Dissertations / Theses on the topic 'Von Willebrand disease (vWD)'

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.

The release and clearance of von Willebrand Factor (VWF) in a group of patients with the quantitative deficiency type 1 von Willebrand Disease (VWD) was investigated. This was done by analysis of circulating VWF and VWF released from the endothelial pool on infusion of a vasopressin analogue. A variety of parameters were investigated pre-and post infusion in order to identify VWF gene linked and non-linked variables that could affect VWF clearance. Increased clearance of plasma VWF in a significant proportion of type 1 VWD patients was shown but this was not consistently associated with steady-state levels of VWF, indicating that circulating plasma VWF levels are not a consistent reflection of the VWF life-cycle in this patient group. An association between galactose exposure and reduced levels of VWF was demonstrated by the increased binding of the lectins Ricinus communis and Erythina crystagalli, this was unrelated to clearance. In addition, no significant ABO blood group effect on VWF clearance was demonstrated. The absolute level of ADAMTS-13, and the susceptibility of VWF to cleavage by ADAMTS-13 were not associated with the clearance rate of VWF in patients with type 1 VWD. Three novel candidate mutations were identified in association with significantly accelerated VWF clearance. Notably, candidate mutations were generally identified in patients with steady-state VWF levels reduced to <20 IUdL"1 and family analysis suggests absolute linkage with the VWF gene. Despite demonstrating an increased rate of clearance in the majority of patients with type 1 VWD, no single underlying common characteristic or variable was predominant within this study group.

von Willebrand disease (VWD) is the most common autosomal bleeding disorder. It is divided into type 1, 2 and 3. Type 2 VWD shows qualitative defects in VWF and is further sub-classified into type 2A, 2B, 2M and 2N, each having different functional defects in VWF. Most of the associated mutations are located at the exons in VWF which encode for the affected functional domains. Diagnosis of VWD is currently based on history and phenotypic tests, which can be difficult often times. Therefore, molecular diagnosis of type 2 VWD is an attractive alternative. There are only a few genotypic characterisation studies of type 2 VWD in Chinese. This study aims to provide genetic data of type 2 VWD in Hong Kong. Archive DNA samples of 21non-type 2N type 2 VWD patients (Group 1), 15 type 2N/mild haemophilia A (HA) patients (Group2) and 35 control subjects were recruited. VWF exon 27, 28 and exon 18, 19, 20, 23, 24 were Sanger sequenced in Group 1 and Group 2 subjects, respectively. All seven exons were sequenced in the control subjects. Seven of 21 Group 1 subjects were found to have pathogenic mutation sin exon 28, with 2being novel. Only 1 Group 2 subject was found to be heterozygous for a novel non-synonymous variation at exon 23, the significance of which could not be ascertained. Sixteen benign polymorphisms were detected from exons sequenced in patients and controls. The low pathogenic mutation detection rate may suggest that the pattern of mutation in Chinese is different from other populations. The possibility of misdiagnosis in a proportion of these patients cannot be excluded in view of the known difficulty in patient ascertainment in VWD and the limited phenotypic diagnostic tools available in Hong Kong. Further studies of other exons are indicated to document the mutation spectrum of type 2 VWD in our Chinese population. RNA work and functional studies are required to fully characterise novel sequence variations found. High throughput mutation detection platforms and better phenotypic characterisation will facilitate the introduction of VWD genotyping into routine clinical diagnostics.
published_or_final_version
Pathology
Master
Master of Medical Sciences

The principal objective of this study was to examine the effect of intense physical exercise on menstrual blood loss in women with Type I von Willebrand disease (vWD). First, we investigated the effect of exercise on the level of the von Willebrand protein (which is deficient in the disease) in a pre-test post-test quasi-experiment conducted on a single group of 40 healthy adult pre-menopausal female volunteers recruited from Sainte-Justine Hospital in Montreal between October and December, 2001. The von Willebrand protein (vWF:Ag), coagulation Factor VIII (FVIII:C), bleeding time (BT), coagulation time (aPTT) and several markers of exercise intensity (sweat sodium, lactate, noradrenaline, adrenaline) were measured before and after a standardized exercise session. The significance of the change in these values with exercise was assessed using a paired Student's t-test. The exercise markers were explored as potential predictors of the exercise-related change in vWF:Ag using multiple linear regression. Results showed that there was an absolute mean increase of 0.30 (95% confidence interval (95% CI) 0.23-0.37) and 0.60 (95% CI 0.44-0.76) in vWF:Ag and FVIILC, respectively, and a significant shortening of the BT and aPTT due to exercise. The change in the sweat sodium collected from patches applied to the forearm during exercise (a marker of exercise intensity) was found to be a significant predictor of the change in vWF:Ag induced by exercise (regression coefficient = 0.05 (95% CI 0.01-0.09). Changes of 1, 5 and 10 units in sodium were associated with average changes of 0.05, 0.26 and 0.52, respectively, in vWF:Ag from baseline (mean 0.83 U/ml). Next, we set out to assess the feasibility and acceptability of a 4-period randomized crossover trial in order to evaluate the effectiveness of exercise in reducing the menstrual blood flow in women with Type I vWD. The methods and protocol of this feasibility study are outlined in this thesis and issues related to patient recruitment, compliance and withdrawals are addressed. The strengths and pitfalls of the crossover design feasibility study are discussed and revisions for the definitive trial are recommended.

Von Willebrand factor (VWF) is a multimeric glycoprotein that critically supports platelet aggregation in hemostasis. Disordered VWF function causes both thrombotic and bleeding disorders, and genetic defects in VWF are responsible for von Willebrand’s disease (VWD), the most common inherited bleeding disorder in humans. Very large VWF multimers exhibit the greatest thrombogenic activity, which is attenuated by ADAMTS13 cleavage in the A2 domain. A2 cleavage is regulated by mechanical force, and pathologically high shear forces are known to enhance proteolysis and cause bleeding in patients. Enhanced cleavage is also described in patients with VWD 2A mutations. In contrast, VWF A2 is stabilized against cleavage by a calcium binding site within A2. Single molecule studies have demonstrated that mechanical unfolding is required for A2 cleavage to expose the scissile bond. In this dissertation, we aim to better understand the mechanosensitivity of A2 cleavage by characterizing the force sensitivity of A2 unfolding and refolding. We first characterized the interaction between VWF A2 and calcium using bulk isothermal calorimetry and thermal denaturation assays. In parallel, we used single molecule optical tweezers to characterize A2 unfolding and refolding. Calcium was found to bind A2 with high affinity, stabilize A2 against thermal denaturation, and enhance domain refolding. In contrast, we found that VWD 2A mutations destabilize the A2 domain against thermal denaturation. R1597W, the most common VWD 2A mutation, lies within the calcium binding loop and exhibited diminished calcium stabilization against thermal denaturation. Using optical tweezers, we found that R1597W also diminished A2 refolding. R1597W refolding in the presence of calcium was similar to that of wild-type A2 in the absence of calcium, suggesting that loss of calcium stabilization contributes to the disease mechanism of R1597W. Other VWD 2A mutations lying outside the calcium binding loop also destabilized A2, but retained calcium mediated stabilization. These studies provide a better understanding of VWD 2A pathophysiology and offer structural insights into A2 unfolding and refolding pathways. By exploring the role of mechanical force in regulating VWF cleavage, this work moves towards a better understanding of how hydrodynamic forces within the vasculature regulate VWF function in hemostasis and thrombosis.

