bolsas, estas foram submetidas ao procedimento de extração do crioprecipitado. Os crioprecipitados das bolsas dos dois grupos foram submetidos à determinação do TP, TTPA, FVIII, FvW e fibrinogênio, no momento zero e após seis meses de estocagem. Para a realização das coletas, foram utilizadas bolsas sanguíneas triplas de plástico, com anticoagulante CPDA-1, sendo a bolsa principal com capacidade para 450 mL de sangue total (JP Indústria Farmacêutica®). Após o crioprecipitado devidamente pronto, uma alíquota de aproximadamente 5 mL da bolsa de crioprecipitado foi separada em criotubos para análise da amostra pré-estocagem e seis meses pós estocagem. As amostras obtidas em cada momento foram congeladas à -80ºC até o momento do processamento. Os resultados mostraram um decréscimo significativo dos fatores e ... / The factor VIII (FVIII), the von Willebrand factor (vWF) and the fibrinogen are extremely important to the blood clotting process, with various physiological functions. Because it contains high concentrations of these factors and fibrinogen, transfusing cryoprecipitate is a therapy mainly used in patients who have von Willebrand disease, Hemophilia A (FVIII deficiency), or who suffer from hypo/dysfibrinogenemia. This hemocomponent is a precipitate obtained after the partial thawing process (between 1 and 6ºC) of fresh frozen plasma, and which is also known as the anti-hemophilic factor. Studies have demonstrated that the cryoprecipitate freezing and storage protocol affects the product quality as well as these factors viability. In order to evaluate the canine cryoprecipitate viability in different freezing and storage protocols, two groups containing 10 units of canine cryoprecipitate (n=20) were evaluated. Following the blood centrifugation, the fresh plasma was frozen at -80ºC (group I) and at -20ºC (group II). Twenty-four hours after freezing the blood bags, they were submitted to the cryoprecipitate extraction procedure. The cryoprecipitate from both groups of blood bags were submitted to the TP, TTPA, FVIII, FvW and fibrinogen determination process, at time zero and after six months of storage. During the collections, triple plastic blood bags were used, along with the anticoagulant CPDA-1, being the main bag capacity of 450 mL of whole blood (JP Indústria Farmacêutica®). After having the cryoprecipitate properly ready, an approximately 5 mL aliquot of cryoprecipitate was separated into cryovials to be analysed pre-storage and six months after storage. However, there was no significant difference between treatments, demonstrating that the difference in initial freezing temperature did not influence the decrease of the factors after six months storage at -20°C / FAPESP: 2012/13677-6

Orientador: Regina Kiomi Takahira / Banca: Luiz Henrique de Araújo Machado / Banca: Simone Gonçalves Rodrigues Gomes / Resumo: O fator VIII (FVIII), o fator de von Willebrand (FvW) e o fibrinogênio são de suma importância na coagulação sanguínea, com diferentes funções fisiológicas. Por conter altas concentrações destes fatores a transfusão de crioprecipitado é uma terapia utilizada principalmente em pacientes que apresentam Doença de von Willebrand, Hemofilia A (deficiência do FVIII), ou pacientes que sofrem de hipo ou disfibrinogenemia. Este hemocomponente é um precipitado obtido após o descongelamento parcial (entre 1 e 6°C) do plasma fresco congelado, e também é conhecido como fator anti-hemofílico. Estudos têm demonstrado que o protocolo de congelamento e armazenamento do crioprecipitado afeta a qualidade do produto e a viabilidade destes fatores. Com o objetivo de avaliar a viabilidade do crioprecipitado canino em diferentes protocolos de congelamento e armazenamento foram avaliados dois grupos compostos de 10 unidades de crioprecipitado canino (n=20). Após a centrifugação das bolsas de sangue, o plasma fresco foi congelado a -80ºC (grupo I) e a -20ºC (grupo II). Vinte e quatro horas após o congelamento das bolsas, estas foram submetidas ao procedimento de extração do crioprecipitado. Os crioprecipitados das bolsas dos dois grupos foram submetidos à determinação do TP, TTPA, FVIII, FvW e fibrinogênio, no momento zero e após seis meses de estocagem. Para a realização das coletas, foram utilizadas bolsas sanguíneas triplas de plástico, com anticoagulante CPDA-1, sendo