A doença de von Willebrand (DvW) é uma coagulopatia hereditária, causada por defeitos qualitativos ou quantitativos do fator de von Willebrand. O tratamento e a prevenção das intercorrências da DvW são bastante dispendiosos e, em geral, se baseiam na administração de concentrado de Fator VIII/FvW (CFVIII/FvW) e/ou da Desmopressina (DDAVP). Em muitas situações, o DDAVP é um tratamento eficaz que não expõe os pacientes aos riscos de contaminação viral e apresenta custo inferior quando comparado ao CFVIII/FvW. No entanto, a dificuldade de diagnóstico e classificação da DvW, bem como o baixo número de pacientes que se submetem ao teste para avaliação da resposta ao DDAVP, restringem a indicação do DDAVP como alternativa terapêutica para esses pacientes. O objetivo deste estudo foi avaliar retrospectivamente a indicação, o uso e o custo dos medicamentos no tratamento de pacientes com DvW com DDAVP e CFVIII/FvW no Hemocentro de Belo Horizonte no período entre 2011 a 2013. Este estudo incluiu 124 (24,22%) pacientes com DvW atendidos no hemocentro. Em 18 pacientes (14,52%) o diagnóstico de DvW não pode ser confirmado. Doze pacientes (9,68%) não puderam ser classificados e 73 foram classificados como tipo 1, 19 como tipo 2 e 2 pacientes como tipo 3. Oitenta e um pacientes fizeram o teste de DDAVP, sendo que 87,65% foram considerados responsivos. Nos pacientes tipo 1, a taxa de resposta ao DDAVP foi de 92%. Quase 32% dos pacientes tipo 1 não realizaram o teste. No período avaliado, foram utilizadas 3.794mcg de DDAVP (R$13.165,18) e 1.582.250 UI de CFVIII/FvW (R$1.075.930,00). Vinte por cento dos pacientes responsivos ao DDAVP utilizaram CFVIII/FvW em indicações onde o DDAVP poderia ter sido considerado (69.200UI de CFVIII/FvW versus 131 ampolas de DDAVP). Nos pacientes potencialmente responsivos ao DDAVP 108.700UI de CFVIII/FvW (R$73.916,00) poderiam ter sido substituídas por 247 ampolas de DDAVP (R$3.428,36). A escolha do DDAVP nessas situações poderia representar uma economia de 95,7% do valor gasto no tratamento do grupo de 27 pacientes responsivos e potencialmente responsivos ao DDAVP e 10,6% do valor total gasto para todo o tratamento dos pacientes no período do estudo. Estudos mais complexos de farmacoeconomia serão necessários para avaliar a magnitude da economia gerada com esse uso. O presente estudo mostrou que o DDAVP é uma alternativa terapêutica de menor custo, cuja indicação e utilização podem ser ampliadas no tratamento dos pacientes com DvW. Dessa maneira, a implementação de estratégias visando melhorar o diagnóstico, a classificação da doença, o acesso à testagem quanto à resposta ao DDAVP, bem como a conscientização dos profissionais de saúde e pacientes, quanto ao custo e segurança do DDAVP podem contribuir para o uso racional dos recursos destinados a essa parcela da população. / Von Willebrand disease (VWD) is a hereditary coagulopathy caused by qualitative or quantitative defects on von Willebrand factor. The treatment and the prevention of VWD complications is quite expensive and is generally based on the administration of vWF:FVIII Concentrates and/or Desmopressin (DDAVP). In many situations, DDAVP is an effective treatment that does not expose patients to viral contamination risks and presents a lower cost when compared to vWF:FVIII concentrates. However, the difficulty of diagnosis and classification of VWD, as well as the low number of patients tested to their responsiveness to DDAVP, restrict the use of DDAVP as an alternative treatment for these patients. The aim of this study was to evaluate retrospectively the clinical indications, the use and the cost of treatment of VWD patients with DDAVP and vWF:FVIII concentrates in the Blood Center of Belo Horizonte between 2011 and 2013. This study enrolled 124 (24.22%) VWD patients attended at the Blood Center.For18 (14.52%) patients, the diagnosis of VWD could not be confirmed. Twelve patients (9.68%) could not