Thesis (Ph. D.)--University of Iowa, 2009. / Thesis supervisors: Kai Wang, Trudy Burns. Includes bibliographical references (leaves 132-136).

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

In humans, von Willebrand factor (vWF) is a key component in hemostasis and acts as a 'cellular adhesive' by letting the circulating platelets bind to exposed subendothelium. It also acts as a carrier and stabilizer of factor VIII (FVIII). A dysfunction or reduction of vWF leads to von Willebrand disease (vWD), resulting in bleeding phenotype which affects 1% of the population. Currently there are a variety of animal models used for the study of vWF and vWD; however, they do not possess the advantages found in zebrafish. Therefore, we set out to establish zebrafish as a model for the investigation of vWF and vWD through the use of bioinformatics and various molecular techniques. Using bioinformatics we found that the vWF gene is located on chromosome 18, that the GPIb? protein sequence is conserved. Confirmation of vWF production was shown by means of immunostaining and by RT-PCR, in thrombocytes as well as in veins and arteries. Evidence of vWF involvement in hemostasis and thrombosis was shown using MO and VMO technology to produce a vWD like phenotype, resulting in an increase in TTO and TTA, as well as a reduction in FVIII when blood was tested using the kPTT assay, coinciding with a decrease in vWF. Stimate treatment provided opposite results of MO and VMO, showing a decrease in TTO and TTA. Investigation of the role of microparticles in hemostasis and their interaction with vWF resulted in a conclusion that the GPIb? receptor should exist on MPs and that it may interact not only with zebrafish vWF but also with human UL-vWF. Agglutination of MPs in the presence of UL-vWF but in the absence of ristocetin and plasma, treatment with ADAMTS-13 abolishing the interaction between MPs and UL-vWF provided evidence that vWF interacts with MPs probably with the GPIb?. We also found that TMPs agglutinate within the vessel wall in vivo when treated with Stimate. In conclusion, this research provided evidence for the presence of vWF in zebrafish and its conserved role in hemostasis. In addition to this we also showed that MPs also participation in hemostasis.

No description available.

Multimerin 1 (MMRN1) is a massive, homopolymeric platelet and endothelial cell protein with functions that are emerging to support platelet adhesive processes. MMRN1 supports platelet adhesion under arterial flow conditions by a mechanism dependent on interactions with von Willebrand factor (VWF). The goals of this thesis were to further define the platelet adhesive functions of MMRN1 by: 1) characterizing the molecular mechanisms of VWF interactions with MMRN1; and 2) investigating if multimerin 1 is important for platelet adhesive functions using mice with and without a selective multimerin 1 (Mmrn1) deficiency. Studies of the mechanism of MMRN1-VWF binding indicated that MMRN1 bound to shear exposed VWF, and that MMRN1 interacted with the A1 and A3 domains in the VWF A1A2A3 region. VWF A1A2A3 also bound to MMRN1 with a physiologically relevant binding affinity, and supported platelet adhesion to MMRN1 at a high shear rate. The selective loss of Mmrn1 in mice had limited effects on tail bleeding times, although it impaired collagen-induced aggregation of washed platelets, as well as high shear platelet adhesion of whole blood on collagen surfaces, in vitro. Additionally, the selective loss of Mmrn1 in mice was associated with impaired and delayed platelet-rich thrombus formation in vivo, in arterioles treated with ferric chloride. These findings provide new insights on platelet adhesive, haemostatic functions at arterial shear rates, and the involvement of the platelet and endothelial cell protein, multimerin 1, to support these processes. / Thesis / Doctor of Philosophy (PhD)

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.

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.

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.

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