a bolsa principal com capacidade para 450 mL de sangue total (JP Indústria Farmacêutica®). Após o crioprecipitado devidamente pronto, uma alíquota de aproximadamente 5 mL da bolsa de crioprecipitado foi separada em criotubos para análise da amostra pré-estocagem e seis meses pós estocagem. As amostras obtidas em cada momento foram congeladas à -80ºC até o momento do processamento. Os resultados mostraram um decréscimo significativo dos fatores e ... / Abstract: The factor VIII (FVIII), the von Willebrand factor (vWF) and the fibrinogen are extremely important to the blood clotting process, with various physiological functions. Because it contains high concentrations of these factors and fibrinogen, transfusing cryoprecipitate is a therapy mainly used in patients who have von Willebrand disease, Hemophilia A (FVIII deficiency), or who suffer from hypo/dysfibrinogenemia. This hemocomponent is a precipitate obtained after the partial thawing process (between 1 and 6ºC) of fresh frozen plasma, and which is also known as the anti-hemophilic factor. Studies have demonstrated that the cryoprecipitate freezing and storage protocol affects the product quality as well as these factors viability. In order to evaluate the canine cryoprecipitate viability in different freezing and storage protocols, two groups containing 10 units of canine cryoprecipitate (n=20) were evaluated. Following the blood centrifugation, the fresh plasma was frozen at -80ºC (group I) and at -20ºC (group II). Twenty-four hours after freezing the blood bags, they were submitted to the cryoprecipitate extraction procedure. The cryoprecipitate from both groups of blood bags were submitted to the TP, TTPA, FVIII, FvW and fibrinogen determination process, at time zero and after six months of storage. During the collections, triple plastic blood bags were used, along with the anticoagulant CPDA-1, being the main bag capacity of 450 mL of whole blood (JP Indústria Farmacêutica®). After having the cryoprecipitate properly ready, an approximately 5 mL aliquot of cryoprecipitate was separated into cryovials to be analysed pre-storage and six months after storage. However, there was no significant difference between treatments, demonstrating that the difference in initial freezing temperature did not influence the decrease of the factors after six months storage at -20°C / Mestre

Orientador: Joyce Maria Annichino-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T16:00:11Z (GMT). No. of bitstreams: 1 Fonseca_BrunadeMoraesMazetto_M.pdf: 657024 bytes, checksum: 715789e040378f27c5de278879f4535b (MD5) Previous issue date: 2011 / Resumo: Níveis elevados de marcadores inflamatórios e fatores de coagulação têm sido relacionados com a patogênese do TEV. Particularmente, a relação inversa entre o FVW e a atividade da ADAMTS13 já foi previamente descrita em pacientes com trombose arterial. Níveis de FVW também mostraram-se elevados durante processos inflamatórios e portanto, poderiam desempenhar um papel de ligação entre inflamação e coagulação nos pacientes com TEV. Objetivo: Avaliar a atividade da ADAMTS13 e do FVW e sua associação com marcadores inflamatórios e evolução clínica pós-trombótica em pacientes com TEV. Pacientes e Métodos: Setenta e sete pacientes com TEV, entre sete meses e seis anos após o episódio agudo, atendidos no Hemocentro de Campinas - UNICAMP foram incluídos neste estudo e 77 indivíduos normais foram selecionados como controles, pareados por idade, gênero, etnia e grupo sanguíneo. A atividade da ADAMTS13 e do FVW foram avaliados pela ligação do FVW ao colágeno, o dímero-D por turbidimetria, a PCR por nefelometria, TNF-'alfa', IL-6, IL-8, antígeno do FVW e da ADAMTS13 foram determinados por ELISA. A presença de trombo residual foi avaliada por ultrassom com Doppler e a SPT através da escala Villalta. Resultados: Trinta pacientes (39%) tiveram TEV causado por fatores de risco transitórios, especialmente pelo uso de anticoncepcional e 47 pacientes tiveram TVE espontâneo. A atividade inflamatória estava aumentada nos pacientes em comparação aos controles, demonstrada pelo aumento significativo dos níveis séricos de TNF-'alfa' and IL-6 nos primeiros (mediana= 2,25 vs 1,59pg/mL, P?0,001; 1,16 vs 0,98pg/ml, P=0,013, respectivamente). Os níveis de IL-8 e PCR foram similares entre os 2 grupos (mediana= 18,3 vs 18,27pg/mL, P=0,47; 0.21 vs 0,17mg/dL, P=0,29, respectivamente). Trinta e dois pacientes (42,8%) foram definidos como tendo um aumento da atividade coagulante, expressa pelo dímero-D > 0,55mg/dL. Nesse grupo de pacientes todos os marcadores inflamatórios como TNF-'alfa', IL-6, IL-8 and PCR, estavam significativamente aumentados quando comparados aos pacientes com dímero-D ? 