be classified and 73patients were classified as type 1, 19 as type 2 and 2 as type 3. Eighty-one patients were tested for DDAVP response and 87.65% (n=71) were considered responsive for the treatment. For type 1 VWD patients, the response rate to DDAVP was 92%. Almost 32% of type 1 VWD patients were not tested. In the period evaluated, 3,794mcg of DDAVP (R$ 13,165.18) and 1,582,250 IU of vWF:FVIII concentrates (R$ 1,075,930.00) were used. Between the cases with clinical indication of DDAVP use, 20% patients used vWF:FVIII concentrates (69.200UI of vWF:FVIII versus 131ampoules of DDAVP). In patients with good responsive to DDAVP, 108,700 IU of vWF:FVIII concentrates used (R$ 73,916.00) could be replaced by 247 ampoules of DDAVP (R$ 3,428.36). The choice of DDAVP in these situations could represent an economy of 95.7% of the value spent on the treatment of the 27 responsive and potentially responsive patients to DDAVP and 10.6% of the total value spent for the entire treatment of patients in the study period. More detailed studies of pharmacoeconomics are necessary to assess the magnitude of the economy generated by the use of DDAVP. This study demonstrated that DDAVP is a lower cost therapeutic alternative whose indication and use can be enhanced in the treatment of VWD patients. In this context, adoption of strategies to improve the differential diagnosis, expand the DDAVP responsiveness test, and aware health professionals and patients about the costs and safety use of DDAVP, could contribute to the rational use of resources designated to treatment of VWD.

Understanding the differential bonding mechanics underlying bleeding disorders is of crucial importance to human health. In this research insight is provided into how four of these bleeding disorders (each with somewhat similar clinical characteristics), work at the molecular bond level. The bleeding diseases studied here can result from defects in the platelet glycoprotein (GP) Ibα the von Willebrand factor (vWF) molecule, or the ADAMTS-13 enzyme. Types 2B and 2M von Willebrand Disease (VWD) result in excess bleeding, yet type 2B has increased binding affinity between platelet GPIbα and vWF, while type 2M has decreased binding affinity between these two molecules. Platelet type VWD (pt-VWD) causes mutations in the GPIbα molecule and has similar characteristics to type 2B VWD. Further, in thrombotic thrombocytopenic purpura, bleeding results when there is a lack of active ADAMTS-13 enzyme. Each disease results in patient bleeding, but due to different mechanisms. This dissertation will explore the bonding mechanics between GPIbα and vWF and how they are altered in each disease state. To observe the GPIbα-vWF bonding mechanics, rolling velocities, transient tethering lifetimes, and tether frequency were determined using a parallel plate flow chamber. Data from these experiments suggest that wt-wt interactions are force dependent and have biphasic catch-slip bonding behavior. The data show that the shear stress at which the maximum mean stop time occurs differs between gain-of-function and loss-of-function mutations. Using similar methods, we study the changes resulting from pt-VWD mutations in GPIbα, and find that the catch bond seen for wt-wt interactions is lost for these mutations. Further, the data suggest that interactions with gain-of-function GPIbα mutations may be transport rather than force dependent. Finally, how the GPIbα-vWF tether bond changes for thrombotic thrombocytopenic purpura was also investigated to show that the bond lifetime in the absence of the enzyme is increased presenting a possible rationale for why bleeding occurs in this disease. Overall, the data show how the bonding mechanics of the GPIbα-vWF tether bond differ in four bleeding diseases. Further, these observations offer potential explanations for how these changes in the bonding mechanism may play a role in the observed patient bleeding.

Von Willebrand disease (vWD) is one of the most common inherited bleeding diseases, caused by a qualitative or quantitative deficiency of the von Willebrand factor (vWF). vWF is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells and it is present in the subendothelial matrix, blood plasma, platelets and endothelium. This glycoprotein represents an important role in thrombus formation by initiating platelet adhesion to sites of injury as well as platelet aggregation. The objective of this study was determine the activities of NTPDase (CD39), 5 -nucleotidase (EC 3.1.3.5, CD73) and Ectonucleotide Pyrophosphatase/Phosphodiesterase (E-NPP) enzymes in platelets patients from von Willebrand disease and healthy patients, as well as ristocetin-induced platelet aggregation (RIPA) and polymorphisms of the α2 gene of the α2β1 integrin. The following groups were studied: 14 patients diagnosed with vWD and 14 healthy patients for control group. For ristocetin-induced platelet aggregation was used a Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP), using a final concentration of ristocetin in 1.25mg/mL. The polymorphisms of the α2 gene was analyzed through the Polymerase chain reaction (PCR), used for digestion of the PCR product of the Bgl II restriction site. NTPDase and E-NPP were decreased in platelets of patients with vWD compared to the group control. Moreover, the activity of the enzyme 5'- nucleotidase was not statistically significant. The results of the RIPA were significantly reduced in patients with vWD compared with the control. The allelic frequencies among vWD patients were found to be 78.57% for 807C and 21.43% for 807T. Our results indicate an decreased NTPDase and E-NPP activities in platelets, may be related to the low adhesiveness of platelets in patients with vWD. The allelic frequency 807C predominant suggests, in agreement with other studies, this polymorphism and factor characteristic of hemorrhagic manifestations in patients with DvW. / A doença de von Willebrand (DvW) é uma das mais comuns entre as doenças hemorrágicas, e é provocada por uma deficiência qualitativa ou quantitativa do fator de von Willebrand (FvW). O FvW é uma glicoproteína multimérica sintetizada por megacariócitos e células endoteliais e está presente no matriz subendotelial, no plasma sanguíneo, nas plaquetas e no endotélio. Esta glicoproteína desempenha um papel importante na formação do trombo plaquetário, iniciando a adesão plaquetária ao local do dano vascular, bem como, a agregação plaquetária. O objetivo deste estudo foi determinar a atividade das enzimas NTPDase (EC 3.6.1.5, CD39), 5'-nucleotidase (EC 3.1.3.5, CD73) e Ectonucleotideo pirofasfatase/fosfodiesterase (E-NPP) em plaquetas de pacientes com a DvW e em plaquetas de pacientes saudáveis, bem como agregação plaquetária induzida pela ristocetina (RIPA) e o polimorfismo do gene α2 da integrina α2β1 da superfície de plaquetas. Os grupos foram divididos da seguinte forma: 14 pacientes diagnosticados com DvW e 14 pacientes saudáveis, para o grupo controle. Para a RIPA foram utilizados um Plasma Rico em Plaquetas (PRP) e um Plasma Pobre em Plaquetas (PPP), utilizando-se uma concentração final de ristocetina de 1.25mg/mL. O polimorfismo do gene α2 foi analisado através da reação em cadeia de polimerase (PCR), utilizando para a digestão do produto da PCR a enzima de restrição Bgl II. Constatou-se que a atividade das enzimas NTPDase e E-NPP foram reduzidas em plaquetas de pacientes com DvW comparadas ao grupo controle. Por outro lado, a atividade da enzima 5'-nucleotidase não foi estatisticamente significativa. Os resultados para os RIPA foram significativamente reduzidos em pacientes com DvW comparado com o controle. A freqüência alélica encontrada entre os pacientes com DvW foi de 78,57% para o alelo 807C e de 21,43% para o alelo 807T. Nossos resultados indicam que a redução da atividade da NTPDase e da E-NPP em plaquetas pode estar relacionada à baixa adesividade das plaquetas em pacientes com DvW. A freqüência alélica 807C predominante sugere, de acordo com outros estudos, que este polimorfismo é fator característico das manifestações hemorrágicas em pacientes portadores da DvW.

Orientador: Regina Kiomi Takahira / Banca: Raimundo Souza Lopes / Banca: Paulo Ricardo de Oliveira Paes / Resumo: O fator de von Willebrand (FvW) realiza sua função promovendo a adesão plaquetária ao local de injúria vascular e funcionando como um estabilizador funcional para o fator VIII. As células endoteliais são o maior local de síntese e armazenamento do FvW. Também existe produção pelos megacariócitos, porém, as plaquetas caninas praticamente não armazenam o FvW. A doença de von Willebrand (DvW) é o distúrbio hemostático hereditário mais comum em homens e cães. Estudos mostram uma prevalência de 1 a 2% em humanos, sendo o tipo 1 o mais freqüente (60-80%), seguido pelo tipo 2 (15-30%) e pelo tipo 3 (5-10%). Em cães, a DvW já foi diagnosticada em mais de 54 raças, sendo encontrada com alta prevalência em Dobermanns. Os sinais clínicos mais comuns da DvW são sinais de alteração em hemostasia primária. Em cães, a DvW pode ser dividida em três tipos, de acordo com a fisiopatologia. A doença do tipo 1 é definida como uma deficiência parcial quantitativa do FvW. O tipo 2 consiste em perda desproporcional das formas multiméricas de alto peso molecular e o tipo 3 resulta de uma deficiência quantitativa grave do FvW. Os testes diagnósticos mais utilizados para a DvW são tempo de sangria, dosagem do antígeno do FvW, atividade de cofator da ristocetina, agregação plaquetária induzida pela ristocetina e análise multimérica do FvW. O objetivo deste estudo foi determinar e padronizar o teste padrão para diagnóstico de Resumo cães com Doença de von Willebrand. Os testes estudados foram: Tempo de sangria da mucosa oral (TSMO), Agregação plaquetária induzida pela Ristocetina (RIPA) e Antígeno do fator de von Willebrand (FvW:Ag) ELISA...(Resumo completo, clicar eletrônico abaixo) / Abstract: von Willebrand factor (vWF) perform its function promoting platelet adhesion to the local of vascular injury and is important for the maintenance of FVIII stability and function. Endothelial cells are the major synthesis and storage site. Megakariocytes may also produce vWF, but canine platelets practically don’t store vwF. Clinical signs more commonly seen in vWD are associated to primary hemostatic alterations. von Willebrand disease (vWD) is the most common inherited hemostatic disease in human and dogs. Studies had demonstrated a prevalence of 1 to 2% in human beings, being the type I the most common (60-80%), followed by type 2 (15-30%) and type 3 (5-10%). In dogs, vWD had already been diagnosed in more than 54 breeds, with a high prevalence in Dobermann Pinchers. In dogs, vWD may be divided in three types, according to the pathophysiology. Type 1 is defined as a quantitative partial deficiency of vWF. Type 2 consists of a disproportional loss of high molecular weight multimeric forms and type 3 results from a severe quantitative deficiency of vWF. The most used test for the diagnosis of vWD are the buccal mucosa bleeding time, measurement of vWF antigen, ristocetin cofactor activity test, platelet aggregation induced by ristocetin and multimeric analysis of vWF. The purpose of this work was to establish and standardize the gold standard test for screening test of dogs with von Willebrand disease. The following tests were studied: Buccal mucosa bleeding time (BMBT), Ristocetin induced platelet aggregation Abstract (RIPA) and von Willebrand factor Antigen (FvW:Ag) ELISA...(Complete abstract, click electronic address below) / Mestre

Made available in DSpace on 2014-06-11T19:23:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T19:51:00Z : No. of bitstreams: 1 mattoso_crs_me_botfmvz.pdf: 741864 bytes, checksum: 4f4908ca9c565d80c40053bc4cacf8bb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fator de von Willebrand (FvW) realiza sua função promovendo a adesão plaquetária ao local de injúria vascular e funcionando como um estabilizador funcional para o fator VIII. As células endoteliais são o maior local de síntese e armazenamento do FvW. Também existe produção pelos megacariócitos, porém, as plaquetas caninas praticamente não armazenam o FvW. A doença de von Willebrand (DvW) é o distúrbio hemostático hereditário mais comum em homens e cães. Estudos mostram uma prevalência de 1 a 2% em humanos, sendo o tipo 1 o mais freqüente (60-80%), seguido pelo tipo 2 (15-30%) e pelo tipo 3 (5-10%). Em cães, a DvW já foi diagnosticada em mais de 54 raças, sendo encontrada com alta prevalência em Dobermanns. Os sinais clínicos mais comuns da DvW são sinais de alteração em hemostasia primária. Em cães, a DvW pode ser dividida em três tipos, de acordo com a fisiopatologia. A doença do tipo 1 é definida como uma deficiência parcial quantitativa do FvW. O tipo 2 consiste em perda desproporcional das formas multiméricas de alto peso molecular e o tipo 3 resulta de uma deficiência quantitativa grave do FvW. Os testes diagnósticos mais utilizados para a DvW são tempo de sangria, dosagem do antígeno do FvW, atividade de cofator da ristocetina, agregação plaquetária induzida pela ristocetina e análise multimérica do FvW. O objetivo deste estudo foi determinar e padronizar o teste padrão para diagnóstico de Resumo cães com Doença de von Willebrand. Os testes estudados foram: Tempo de sangria da mucosa oral (TSMO), Agregação plaquetária induzida pela Ristocetina (RIPA) e Antígeno do fator de von Willebrand (FvW:Ag) ELISA... / von Willebrand factor (vWF) perform its function promoting platelet adhesion to the local of vascular injury and is important for the maintenance of FVIII stability and function. Endothelial cells are the major synthesis and storage site. Megakariocytes may also produce vWF, but canine platelets practically don t store vwF. Clinical signs more commonly seen in vWD are associated to primary hemostatic alterations. von Willebrand disease (vWD) is the most common inherited hemostatic disease in human and dogs. Studies had demonstrated a prevalence of 1 to 2% in human beings, being the type I the most common (60-80%), followed by type 2 (15-30%) and type 3 (5-10%). In dogs, vWD had already been diagnosed in more than 54 breeds, with a high prevalence in Dobermann Pinchers. In dogs, vWD may be divided in three types, according to the pathophysiology. Type 1 is defined as a quantitative partial deficiency of vWF. Type 2 consists of a disproportional loss of high molecular weight multimeric forms and type 3 results from a severe quantitative deficiency of vWF. The most used test for the diagnosis of vWD are the buccal mucosa bleeding time, measurement of vWF antigen, ristocetin cofactor activity test, platelet aggregation induced by ristocetin and multimeric analysis of vWF. The purpose of this work was to establish and standardize the gold standard test for screening test of dogs with von Willebrand disease. The following tests were studied: Buccal mucosa bleeding time (BMBT), Ristocetin induced platelet aggregation Abstract (RIPA) and von Willebrand factor Antigen (FvW:Ag) ELISA...(Complete abstract, click electronic address below)

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for the unknown etiologies in Type 1 VWD. Moreover, therapeutic interventions could be designed in the future by modulation of these gene products to control bleeding or thrombosis.Zebrafish has three f9 genes, f9a, f9b, and f9l and the ortholog to human F9 is unknown. RNA analysis showed an age-dependent increase in expression of all three genes from larval stages to adults, comparable to those observed in mice and humans while mass spectrometry and immunohistochemistry confirmed the presence of all three proteins in the fish. Based on coagulation assays performed after individual gene knockdown and immunodepletion, we identified that zebrafish f9a has functional activity similar to human F9 and Fixl is functionally similar to Fx. Thus, the zebrafish could be used to identify factors controlling f9 gene expression with age and for modeling Hemophilia B in the quest to develop gene therapy protocols. In zebrafish, dilute plasma with exogenously added human fibrinogen was used for kinetic coagulation assays. Here, we developed a microkinetic assay using 25% zebrafish or 30% human plasma followed by the addition of coagulation activators and CaCl2. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established human kinetic curves. Moreover, clotting times derived from these kinetic curves were identical to human PT, PTT, and Russel's viper venom time. Thus, the microkinetic assay developed here could measure blood coagulation activity in small animal models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.