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

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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)

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

La Malaltia de von Willebrand (VWD) és la coagulopatia congènita més freqüent a la població general. Consisteix en una diàtesi hemorràgica causada per una deficiència qualitativa i/o quantitativa del factor de von Willebrand (VWF) que es transmet amb caràcter autosòmic dominant o, menys freqüentment, recessiu. El VWF és una glicoproteïna adhesiva present en plaquetes, cèl•lules endotelials i megacariòcits que té diferents funcions donat que participa en l’hemostàsia primària i col•labora al mateix temps en la secundària. És un mediador de l’adhesió de les plaquetes al subendotel•li en el lloc de la lesió vascular i transporta al FVIII, al que protegeix de la degradació proteolítica prematura. El gen del VWF (VWF) s’extén unes 178 kilobases en el genoma i conté un total de 52 exons, sent un dels gens més grans i complexos descrits en humans. Addicionalment existeixen una sèrie de factors que dificulten de manera considerable la caracterització molecular de la VWD i que han fet que la seqüenciació directa no s’hagi considerat el mètode de referència per al seu diagnòstic. En primer lloc, el VWF és un gen altament polimòrfic i, fins al moment, s’hi han descrit 102 SNPs (Build 132) en regió codificant, el que pot dificultar la identificació de les mutacions i, en segon lloc, existeix un pseudogèn parcial al cromosoma 22 d’aproximadament 30 kb molt homòleg (>96%) als exons 23-34 del VWF. Amb l’objectiu de facilitar l’anàlisi genètic de la VWD, s’ha dissenyat un procediment simplificat basat en la seqüenciació completa del gen, que s’ha utilitzat per identificar la mutació en un total de 40 famílies i demostra la seva validesa com a mètode rutinari de diagnòstic molecular. Amb l’aplicació d’aquest mètode s’han identificat un total de 58 mutacions (41 diferents), 19 de las quals no s’havien descrit prèviament a la literatura. Entre els diferents tipus de mutació responsables de la VWD, aquelles que modifiquen la regió codificant del gen tenen un clar efecte deleteri, però les conseqüències de les mutacions que afecten potencialment l’splicing (PSSM) són menys evidents. Amb l’objectiu d’estudiar l’efecte d’aquestes mutacions s’ha desenvolupat un mètode per a la seqüenciació completa del cDNA del VWF en leucòcits i plaquetes que ens ha permès revelar l’efecte de diverses PSSM. L’aparició de les plataformes de seqüenciació de nova generació (NGS), que són fins 200 vegades més ràpides i econòmiques que la seqüenciació tradicional, ha plantejat nous reptes en el diagnòstic molecular de les malalties hereditàries. Per això s’ha desenvolupat una nova estratègia d’amplificació del gen en un total de 14 PCRs llargues i hem adaptat el procediment desenvolupat prèviament per a la seqüenciació completa del VWF a les noves plataformes de NGS. Aquestes estratègies permetran l’anàlisi simultània d’un gran nombre de mostres de pacients i familiars de manera més ràpida i econòmica que per seqüenciació tradicional. Amb l’objectiu de recopilar tota la informació generada a partir del diagnòstic molecular dels pacients amb VWD i fer-la accessible, hem dissenyat un nou apartat dins d’Hemobase (registre de mutacions per a les Hemofílies A i B) dedicat a la VWD (www.vwf.hemobase.com). Aquesta pàgina d’accés lliure per Internet, conté un registre de les mutacions identificades en pacients amb VWD després de la seqüenciació directa del VWF. El registre permet realitzar cerques, relacionar qualsevol mutació amb la base de dades internacional i accedir directament a les publicacions corresponents. Es pretén que l’estudi molecular dels pacients permeti una millor comprensió dels mecanismes implicats en la fisiopatologia de la malaltia i ofereixi una visió més àmplia de l’epidemiologia molecular a la nostra població.
Von Willebrand Disease (VWD) is the most frequent congenital coagulopathy in the general population. It has been proved to be particularly complex due to a series of factors that make difficult the molecular diagnostic of the disease: the von Willebrand Factor gene (VWF) is large and complex; it is very polymorphic; there is a partial pseudogene in chromosome 22 highly homologous (>96%) to a region of the VWF; and the existence of other genes implied in the disease cannot be discarded. All this set of difficulties causes that the molecular study of the VWD remains confined to basic investigation and the application to the clinical routine has been considerably delayed. With the aim to facilitate the genetic study of the VWD, we designed and optimized a procedure for direct sequencing of the VWF, that allowed us to study 40 families identifying 58 mutations (41 different), 19 of which were new. Among the different types of mutation that cause VWD, those affecting the coding region have frequently a clear deleterious effect; however, the consequences of the potential splice site mutations (PSSM) are less predictable. A method for the complete sequencing of the VWF cDNA in leukocytes and platelets has been developed allowing the elucidation of the effect of several PSSM studied. Next Generation Sequencing (NGS) platforms are faster and cheaper that the traditional sequencing. In order to take advantage of this new technology, we have developed an optimized strategy for the amplification of the VWF by LR-PCRs and we have tailored the previously developed short PCR procedure. The objective is to achieve high performance in the molecular characterization of VWD patients and relatives and to establish the basis for a large-scale molecular study approach. Finally, a database of the mutations identified, responsible for the pathology (www.vwf.hemobase.com), has been established in order to correlate molecular and clinical parameters. The development of suitable tools for the molecular diagnosis of VWD will significantly facilitate the clinical diagnosis and will guide the clinician towards the better therapeutic option.

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.

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

A hipóxia é capaz de alterar muitos mecanismos bioquímicos nas células endoteliais. Dentre eles, a indução da expressão endotelial de moléculas de adesão, como o fator de von Willebrand (FVW) que, em resposta ao estímulo, é secretado em sua forma mais ativa na interação com as plaquetas, o que pode resultar em trombose. Nas condições fisiológicas, o padrão multimérico do FVW no plasma é essencialmente determinado pela ADAMTS-13 (uma desintegrina e metaloproteinase com domínios trombospondina). Este estudo teve como objetivo verificar se a atividade da enzima ADAMTS-13, assim como as características do FVW relacionáveis a ela, poderiam estar alteradas na presença de hipoxemia comparativamente à condição de oxigenação normal. Este estudo longitudinal envolveu 56 pacientes portadores de cardiopatias congênitas cianogênicas, em idades entre um e sete anos, candidatos ao tratamento cirúrgico. Os pacientes foram avaliados no pré-cirúrgico (basal), no pós-operatório imediato (pós 48 horas) e após 30 dias de cirurgia, e foram divididos em dois grupos (A e B) baseado na saturação periférica de oxigênio (SpO2) no momento pós 30 dias. Foram determinados o antígeno do FVW e a análise das suas subunidades, a atividade da ADAMTS-13 e a presença de inibidores da ADAMTS-13. Os pacientes de ambos os grupos apresentaram aumento significante da SpO2, da concentração antigênica do FVW e da atividade da ADAMTS-13 nos momentos pós 48 horas e pós 30 dias em comparação com o momento pré (basal). As densidades normalizadas da subunidade principal do FVW (225 kDa) e do fragmento de 176 kDa apresentaram tendência ao aumento nos momentos pós 48 horas e pós 30 dias nos dois grupos. A razão entre a atividade da ADAMTS-13 e o FVW estava menor do que 1 no momento pós 48 horas, indicando consumo da enzima; entretanto, no momento pós 30 dias a razão fica 1:1, e o FVW se aproxima dos valores de referência. Verificamos ainda que 29% destes pacientes apresentaram inibidores contra a ADAMTS-13 no momento pré-operatório. Ainda explorando as variáveis SpO2, FVW:Ag, atividade da ADAMTS-13 e a composição das subunidades do FVW, foi feito um estudo de correlação linear entre estas variáveis. Observamos uma baixa correlação entre a enzima ADAMTS-13 e o FVW:Ag, e da enzima com os fragmentos do FVW de 176 e 140 kDa, principalmente no grupo B. No grupo A, esta correlação no momento pós 48 horas mostrou tendência a ser negativa. A maioria dos pacientes apresentou melhoras na saturação periférica de oxigênio. O aumento das variáveis estudadas no pós-operatório imediato pode ter ocorrido em função da cirurgia, que provavelmente ocasionou um quadro de lesão endotelial com inflamação, indicando que pode existir um equilíbrio entre o FVW e a ADAMTS-13 em níveis fisiológicos. Entretanto, este equilíbrio pode ser quebrado quando ocorre aumento do FVW, provavelmente por consumo da enzima. Parece-nos, portanto, que a ADAMTS-13 pode funcionar como um mecanismo de proteção a estes pacientes com tendência à trombose
Hypoxia has been shown to alter several biochemical mechanisms in endothelial cells. In addition, hypoxia induces the endothelial expression of adhesion molecules, including von Willebrand factor (VWF). Increased release of high-molecular-weight VWF multimers is associated with higher risk for thrombotic events. In physiological conditions, the multimeric pattern of plasma VWF is essentially determined by the action of ADAMTS-13 (a desintegrin and metalloprotease with thrombospondin type 1 domains). The aim of this study was to investigate if ADAMTS-13 activity and VWF subunit fragments were altered by hypoxia in cyanotic congenital heart disease. Fiftysix patients (age 1 to 7 years) with cyanotic congenital heart disease admitted to the Heart Institute for heart surgery were included in this longitudinal study. Patients were evaluated before (baseline) corrective surgery, postoperative 48 hours and postoperative 30 days. Patients were classified in two groups (A and B) based on the peripheral oxygen saturation after 30 days surgery. VWF antigenic concentration, VWF subunit composition, ADAMTS-13 activity and presence of ADAMTS-13 inhibitors were determined. Peripheral oxygen saturation, VWF:Ag and ADAMTS-13 activity were all increased significantly in both groups, in postoperative 48 hours and postoperative 30 days in comparison with baseline moment. Normalized density of VWF main subunit (225 kDa) and proteolytic fragment with 176 kDa tended to increase in postoperative 48 hours and postoperative 30 days in both groups. The rate between ADAMTS-13 activity and VWF:Ag was lower than 1 in postoperative 48 hours, an indicating of enzyme consumption; however, in the postoperative 30 days the rate was 1:1 and VWF:Ag values were near those of reference. 29% of patients presented ADAMTS-13 inhibitors at the baseline moment. A study of correlation among variables as peripheral oxygen saturation, VWF:Ag, VWF subunit composition and ADAMTS-13 was done. It was observed that ADAMTS-13 correlated slightly positively with VWF:Ag and with VWF fragments 176 and 140 kDa, mainly in group B; in group A, the correlation at postoperative 48 hours tended to be negative. Most of the patients improved their peripheral oxygen saturation. The increased value of variables observed in postoperative 48 hours can be explained by the endothelial injury and inflammation caused by the surgery itself. This indicates an equilibrium between VWF:Ag and ADAMTS-13 in physiological conditions. However, this equilibrium could disappear when VWF is increased, probably by enzyme consumption. We conclude that ADAMTS-13 can act as a protective mechanism in these patients with thrombotic tendency

Von Willebrand Disease (VWD) is the most common bleeding disorder. In addition to known major genes, genetic modifiers, such as ABO blood group, affect quantitative outcome measures for VWD. The data consist of an 854-member Amish pedigree with established linkage of VWD to a locus within the Von Willebrand Factor (VWF) gene on chromosome 12. The DNA sequence of the causative mutation is known. Phenotypic information and genotypic data consisting of VWF mutation status and a genome screen of markers are available for 385 pedigree members. Genetic modifiers of the VWF mutation are investigated using known and new conditional linkage methods that search for modifier genes of a major gene with known mutation. The MCMC-based program LOKI was used to conduct multipoint linkage analysis of VWD outcome measures while controlling for the VWF mutation. Adjustment for the mutation did not eliminate the linkage signal on chromosome 12 in the same location as the VWF mutation. Evidence for QTLs was also found on six other chromosomes. Smod, a score statistic that detects evidence of a genetic modifier conditional on linkage to a major gene, was developed for sib pair data. To limit the modifier gene main effect, Smod was developed so that variance due to the modifier locus is bounded above by the variance of the interaction between major gene and modifier gene. The performance of Smod was compared to other published score statistics. Power to detect linkage to the modifier locus depended on major gene and modifier gene risk allele frequencies, relative contribution of the major gene main effect to the interaction effect, and the upper bound on the modifier gene main effect. The Amish pedigree was broken up into sib pair data and analyzed using Smod and other score statistics. Using these statistics, the strongest evidence for QTLs for VWD was also found on chromosome 12 in the region of the VWF mutation. Combined with the LOKI results, further analysis will help determine if intragenic modification is occurring or if linkage disequilibrium between the mutation and analyzed markers is driving results.

La malaltia de von Willebrand (VWD) és la patologia hemorràgica hereditària més freqüent a la població i està causada per dèficits quantitatius o qualitatius del factor von Willebrand (VWF). Segons el defecte, es distingeixen tres grans categories: la VWD de tipus 1 caracteritzada per una reducció parcial del VWF, la VWD de tipus 3 per una absència total de factor i la VWD de tipus 2 per defectes funcionals en aquesta proteïna. Actualment el diagnòstic d’aquesta patologia es basa en manifestacions clíniques de sagnat, una història familiar positiva i la realització d’un conjunt extens de proves de coagulació. Per altra banda, el diagnòstic genètic de la VWD s’ha considerat un test de segona línia degut a l’elevat cost que suposa realitzar l’anàlisi molecular del gen del VWF (VWF) mitjançant la tecnologia de Sanger. Els avenços realitzats en els últims anys en les eines de seqüenciació massiva (NGS) han promogut un interès creixent per la seva aplicació al diagnòstic clínic de malalties causades per gens de gran mida i altament polimòrfics com és el cas de la VWD. L’objectiu general d’aquesta tesi doctoral consisteix en aprofitar la tecnologia de NGS pel desenvolupament i optimització d’un protocol versàtil i econòmic per abordar l’estudi molecular de la VWD. Es pretén aplicar el nou protocol a grans cohorts de pacients per aprofundir en la investigació dels mecanismes genètics de la VWD i determinar la implicació de les variants identificades al VWF en diferents aspectes del fenotip amb rellevància en el curs clínic de la malaltia. En aquest sentit, s’ha dissenyat i optimitzat un protocol eficaç i econòmic que permet amplificar el VWF en 48 pacients simultàniament i seqüenciar-los posteriorment per NGS. El desenvolupament d’aquesta estratègia ha tingut especial rellevància dins del projecte “Perfil Clínic i Molecular de la malaltia de von Willebrand: Registre Espanyol” (PCM-EVW-ES), l’objectiu del qual és el d'establir un registre nacional per a la VWD i en el qual han participat la majoria dels grans hospitals del país reclutant 556 individus. L’aplicació del procediment en aquest registre ha permès identificar un elevat número de mutacions diferents al VWF i ha estat essencial per a la confirmació i la classificació de la VWD en 480 pacients dels 556 inicialment inclosos, establint-se una correlació genotip-fenotip en el 94,6% dels casos. Tanmateix, l’aplicació del protocol en l’estudi retrospectiu d’una cohort portuguesa de 92 pacients amb VWD, ha demostrat la seva potència, eficàcia, rapidesa i reducció de costos, en comparació amb la tecnologia de Sanger, pel diagnòstic molecular d’aquesta patologia. A continuació, derivat de l’estudi genètic d’aquestes cohorts s’han seleccionat varies mutacions potencials d’splicing (PSSM) per a investigar el seu efecte patogènic mitjançant estudis transcripcionals in vivo. Els resultats obtinguts han permès caracteritzar l’efecte patogènic en el processament de l’mRNA del VWF de 8 PSSM i posen de manifest que mutacions de tipus missense i sinònimes també poden actuar alternat l’splicing. Per últim, s’ha investigat la participació dels polimorfismes en la modulació de la gravetat i les manifestacions clíniques de la VWD. D’aquesta manera s’ha estimat que quatre polimorfismes poden arribar a explicar de l’1,5% al 2,6% de la variància fenotípica de diferents mesures del VWF (VWF:Ag, VWF:RCo i/o VWF:CB). En resum, l’anàlisi genètica del VWF mitjançant la tecnologia NGS ha demostrat la seva vàlua en el diagnòstic de la VWD i ha aportat dades per a comprendre i esclarir la base molecular d’aquesta patologia.
Von Willebrand's disease (VWD) is the most common inherited bleeding disorder and is caused by a deficiency or dysfunction of von Willebrand factor (VWF). There are three major categories: type 1 resulting from a partial reduction of the VWF, VWD type 3 resulting from a complete or near complete absence of this factor and VWD type 2 characterized by qualitative deficiency. Currently, the diagnosis of this pathology is based on clinical and phenotypic information. The molecular diagnosis of VWD has been considered as a second line test due to the high cost to perform the molecular analysis of the VWF gene (VWF) by Sanger. Recently, the advances in high throughput sequencing methologies (NGS) have promoted a growing interest in its application to the clinical diagnosis of monogenic diseases. The main objective of this doctoral thesis is to develop and optimize a versatile and economic protocol based on NGS to address the molecular study of the VWD. It is intended to apply the new protocol to large patient cohorts to study in depth the molecular mechanisms of the VWD and determine the implication of VWF variants in the VWD phenotype. In this sense, an effective and economical protocol has been designed and optimized that allows the amplification of the VWF in 48 patients simultaneously and sequencing by NGS. The development of this strategy has been of particular relevance in the "Clinical and Molecular Profile of von Willebrand's Disease: Spanish Registry" (PCM-EVW-ES), whose purpose is to establish a national registry of the VWD. The majority of Spanish hospitals participated in this project and recruited a total of 556 individuals. The application of the developed procedure in this registry has prompted the identification of a high number of VWF mutations and has been essential for confirmation and classification of the VWD in 480 patients of the 556 initially included, establishing a genotype-phenotype correlation in 94.6% of cases. Additionally, the application of this protocol in the retrospective study of a Portuguese cohort of 92 VWD patients has demonstrated its power, efficiency, and cost reduction, compared to Sanger's technology, in molecular diagnosis of VWD. Derived from the genetic study of these cohorts, several potential splice site mutations (PSSM) have been selected to investigate their pathogenic effect through transcriptional studies in vivo. The results obtained demonstrate the pathogenic effect of 8 VWF PSSM in the mRNA processing and prove that missense and synonymous mutations could also affect splicing. Finally, it has been investigated the participation of VWF polymorphisms in the modulation of the severity and the clinical manifestations of the VWD. In this way, it has been estimated that four polymorphisms can account from 1.5% to 2.6% of the phenotypic variance of different VWF measurements (VWF: Ag, VWF: RCo and / or VWF: CB). In summary, genetic analysis of the VWF by means of NGS technology has proven its worth in the diagnosis of the VWD and has provided data to understand and elucidate the molecular basis of this pathology.

Platelet function in dogs with chronic liver disease Ashley R. Wilkinson Background: Dogs with acquired chronic liver disease often have hemostatic derangements. It is currently unknown whether dogs with acquired chronic liver disease have decreased platelet function and alterations in von Willebrand factor (vWF) that may contribute to hemostatic abnormalities. Hypothesis: Dogs with chronic liver disease have prolonged platelet closure time (CT), assessed with the PFA-100®, and buccal mucosal bleeding time (BMBT), and increased vWF concentration compared to healthy dogs. Animals: Eighteen dogs with chronic acquired liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy and eighteen healthy age-matched control dogs. Methods: Prospective study. BMBT, CT using the PFA-100®, and vWF antigen were measured in dogs with chronic liver enzyme elevation undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy. After undergoing ultrasound-guided needle biopsy, dogs were monitored for hemorrhage with serial packed cell volume measurements and focused assessment with sonography. An unpaired t-test was used for normally distributed data and the Mann-Whitney test was used when non-Gaussian distribution was present. The level of significance was set at P <0.05. Results: The CT was not different between the two groups (P = 0.27). The BMBT was significantly longer in the liver disease group compared to the control group (P = 0.019). There was no difference in the mean vWF antigen of the two groups (P = 0.077). Conclusions and clinical relevance: These results demonstrate mild impairment of primary hemostasis in dogs with chronic liver disease based on prolongation of BMBT.
Master of Science
Background: Dogs with chronic liver disease often have abnormal blood clotting activity. It is currently unknown whether dogs with chronic liver disease have decreased platelet function and alterations in von Willebrand factor (vWF) that may contribute to blood clotting abnormalities. Platelet function can be assessed using the PFA-100®, which measures platelet closure time (CT), and buccal mucosal bleeding time (BMBT). The PFA-100 simulates blood in circulation to assess platelet function. BMBT is a crude but readily available test to assess platelet function in practices without sophisticated methods of assessing platelet function. Hypothesis: Dogs with chronic liver disease have prolonged CT and BMBT, which both suggest platelet dysfunction. Additionally, dogs with chronic liver disease have increased vWF concentration compared to healthy dogs. Animals: Eighteen dogs with chronic acquired liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy and eighteen healthy age-matched control dogs. Methods: Prospective study. BMBT, CT, and vWF antigen were measured in dogs with chronic liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy. After undergoing ultrasound-guided needle biopsy, dogs were monitored for hemorrhage. Results: The CT was not different between the two groups but the BMBT was significantly longer in the liver disease group compared to healthy dogs. There was no difference in the mean vWF antigen between the two groups. Conclusions and clinical relevance: These results demonstrate mild impairment of blood clotting activity in dogs with chronic liver disease based on prolongation of BMBT compared to healthy dogs. Prolongation of BMBT compared to healthy dogs is suggestive of endothelial dysfunction and/or platelet dysfunction in dogs with chronic liver disease.

Variantes genéticas trombogênicas podem aumentar o risco de eventos adversos em pacientes com coronariopatia crônica. Estudos prévios demonstraram que o Haplótipo H2 do gene do receptor P2Y12 apresenta uma maior agregação plaquetária e está associado com a presença de isquemia arterial periférica. A metaloprotease ADAMTS13 é responsável pela clivagem do fator de von Willebrand e recentemente foi associada com doença isquêmica coronariana. O objetivo deste trabalho foi avaliar o efeito das variantes genéticas funcionais trombogênicas dos Haplótipos H1 e H2 do receptor plaquetário P2Y12 e dos polimorfismos C1342G (Q448E), C1852G (P618A) e C2699T (A900V) da metaloprotease ADAMTS13 em 611 pacientes com doença arterial coronariana multiarterial com função ventricular preservada, acompanhados por um período de 05 anos no ensaio clínico do projeto MASS II (Medical, Angioplasty, or Surgery Study II) em relação aos eventos morte, infarto agudo do miocárdio, angina refratária necessitando um novo procedimento e acidente vascular cerebral. Neste estudo, a avaliação dos Haplótipos H1 e H2 nos pacientes do MASS II não encontrou diferença entre estes haplótipos e os eventos estudados. A análise dos polimorfismos da ADAMTS13 não encontrou associação entre os polimorfismos e os eventos estudados, exceto para a variante genética T2699 (Val900) que está associada com o evento morte (OR: 1,67 95%IC: 1-2,78, p= 0,049) e morte por causa cardiovascular (OR: 2,23 95%IC: 1,2-3,94, p=0,004) e apresenta uma diminuição na sobrevida livre de morte por causa cardíaca para os portadores do genótipo TT relacionado à este polimorfismo. A análise dos haplótipos e das combinações alélicas destes polimorfismos não apresentou associação com eventos ou com a sobrevida livre dos eventos nestes pacientes.
Thrombotic genetic variants could improve the risk of adverse events related to coronary arterial disease (CAD). P2Y12 platelet receptor H2 haplotype showed higher aggregation index and a positive association was described between such genetic variant and peripheral artery disease. DAMTS13 is a metaloprotease responsible to von Willebrand factor cleavage recently found correlated to CAD. We tested the genetic variants P2Y12 receptor H1 and H2 haplotypes and ADAMTS13 polymorphisms C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 611 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function in a follow up period of 05 years. The incidence of the end points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for P2Y12 H1 and H2 haplotypes and ADAMTS polymorphisms. In our study, we did not disclose any association between H1 or H2 haplotype groups regarding the incidence of any of the studied cardiovascular end-points. The association of ADAMTS13 genotypes and cardiovascular events did not showed any association between C1342G (Q448E), C1852G (P618A) variants and cardiovascular end points. Our date provide a strong association between T2699 variant and increased risk to death (OR: 1,67 CI: 1-2,78, p= 0,049) and cardiac death (OR: 2,23 CI: 1,2-3,94, p=0,004) in a population with CAD. The allelic combinations and haplotypes obtained from ADAMTS13 polymorphisms were not associated to cardiac end points and survival differences between MASS II patients.

La maladie d'Alexander est une maladie neurodégénérative due à des mutations hétérozygotes du gène GFAP codant le principal filament intermédiaire des astrocytes matures. Nous avons étudié l'effet des mutations GFAP dans l'hippocampe d'un patient avec AxD infantile et de deux souris knockin, l'une portant une mutation dans le rod domain (p.R85C) et l'autre dans le tail domain (p.T409I). Chez le patient, nous décrivons pour la première fois: (i) des changements morphologiques sévères des cellules GFAP+ dans la zone subgranulaire du gyrus denté, qui ont perdu la plupart de leurs processus radiaux; (ii) une réactivité microgliale; (iii) et un déficit de la neurogénèse hippocampique postnatale. Nous avons trouvé des anomalies similaires dans les deux souris knockin, plus sévères chez les homozygotes. La comparaison de ces modèles a montré que ces anomalies prédominent chez les souris GFAPT409I, tandis que l’accumulation de GFAP est supérieure chez les souris GFAPR85C. La comparaison des deux modèles de souris a montré que les conséquences pathologiques dépendent la localisation de la mutation dans la GFAP. Ces résultats suggèrent qu'en plus du gain évident de fonction, d'autres dysfonctions astrocytaires dans peuvent être dues à une perte de fonction. De plus, nous avons traité les souris mutantes avec de la ceftriaxone, connu pour son effet neuroprotecteur, mais nous n'avons observé aucun effet significatif. Enfin, la mégalencéphalie étant fréquente chez les patients AxD, nous avons mesuré la quantité d'eau cérébrale chez les souris mutantes GFAP. Nous avons trouvé une augmentation significative de la teneur en eau chez les souris GFAPR85C/R85C âgées d'un an. Nous avons observé une localisation anormale de l'AQP4 dans les astrocytes des asouris mutées, pouvant participer au déséquilibre hydrique cérébral
Alexander disease is a neurodegenerative disorder caused by heterozygous mutations of GFAP gene coding the major intermediate filament of mature astrocytes. We studied the effect of GFAP mutation in the hippocampus of infantile onset AxD patient and two novel knockin mouse models, one bearing a mutation located in the rod domain (p.R85C), and the other bearing a mutation located in the tail domain (p.T409I) of mouse Gfap. In the AxD patient, we describe for the first time: (i) obvious morphological changes of GFAP+ cells in the subgranular zone of the dentate gyrus, which have lost most of their radial processes; (ii) microglial reactivity; (iii) and deficit in postnatal hippocampal neurogenesis. We found similar abnormalities in the two knockin mouse lines, more obvious in homozygous mice. A comparison of these mouse models showed that pathological findings predominated in the GFAPT409I mice, whereas GFAP accumulated in larger amounts in the GFAPR85C mice. The comparison of the two mouse models showed that their pathological consequences depend on the location of the mutated residues in GFAP. These findings suggest that in addition to the evident gain of GFAP function, other astrocyte dysfunctions in this disease may be due to a loss of function of GFAP. In addition, we treated the mice mutants with ceftriaxone, which has been reported to have a neuroprotective effect, but we observe no significant effect. Finally, AxD patients have often megalencephaly, therefore we measured the brain water content in AxD mouse models. We found a significant increase in brain water content in the one year old GFAPR85C/R85C mice vs controls. We observed mislocalization of AQP4 in mutant mice astrocytes that can participated to water imbalance in brain

A disfunção microvascular, envolvendo células endoteliais, plaquetas e leucócitos, está presente na hipertensão arterial pulmonar (HAP), associando-se a risco aumentado de trombose e menor sobrevida. Estudos sobre disfunção microvascular são escassos em outras formas da doença que não a idiopática. Os objetivos do estudo foram: caracterizar a disfunção microvascular em diferentes formas de HAP através da dosagem de marcadores bioquímicos, avaliando possíveis correlações com índices de gravidade; investigar os efeitos da administração de rosuvastatina em níveis circulantes de marcadores de disfunção microvascular nesses pacientes; e investigar possível associação entre o nível plasmático dos marcadores e prognóstico. Foram incluídos sessenta pacientes: 14 com HAP idiopática ou hereditária, e 46 com HAP associada a cardiopatia congênita (HAPCCg) sem hipoxemia (N=18) ou com hipoxemia (N=28), com idades entre 13 e 60 anos. Foram dosados os níveis plasmáticos circulantes do antígeno do fator de von Willebrand (vWF:Ag), ativador tecidual do plasminogênio (t-PA); inibidor do ativador do plasminogênio (PAI-1), fator de necrose tumoral (TNF-), proteína C reativa (PCR), selectina-P; interleucina-6 (IL-6); e interleucina-10 (IL -10), na condição basal e após 30, 60 e 180 dias de tratamento, por método imunoenzimático. Após randomização, administrouse placebo (N=30) ou dose única oral diária (10mg) de rosuvastatina (N=30), por seis meses. Dados demográficos e funcionais como idade, distância caminhada em seis minutos, saturação periférica de oxigênio em repouso e após esforço, bem como hematócrito, também foram registrados. Pacientes com HAPCCg foram acompanhados por um período de 0,7 a 4,0 anos (mediana de 3,6 anos). Na condição basal, excetuando-se TNF- e PCR, todas as proteínas apresentaram-se significantemente elevadas em relação aos controles (p<0,001), havendo correlação com índices de gravidade clínica. No estudo com rosuvastatina, houve redução significante nos níveis de selectina-P em relação ao placebo (p=0,037), ao longo do tratamento. Houve melhora na saturação periférica de oxigênio após seis minutos de caminhada, no grupo estatina, em pacientes com HAPCCg com hipoxemia, em relação ao placebo. Considerando-se o período de acompanhamento, em portadores de HAPCCg, níveis plasmáticos persistentemente elevados do vWF:Ag (média de quatro determinações), acima do nível correspondente ao percentil 95 dos controles (139 U/d/L) associaram-se maior risco de morte (razão de risco 6,56, IC 95% 1,46 a 29,4, p=0.014), sem alteração após ajustamento para variáveis demográficas, funcionais e de tratamento, à análise multivariada. Assim, a disfunção microvascular está presente em indivíduos com HAP idiopática, hereditária ou associada a cardiopatias congênitas. Na HAP, o uso crônico de rosuvastatina em dose baixa associase à redução do nível circulante de selectina-P, e propicia aumento na saturação periférica de oxigênio ao final do exercício, em indivíduos com HAPCCg e hipoxemia. Em indivíduos portadores de HAPCCg, níveis plasmáticos persistentemente elevados do vWF:Ag são indicativo de pior prognóstico
Microvascular dysfunction, involving endothelial cells, platelets and leukocytes, is present in pulmonary arterial hypertension (PAH), and is associated to higher risk to thrombotic complications and mortality. Most data about microvascular dysfunction in PAH do not include other forms of the disease beyond idiopathic PAH. The present study was planned to measure plasma levels microvascular dysfunction markers in two different forms of PAH, and investigate possible correlations with indices of severity of the disease; to investigate the effects of chronic rosuvastatin administration versus placebo on the circulating levels of these markers; and to investigate possible associations between levels of these parameters and prognosis. Sixty patients (aged 13 to 60 years) were included, 14 with idiopathic or hereditary PAH, and 46 with congenital heart disease-associated PAH (CHDPAH), in the absence (N=18) or presence (N=28) of hypoxemia. Plasma levels of von Willebrand factor antigen (vWF:Ag), tissue-plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor alpha (TNF-), reactive C protein (RCP), P-selectin, interleukin-6 (IL- 6), and interleukin-10 (IL-10) were measured before treatment and 30, 90, and 180 days on treatment using high-sensitivity enzyme-linked immunosorbent assay kits. Patients were randomly assigned to placebo (N=30) or a single oral dose of rosuvastatin (N=30), 10mg/day, for six months. Demographic and functional data such as age, six-minute walk distance, peripheral oxygen saturation at rest and at the end of the six-minute walk, as well as the hematocrit, were recorded. Patients with CHDPAH were followed-up for 0.7 to 4.0 years (median 3.6 years). At baseline, levels of all proteins (except TNF- and RCP) were significantly increased in patients versus controls (p<0,001), and correlated significantly with indices of severity of the disease. P-selectin level was lower in the rosuvastatin group compared with placebo throughout the treatment (p = 0.037). In hypoxemic CHDPAH patients, the peripheral oxygen saturation, at the end of the six-minute walk, was higher in the rosuvastatin group, compared with placebo. During the follow-up of patients with CHDPAH, an average vWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was associated with a high risk of death (hazard ratio 6.56, 95% CI 1.46 to 29.4, p=0.014). This was not modified after adjustment for demographic, functional and treatment-related variables in multivariate analysis. In conclusion, microvascular dysfunction is present in individuals with idiopathic, hereditary and the congenital heart disease-associated PAH. The chronic use of low-dose rosuvastatin is associated to reduction of circulating levels of P-selectin. In patients with CHDPAH with hypoxemia, rosuvastatin also increases peripheral oxygen saturation during exercise. In CHDPAH patients, a sustained increase in plasma vWF:Ag is indicative of poor prognosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease, which primarily affects middle-aged women. The liver histology is characterized by inflammation and destruction of the intrahepatic bile ducts as well as a high frequency of granuloma. Although the etiology is unknown, the occurrence of associated multiorganic abnormalities such as Sjogren's syndrome, scleroderma, rheumatic disorders and thyroid gland diseases have been cited as evidence favouring an autoimmune background. Addison and Gull in 1851 described the first patient with jaundice and xanthomatosis. PBC was first mentioned in 1876 as an entity by Hanot. PBC was considered to be a rare disease until in 1973 Sherlock and Scheuer described 100 patients. Since then a greater awareness of the disease combined with a wider use of laboratory screening methods has led to the discovery of an increasing number of patients with PBC. In an epidemiological investigation of PBC in the northern part of Sweden a point prevalence of 151 per 106 was found, which is the highest so far reported, and the mean annual incidence amounted to 13.3 per 106. Asymptomatic PBC was present in more than one third of the patients which is consistent with the finding in other epidemiological investigations and is supposed to explain the higher prevalence of PBC and the better prognosis. Nevertheless 25 patients died during the study period, 14 as a direct consequence of the liver disease. Chronic intrahepatic cholestasis has been reported in sarcoidosis and, moreover, a high frequency of liver granuloma is found. The implication of the present study is that a negative Kveim test in combination with positive mitochondrial antibodies is accurate in differentiating PBC from sarcoidosis. Multisystem involvement is frequently observed in PBC and the present study confirms this. In the prospective investigation of 26 PBC patients 50 % had arthropathy considered to be associated with PBC. Rheumatoid arthritis was found in 5 patients, who all had symptoms of liver disease in addition. Lung function impairment was present in 56% (1 asymptomatic PBC). Most commonly a reduced diffusion capacity was found (36%). Bronchial asthma was present in three patients, and severe lung emphysema in one. Features of Sjogren's syndrome was found in 73% (3 asymptomatic PBC). In 6 patients keratoconjunctivitis sicca (KCS) was evident with the rose bengal test demonstrating corneal staining and a Schirmer test of less than 5 mm. Radiological findings of sialectasia were demonstrated in 6 patients, of whom 5 had KCS as well. The ultimate treatment in PBC is liver transplantation and to calculate the need for that, good epidemiological surveys are needed, and also indicators of hepatocellular function. The present investigation indicates that determination of the von Willebrand factor could be used for this purpose.

Härtill 6 uppsatser

digitalisering@umu

Les thrombopathies congénitales sont des pathologies au phénotype hémorragique hétérogène dues à des anomalies fonctionnelles des plaquettes sanguines parfois associées à des anomalies morphologiques et/ou à une thrombopénie. Les outils diagnostic disponibles permettent de corréler une anomalie fonctionnelle plaquettaire à un déficit protéique voire à une mutation d’un gène spécifique. Cependant, en 2015, au moins 50% des thrombopathies restent non étiquetées. Dans ce contexte, le laboratoire d’hémostase de l’EFS Alsace et l’unité INSERM UMR S949 travaillent de concert à l’identification et à la caractérisation de ces pathologies. Ainsi, nous avons mis en évidence trois nouvelles mutations du récepteur P2Y12 dans trois familles différentes. La mutation p.His187Gln a été entièrement caractérisée sur des plaquettes fraiches du patient et dans un modèle cellulaire adapté. La reproduction des deux autres mutations (p.Tyr259Cys et p.Phe95Ser) est en cours. Nous avons par ailleurs identifié une famille dont trois membres sont atteints d’une maladie du pool vide non syndromique et nous suspectons grâce au séquençage d’exome effectué que le transporteur de nucléotides VNUT est impliqué dans la pathologie
Inherited platelets disorders (IPD) are pathologies associated with heterogeneous bleedingphenotypes. They are due to functional platelets deficiency that may come with morphological abnormalities and/or thrombocytopenia. Available diagnosis tools are used to link a functional deficiency to a protein defect and in some instances to a specific genetic mutation. However, in 2015 at least 50% of IPD are still not identified. In this context, the haemostasis laboratory of the EFS Alsace together with INSERM UMR S949 team are working to diagnose and characterize these pathologies. Thus, we found three new P2Y12 receptor variants in three different families. The mutation p.His187Gln has been fully characterized on blood fresh platelets coming from one patient and in an appropriate cellular model. The reproduction of the two other mutations (p.Tyr259Cys and p.Phe95Ser) is currently ongoing. We also studied a family in which three members are carrying a delta storage pool disease. The full exome sequencing analysis leads us to suspect the involvement of the nucleotides transporter VNUT in this pathology

von Willebrand Disease (VWD) is the most common inherited bleeding disorder in humans, resulting from quantitative or qualitative deficiencies of von Willebrand factor (VWF). Type 3 VWD is the rarest and most severe form of the disease. This thesis characterizes the phenotype-genotype correlations of a cohort of Canadian type 3 VWD patients and their family members. Three main findings are highlighted: 1) 50% of families showed evidence of co-dominant inheritance as opposed to recessive, 2) 42% of mutations identified were located in the VWF propeptide region (VWFpp), 3) index cases (IC) with mutations in the VWFpp had a more severe bleeding diatheses than IC with mutations elsewhere. We investigated two of the identified VWFpp mutations (ex4-5del and Cys633Arg) to elucidate their molecular mechanisms using two cellular models. Patient-derived blood outgrowth endothelial cells (BOEC) are ideal for studying the underlying molecular mechanism of VWF mutations as they represent the native vascular endothelium. BOEC were isolated from type 3 VWD IC and family members with the mutations of interest. A heterologous cellular system was also used to study the VWF mutations in vitro. The VWFpp mutations caused impaired VWF secretion, defective multimerization, qualitative and quantitative defects in Weibel-Palade body (WPB) formation, and resulted in VWF retention within the endoplasmic reticulum. We attempted to restore secretion and multimerization by co-transfecting each mutant with the wild-type VWF propeptide (VWFpp), which was unsuccessful. Additionally, we investigated a third mutation, c.8419_8422dupTCCC, which is unique to the Canadian VWD population and is found at a high frequency in a specific geographic population. While we hypothesized that this mutation would disrupt dimerization due to its location in the C-terminal cysteine knot (CK) domain of VWF we did not find this to be true. The results presented within this thesis provide new insight into the genetics and pathobiology of type 3 VWD, the functional contribution of the VWFpp to type 3 VWD and highlight the utility of BOEC as a cellular model for evaluating the pathogenic mechanisms of VWF mutations.
Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-10-17 21:15:37.685

Tese de doutoramento em Biociências, no ramo de Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Haemostasis is a normal defence mechanism that requires the combined activity of vascular, platelet and plasma factors. Under physiologic conditions, a haemostatic balance is achieved through the effects of natural procoagulant and anticoagulant factors which, in equilibrium with each other, provide haemostasis at the sites of vascular injury. Abnormalities of these haemostasis factors can lead to excessive bleeding or thrombosis. One of the key players in both processes is the von Willebrand factor (VWF), an adhesive glycoprotein of large dimensions with crucial functions in haemostasis derived from its ability to organise in multimers. The high-molecular-weight multimers (HMWM) of VWF are essential for primary haemostasis, mediating a molecular endothelium–platelet bridge for binding collagen and the platelet receptors glycoprotein (GP), GPIb and GPIIb/IIIa. In addition, VWF binds and stabilises factor VIII (FVIII) in the circulation, protecting it from rapid proteolytic degradation while delivering it to sites of vascular damage. Once secreted into the blood, VWF multimers are subject to competing processes of clearance and of proteolysis by ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13). The unusually large multimers of VWF are, under normal conditions, cleaved by ADAMTS13 to smaller, less adhesive multimers. As a result of this physiological process, VWF insufficiencies may cause haemorrhage by reducing platelet function, or by reducing the FVIII concentration. Abnormalities in VWF secretion, intravascular clearance of VWF, the assembly of VWF multimers, or exaggerated proteolytic degradation by ADAMTS13 can cause diverse forms of von Willebrand disease (VWD). Conversely, defects in proteolysis of VWF by ADAMTS13 can cause thrombotic thrombocytopenic purpura (TTP), a disease caused by the clumping of platelets by ultra-large VWF and defined clinically by microangiopathic hemolytic anaemia and thrombocytopenia. There are two main forms of thrombotic microangiopathies (TMAs) that have overlapping clinical phenotypes: TTP and atypical haemolytic uraemic syndrome (aHUS). Conceptually, TTP has been distinguished from aHUS by more common neurological manifestations, whereas in aHUS, renal involvement is more pronounced. However, this rule is not always reliable; some aHUS patients have neurological complications and some TTP patients have renal failure. Over the last decade, noteworthy progress has been made to improve the clinical and laboratory approaches to predict the expected signs and symptoms, patient outcome and genotype-phenotype correlations. Despite this, the differential diagnosis of TTP/aHUS remains challenging. While TTP is characterised by a severe deficiency of ADAMTS13, aHUS is characterised by hyperactivation of the alternative complement pathway resulting from either a loss-of-function mutation in a regulatory gene, CFH, CFI, CD46 (MCP) or THBD, or a gain-of-function mutation in an effector gene, CFB or C3. The molecular analysis of VWF, ADAMTS13 and complement genes are very useful in the diagnosis of VWD and differential diagnosis of TTP/aHUS. To better understand the clinical variability, which is found even within families, it is necessary to characterise the mutational profile. Nevertheless, up to now, the molecular analysis of VWF and genes implicated in TMAs was not affordable by diagnostic laboratories due to the high costs involved in the study of multiple genes by conventional Sanger DNA sequencing. The advent of next-generation sequencing (NGS) is changing this paradigm since NGS allows for simultaneous sequencing of large gene panels and generates competitive results at a lower cost and in a shorter period of time. With this in mind, we conducted a study in a haemorragic disease (VWD) and a thrombotic disease (TTP/aHUS) with two main objectives: first, to expand our understanding of the molecular basis of different types of VWD, TTP and aHUS and establish phenotype-genotype correlations; second, to develop a diagnosis workflow based on VWF/ADAMTS13 activity and screening of mutations using a custom NGS gene panel. This approach allowed for the creation of a new algorithm that uses an efficient and cheaper methodology to establish the diagnosis, prognosis and more accurate treatment for these diseases. First, we conducted a study to determine the correlations between genotype and phenotype in 92 Portuguese individuals from 60 unrelated families with VWD; therefore, we directly sequenced VWF. We compared the classical Sanger sequencing approach and NGS to assess the value-added effect on the analysis of the mutation distribution in different types of VWD. Sixty-two different VWF mutations were identified, 27 of which had not been previously described. NGS detected 26 additional mutations, contributing to a broad overview of the mutant alleles present in each VWD type. Twenty-nine probands (48.3%) had two or more mutations; in addition, mutations with pleiotropic effects were detected, and NGS appropriately classified seven of them. Furthermore, the differential diagnosis between VWD 2B and platelet type VWD (n = 1), Bernard-Soulier syndrome and VWD 2B (n = 1), and mild haemophilia A and VWD 2N (n = 2) was possible. Next, we evaluated two VWF dysfunctions that could be underdiagnosed: VWF collagen-(types III and VI) binding defects and acquired von Willebrand syndrome (AVWS) in hematologic malignancies. The functional and molecular studies focused on VWF collagen-binding defects reported VWF sequence variations that may not interfere with VWF functional screening assays showing normal values, and can only be detected using type VI collagen. This analysis in our cohort of nine patients, albeit small, indicated that a laboratory approach based on the correlation of type III and type VI collagen-binding assays and molecular studies is indispensable for a more accurate diagnosis of type 2M VWD. Moreover, the detection of the specific type VI collagen-binding defect may contribute to the correct diagnosis of patients with mild bleeding disorders that are often classified as having an undefined cause. AVWS is a relatively uncommon and under-diagnosed acquired bleeding disorder, and it occurs in individuals with no personal or familial history of bleeding diathesis. AVWS is usually associated with an underlying disorder, namely lymphoproliferative disorders, cardiovascular diseases, myeloproliferative neoplasms, other cancers and autoimmune diseases. The diagnosis is challenging because it greatly depends on clinical suspicion. On the other hand, the tests used to assess AVWS are the same as those for VWD, and the differential diagnosis between both can sometimes be difficult. We studied six patients with AVWS and different underlying diseases: Waldenstrom’s Macroglobulinemia (WM), Marginal Zone Lymphoma (MZL), Chronic Myeloid Leukaemia (CML) and Essential Thrombocythemia (ET). The clinical‐laboratory correlation in the six cases led to the diagnosis of AVWS. The detection of low VWF levels in two patients came before the diagnosis of the underlying lymphoproliferative disease. In another patient, the detection of the VWF variant could have led to a misdiagnosis, in this case, a congenital VWD. However, the discordant genotype-phenotype correlation and late haemorrhagic manifestation led to the suspicion of simultaneous AVWS. Including an evaluation of VWF analysis in late haemorrhagic episodes and/or in AVWS-associated disorders enhanced the efficacy of AVWS diagnosis, leading to a more tailored management approach in each patient. Finally, we analysed genotype-phenotype correlations in 45 patients (11 TTP and 34 aHUS) based on the impact of the predicted pathogenicity of each variant found and the co-inherited known genetic risk factors for TMAs. In total, 33 different rare variants, eight of which novel (in ADAMTS13, CFH and CD46), were identified across seven genes. The eleven TTP patients were homozygous (n = 6), compound heterozygous (n = 2) and heterozygous (n = 3) for 10 ADAMTS13 variants (six pathogenic mutations). Among the 34 aHUS patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause aHUS only when combined with other variants and/or CFH and CD46 risk haplotypes or CFHR1-3 deletion. Our study provides evidence of the usefulness of the NGS panel as an excellent technology that enables more rapid diagnosis of TMAs, and is a valuable asset in clinical practice to discriminate between TTP and aHUS. The custom NGS panel has made it easier to study VWF, ADAMTS13 and the complement genes. Moreover, this technology has changed the paradigm of routine molecular studies: in the face of multiple genetic changes found in every patient, the critical challenge became discriminating disease-associated variants from the broader background of variants present in all patients’ genomes. This analysis led us to a well-established bioinformatics pipeline according to NGS guidelines, and evidences that a clinical-laboratory approach for each patient’s genotypic data must be evaluated with consideration of their specific and differential clinical manifestations. In conclusion, the study of these 144 patients contributes to a better understanding of the molecular genetics of VWF, ADAMTS13 and complement gene-related phenotypes. Moreover, it provides evidence of the usefulness of the NGS panel as an excellent and advantageous technology that enables more rapid and cost-effective diagnosis. These findings show that this is a valuable asset in clinical practice given that a correct diagnosis is essential for determining the most effective treatment for each patient with these complex diseases.
A hemostase é um mecanismo de defesa que requer a atividade combinada do endotélio, plaquetas e fatores plasmáticos. Sob condições fisiológicas, o equilíbrio hemostático é conseguido através da atividade dos fatores procoagulantes e anticoagulantes naturais que, em equilíbrio entre si, proporcionam a formação de um tampão hemostático nos locais de lesão vascular. Alterações destes fatores hemostáticos podem dar origem a hemorragia ou trombose. Um dos principais intervenientes em ambos os processos é o factor von Willebrand (FVW), uma glicoproteína adesiva de grandes dimensões com funções cruciais na hemostase devido à sua capacidade de se organizar em multímeros. Os multímeros de alto peso molecular (MAPM) do FVW são essenciais para a hemostase primária, ao mediar uma ponte molecular de endotélio-plaqueta para ligação ao colagénio e aos recetores plaquetares glicoproteína (GP) Ib e GPIIb/IIIa. Além disso, o FVW liga e estabiliza o factor VIII (FVIII) na circulação, protegendo-o da rápida degradação proteolítica, ao mesmo tempo que o distribui para os locais de lesão vascular. Uma vez na corrente sanguínea, os multímeros do FVW são submetidos a processos de clearance e de proteólise pela enzima ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13). Os multímeros do FVW de grandes dimensões são, em condições normais, clivados pela ADAMTS13 em multímeros mais pequenos e menos adesivos. Como resultado deste processo fisiológico, deficiências no FVW podem causar hemorragia por redução da função plaquetar ou por redução da concentração do FVIII. Anomalias na secreção do FVW, clearance intravascular, formação dos multímeros, ou a degradação proteolítica excessiva pela ADAMTS13 podem dar origem a diversas formas da doença de von Willebrand (DVW). Por outro lado, as deficiências na proteólise do FVW pela ADAMTS13 podem originar púrpura trombocitopénica trombótica (PTT), uma doença causada por aglomerados de plaquetas e MAPM do FVW, e que se apresenta clinicamente com trombocitopenia e anemia hemolítica microangiopática. Existem duas formas principais de microangiopatias trombóticas (MATs) que têm fenótipos clínicos sobrepostos: PTT e síndrome hemolítico urémico atípico (SHUa). Conceitualmente, a PTT tem sido diferenciada do SHUa por ter manifestações neurológicas mais frequentes, enquanto no SHUa o envolvimento renal é mais pronunciado. No entanto, esta regra nem sempre é fiável: alguns doentes com SHUa, apresentam complicações neurológicas e alguns doentes com PTT apresentam insuficiência renal. Ao longo da última década foram feitos progressos notáveis para melhorar a abordagem clinico-laboratorial, para identificar sinais e sintomas, estabelecer o prognóstico dos doentes e analisar a correlação genótipo - fenótipo. Apesar disso, o diagnóstico diferencial PTT/SHUa ainda é um desafio. Embora a PTT seja caracterizada por uma deficiência grave de ADAMTS13, o SHUa resulta da hiperactivação da via alternativa do complemento devida a uma mutação com perda-de-função num dos genes reguladores, CFH, CFI, CD46 (MCP) ou THBD, ou de uma mutação com ganho-de-função num gene efetor, CFB ou C3. A análise molecular do VWF, ADAMTS13 e genes do complemento é muito útil no diagnóstico da DVW e diagnóstico diferencial de PTT/SHUa. Para melhor compreender a variabilidade clínica, inclusivé dentro da mesma família, é necessário caracterizar o perfil mutacional. Contudo, até agora, a análise molecular do VWF e dos genes implicados nas MATs não estava acessível aos laboratórios de diagnóstico devido aos elevados custos envolvidos no estudo de múltiplos genes pelo método convencional de sequenciação do DNA (Método de Sanger). O aparecimento da sequenciação de nova geração (NGS-next generation sequencing) está a alterar este paradigma, uma vez que a NGS permite a sequenciação simultânea de grandes painéis de genes e gera resultados competitivos a um custo mais baixo e num curto período de tempo. Baseados nestes dados, realizámos um estudo numa doença hemorrágica (DVW) e numa doença trombótica (PTT/SHUa) com dois objetivos principais: 1) alargar o nosso conhecimento sobre a base molecular dos diferentes tipos de DVW, PTT e SHUa e estabelecer correlações genótipo – fenótipo; 2) desenvolver uma estratégia de diagnóstico com base na atividade de FVW/ADAMTS13 e desenhar um painel de genes para efetuar a pesquisa de mutações por NGS. Esta abordagem permitiu-nos criar um novo algoritmo de estudo, que utiliza uma metodologia eficiente e mais barata, e que permite estabelecer o diagnóstico e o prognóstico e contribui para uma melhor definição da estratégia terapêutica destas patologias. Em primeiro lugar, realizámos um estudo para determinar as correlações entre o genótipo e o fenótipo em 92 indivíduos Portugueses, de 60 famílias não relacionadas com DVW; assim, sequenciámos diretamente o VWF. Comparámos a abordagem clássica de sequenciação de Sanger e a metodologia NGS para avaliar a mais-valia na análise de mutações nos vários tipos de DVW. Foram identificadas 62 mutações diferentes no VWF, 27 das quais não tinham sido previamente descritas. A NGS detetou 26 mutações adicionais, contribuindo para uma perspetiva alargada dos alelos mutados presentes em cada tipo de DVW. Vinte e nove propósitos (48,3%) apresentavam uma ou mais mutações; além disso, foram detetadas mutações com efeitos pleiotrópicos, tendo a NGS classificado adequadamente sete delas. Foi ainda possível o diagnóstico diferencial entre DVW 2B e DVW tipo plaquetar (n=1), síndrome de Bernard-Soulier e DVW 2B (n=1) e hemofilia A ligeira e DVW 2N (n=2). De seguida, avaliamos duas disfunções do FVW que poderiam estar a ser subdiagnosticadas: defeitos de ligação FVW-colagénio (tipos III e VI) e síndrome de Von Willebrand adquirida (SVWA) em doenças hemato-oncológicas. Os estudos funcionais e moleculares incidiram nas alterações de ligação ao colagénio associados a variações da sequência do VWF que podem não interferir nos testes funcionais de screening, que apresentam valores normais, e que só podem ser detetados utilizando colagénio tipo VI. Esta análise no nosso grupo de nove doentes, apesar de pequeno, indicou que uma abordagem laboratorial baseada na correlação entre os ensaios de ligação ao colagénio tipo III e tipo VI e os estudos moleculares é indispensável para um diagnóstico mais preciso da DVW tipo 2M. Além disso, a deteção dos défices específicos da ligação ao colagénio tipo VI pode contribuir para o diagnóstico de doentes com perturbações hemorrágicas ligeiras que muitas vezes são classificados com tendência hemorrágica de causa desconhecida. SVWA é uma patologia hemorrágica adquirida, subdiagnosticada e relativamente pouco frequente, que ocorre em indivíduos sem história pessoal ou familiar de diátese hemorrágica. SVWA está geralmente associado com uma patologia subjacente, nomeadamente doenças linfoproliferativas, doenças cardiovasculares, neoplasias mieloproliferativas, outros cancros e doenças autoimunes. O diagnóstico é desafiador porque depende muito da suspeita clínica. Por outro lado, os testes usados para diagnosticar o SVWA são os mesmos utilizados para o diagnóstico da DVW, e a distinção entre as duas patologias pode ser difícil. Avaliámos seis doentes com SVWA e diversas patologias subjacentes: Macroglobulinemia de Waldenstrom (MW), Linfoma de Zona Marginal (LZM), Leucemia Mielóide Crónica (LMC) e Trombocitémia Essencial (TE). A correlação clínica – laboratorial nos seis casos permitiu o diagnóstico de SVWA. A deteção de níveis baixos de FVW em dois doentes ocorreu antes do diagnóstico da doença linfoproliferativa subjacente. Noutro doente, a deteção da variante de VWF poderia ter levado a um diagnóstico incorrecto de DVW congénita. No entanto, a correlação genótipo - fenótipo discordante e a manifestação hemorrágica tardia, levou à suspeita da presença concomitante de SVWA. Incluir uma avaliação da análise do VWF em episódios hemorrágicos tardios e/ou em doenças associadas com SVWA aumentou a eficácia do diagnóstico de SVWA, e permitiu uma estratégia terapêutica mais individualizada. Finalmente, analisámos as correlações genótipo-fenótipo em 45 pacientes (11 PTT e 34 SHUa) com base no impacto da patogenicidade prevista para cada variante encontrada e dos fatores hereditários de risco genético para MATs. No total, foram identificadas 33 variantes raras diferentes em sete genes, oito das quais descritas pela primeira vez (em ADAMTS13, CFH e CD46). Os onze doentes com PTT eram homozigóticos (n=6), heterozigóticos compostos (n=2) e heterozigóticos (n=3) para 10 variantes ADAMTS13 (seis mutações patogénicas). Entre os 34 doentes com SHUa, 17 eram heterozigóticos para 23 variantes nos diferentes genes do complemento com consequências distintas, desde mutações patogénicas únicas associadas com penetração completa da doença, a variantes benignas que apenas causam SHUa quando associadas a outras variantes e/ou haplótipos de risco CFH e CD46 ou delecção CFHR1-3. O nosso estudo evidencia a utilidade do painel NGS como uma excelente tecnologia que permite um diagnóstico mais rápido de MATs, e uma mais-valia na prática clínica para discriminar entre PTT e SHUa. O painel NGS personalizado tornou mais fácil o estudo do VWF, ADAMTS13 e dos genes do complemento. Além disso, esta tecnologia mudou o paradigma dos estudos moleculares de rotina: face às múltiplas alterações genéticas encontradas em cada doente, o desafio principal foi discriminar as variantes associadas com a doença de entre as variantes presentes no genoma de todos os doentes. Esta análise levou-nos a um algoritmo bioinformático bem estabelecido, de acordo com as guidelines NGS, e à demonstração de que os dados do genótipo de cada doente devem ser avaliados numa abordagem clínico – laboratorial, tendo em conta as suas manifestações clínicas específicas. Em conclusão, o estudo destes 144 doentes contribui para um melhor entendimento da genética molecular de VWF, ADAMTS13 e dos genes do complemento, assim como dos fenótipos associados. Além disso, evidencia a utilidade do painel NGS como uma tecnologia excelente e vantajosa que permite um diagnóstico mais rápido e mais económico. Estes resultados mostram ainda que esta estratégia é uma mais-valia na prática clínica dado que um diagnóstico correto é determinante na escolha da melhor estratégia terapêutica para cada um destes doentes com patologias tão complexas.
Forum Hematológico

Von Willebrand factor (VWF) promotes platelet adhesion and aggregation at sites of vascular damage. This function is directly related to the multimer size of VWF. The VWF-specific metalloprotease ADAMTS13 decreases VWF multimer size by cleaving at Y1605-M1606 in the VWF A2 domain. This thesis examined the sensitivity of ADAMTS13 cleavage to mutagenesis of the full-length multimerized VWF substrate, and a small VWF A2 domain fragment, VWF115. The ADAMTS13 cleavage site at Y1605-M1606 was mutated with the most severe loss of cleavage observed in Y1605A/M1606A. In addition, 4 single nucleotide polymorphisms were examined, with D1472H, Q1571H, P1601T proteins all showing increased resistance to cleavage. In contrast, G1643S has enhanced cleavage in the full-length VWF substrate but shows cleavage resistance in VWF115. Three von Willebrand disease mutations were also examined. In patients, R1597W has enhanced ADAMTS13 cleavage and a loss of high molecular weight multimers, while R1205H has enhanced protein clearance resulting in very low VWF levels and Y1584C patients have moderately low VWF levels. R1597W has enhanced cleavage of full-length VWF, while a slight cleavage increase is observed in VWF115 for Y1584C, and no change is seen with R1205H. The VWF mutations R1597W, Y1605A/M1606A, R1205H and Y1584C were further examined in the VWF knockout mouse using recombinant VWF protein infusion and hydrodynamic delivery of VWF cDNA to determine the effects these mutations produce on VWF antigen levels, multimer structure, secretion, clearance and function in a thrombotic injury model. All four mutations had different pathogenic mechanisms. R1597W showed accelerated clearance with loss of multimer structure, and greatly increased time to thrombotic occlusion. Y1605A/M1606A showed accelerated clearance with normal or supranormal multimer structure, a loss of thrombotic occlusion but increased platelet accumulation. Y1584C showed no change in protein clearance, with decreased VWF antigen level, reduced multimer structure, and reduced thrombotic potential. R1205H demonstrated a synthetic defect in vitro and in vivo increased clearance with a decrease in VWF antigen levels and normal multimer structure and a variable thrombotic potential. These results validate the use of the genetically-modified VWF knockout mouse model for evaluating the pathogenic mechanisms of putative VWF mutations.
Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-07-28 10:24:40.654