0,55 mg/L (P=0,0057; 0,001; 0,0093 e 0,0075; respectivamente). A presença de SPT e trombo residual não foram associados ao aumento da atividade coagulante. A atividade da ADAMTS13 e os níveis séricos de IL-8 estavam aumentados em pacientes com SPT quando comparados aos pacientes sem SPT. Todos os marcadores inflamatórios e parâmetros da coagulação estudados foram similares em pacientes independentemente da presença do trombo residual. Conclusão: Este estudo sugere que exista atividade inflamatória e procoagulante nos pacientes mesmo após o episódio agudo do TEV, que, entretanto, não se mostrou estar relacionada com a persistência das seqüelas clínicas e radiológicas da TVP. Além disso, o aumento do FVW nos pacientes corrobora a hipótese de ativação crônica da inflamação. Neste contexto, o aumento observado da ADAMTS 13 poderia ser compensatório frente ao aumento crônico do FVW e poderia inclusive atuar com um mecanismo protetor contra a atividade pró-trombótica observada nestes pacientes / Abstract: Introduction: Increased levels of inflammatory markers and clotting factors have been related to the pathogenesis of VTE. Particularly, the inverse relation between VWF and ADAMTS13 activity has been previously described in patients with arterial thrombosis. VWF levels are also known to be increased during inflammatory processes and therefore could play a role linking the inflammatory and coagulation systems activities in patients with VTE. Objective: To evaluate the activity of ADAMTS13 and VWF in patients with VTE and its association with inflammatory markers and clinical outcome of post-thrombotic syndrome. Patients and methods: Seventy-seven patients with VTE, 7 months to six years after the acute episode, attended at the Hemocentro of Campinas - UNICAMP, were included in this study and 77 normal subjects were selected as controls, matched by gender, age, ethnicity and ABO blood group. The activity of ADAMTS 13 was performed by VWF collagen binding, D-dímer by turbidimetry, CRP by nephelometry , and TNF-'alpha', IL-6 and IL-8, VWF and ADAMTS13 antigen by ELISA. The presence of RVO was investigated by duplex examination and PTS by Villalta scale. Results: Thirty patients (39%) had VTE caused by transient risk factors, mainly the use of oral contraceptives, and 47 patients had spontaneous VTE. Serum levels of TNF-'alpha' and IL-6 were significantly increased in patients when compared to controls (median= 2.25 vs 1.59pg/mL, P?0.001; 1.16 vs 0.98pg/ml, P=0.013, respectively) whereas levels of IL-8 and CRP were similar among the groups (median= 18.3 vs 18.27pg/mL, p=0.47; 0.21 vs 0.17mg/dL, P=0.29, respectively). Thirty-two patients (42,8%) had D-dimer > 0.55 mg/L and were defined as having increased coagulation activity. Inflammatory markers, such as TNF-'alpha', IL-6, IL-8 and CRP, were significantly higher in those patients, comparing to patients with D-dimer ? 0.55 mg/L (P=0.0057, 0.001, 0.0093 and 0.0075, respectively). The presence of PTS or RVO were not associated with increased inflammatory or coagulation activity. Only ADAMTS13-CBA and plasma levels of IL-8 were higher in patients with PTS comparing to patients without PTS. All inflammatory markers and coagulation parameters studied were similar in patients regardless the presence of RVO. Conclusion: Our findings suggest that there is an inflammatory and pro-coagulant activity in patients even after the acute episode of VTE, however, these activities were not related to the persistence of clinical and radiological sequels of DVT. Moreover, the increasing levels of VWF, observed in patients, support the hypothesis that the inflammation is chronically activated. In this context, the increasing levels of ADAMTS13, also observed in patients, could be explained as a compensatory mechanism and maybe act as a protection against pro-thrombotic activity seen in these patients / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica