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.

Acute necrotizing pancreatitis (ANP) is a common gastrointestinal cause of emergency admissions in dogs and humans and can lead to a systemic inflammatory response syndrome resulting in multiple organ dysfunction syndrome. Among the various complications associated with ANP, acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome (ARDS), are major contributors leading to high mortality rates associated with severe acute pancreatitis (AP) in human patients. The incidence of ALI/ARDS in ANP dogs is not well characterized. However, signs of respiratory complications have been reported clinically in dogs suffering from AP. The pathophysiology of ANP and its systemic complications in dogs and humans are not well understood. Most of the data related to AP comes from rodent models of AP, which may not always represent the true mechanisms occurring in the lungs of dogs or humans with ANP. I decided to undertake evaluation of pancreas and lungs from dogs (N=21) that died of ANP. The cases were selected through the search of the medical records of the Veterinary Medical Center of the Western College of Veterinary Medicine (WCVM). Six healthy SPCA dogs were used as controls. The histology of pancreas was first graded to record the range of ANP severities within dog cases included in this study. Then, characterization of lung inflammation was done with histological grading and qualitative analysis of immunohistochemical staining for von Willebrand Factor (vWF), Toll-Like Receptor-4 (TLR4), interleukin-6 (IL6), and inducible nitric oxide synthase (iNOS). Quantification of the recruitment of septal macrophages in the lungs, designated as pulmonary intravascular macrophages (PIMs), in ANP dogs was achieved by counting the number of positive cells in alveolar septa using a macrophage antibody (MAC387). The results revealed that dogs suffering from ANP have variable lung inflammation, which was characterized by a significant infiltration of mononuclear phagocyte cells in the alveolar septa of all ANP dogs (median, 138; range 31-935) compared to control dogs (median: 1.5; range 0-16; p < 0.001), which suggested that PIMs are induced in ANP. In addition, robust staining for vWF in alveolar septal capillaries in lungs of ANP dogs suggested a strong microvascular inflammatory response. Finally, TLR4, IL6, and iNOS expression was increased in lungs of ANP dogs compared to control dogs. The second study was to investigate whether PIMs are induced in a mouse model of L-arginine-induced ANP. Therefore, lungs of L-arginine treated mice (n=7 per time point) were evaluated at various time points (24 hours, 72 hours and 120 hours) using histology and immunohistochemical staining for CD68 cells and vWF. Nine control mice were used. Counting of CD68-positive cells in the lungs of mice treated with L-arginine showed increased numbers of mononuclear phagocytes in alveolar septa at every time point (p<0.001). Also, the lung’s vasculature from L-arginine-treated mice showed increased vWF staining. Taken together, the data showed that ANP in dogs caused significant recruitment of PIMs, increased expression of vWF, TLR4, IL-6, and iNOS suggesting presence of lung inflammation. The mouse model of L-arginine-induced ANP also showed recruitment of PIMs and increased vascular expression of vWF suggesting that this model may be relevant to study the mechanisms of PIMs recruitment and their functions in lung physiology associated with ANP.

Resumo: A Doença de von Willebrand (DvW) é o distúrbio hemostático hereditário mais comum nos homens e também nos cães. A DvW já foi diagnosticada em mais de 54 raças de cães nos EUA. A DvW é causada por um defeito quantitativo e/ou qualitativo do Fator de von Willebrand (FvW), e nos cães é dividida em 3 tipos, tipo I, II e III. O diagnóstico da DvW está baseado na quantificação do FvW plasmático e testes de função plaquetária dependentes do FvW. Esse estudo foi dividido em duas partes, avaliação da prevalência da DvW (parte I) e avaliação das alterações do FvW em cadelas gestantes, e durante diferentes estágios do ciclo reprodutivo, em cadelas não gestantes (parte II). Os objetivos da parte I foram determinar a prevalência da Doença de von Willebrand (DvW) em cães da região de Botucatu, São Paulo, Brasil, e avaliar os testes laboratoriais para diagnóstico desta doença. Foram utilizados 350 cães de variadas idades e raças, de ambos os sexos. Os animais utilizados no estudo não tinham histórico ou evidências clínicas de alterações hemostáticas. Foram realizados os seguintes testes: antígeno do Fator de von Willebrand (vWF:Ag), Tempo de sangramento da Mucosa Oral (TSMO), Tempo de tromboplastina parcial ativada (TTPa) e atividade do Fator VIII (FVIII). A prevalência encontrada para Doença de von Willebrand em cães da região de Botucatu foi de 1,43%. A determinação do vWF:Ag foi o melhor teste laboratorial para diagnosticar a DvW. Os objetivos da parte II foram a avaliação das alterações encontradas na concentração do FvW em cadelas gestantes, e também durante diferentes estágios do ciclo reprodutivo, observados após a gestação, em animais portadores e não portadores para a DvW, além da avaliação de correlação entre FvW e cortisol. Foram utilizadas 7 cadelas portadoras (GI) e 9 não portadoras (GII) para DvW. Os animais foram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Von Willebrand disease (vWD) is the most common inherited hemostatic disturbance affecting men and also dogs. It has already been diagnosed in more than 54 dog breeds in the USA. vWD is caused by a quantitative and/or qualitative defect in von Willebrand Factor (vWF); in dogs, it is divided into types I, II and III. The diagnosis of vWD is based on plasma vWF quantification and vWF-dependent platelet function tests. This study was divided in two sections, evaluation of vWF prevalence (Section I) and evaluation of vWF concentrations in pregnant bitches, and during various reproductive cycle stages in nonpregnant bitches (section II). The aims of (section I) were to assess the prevalence of von Willebrand disease in dogs from the region of Botucatu, São Paulo State, Brazil, and to evaluate diagnostic tests. The study included 350 dogs of various ages, different breeds, and both sexes. Dogs included in the study had no historical or clinical evidence of abnormal bleeding. von Willebrand factor antigen (vWF:Ag), Buccal mucosal bleeding time (BMBT), Activated partial thromboplastin time (aPTT), and Factor VIII activity (FVIII) were evaluated in their ability to diagnose vWD. The prevalence of vWD in dogs was 1.43% in the Botucatu region of Brazil. Determination of vWF:Ag was the best laboratory test to diagnose vWD. The aims of section II were the evaluation of vWF concentrations in pregnant bitches, and during various reproductive cycle stages in non-pregnant bitches, observed after the pregnancy in vWD affected and unaffected bitches, over there of the correlation among vWF and cortisol. Were used seven bitches affected (GI) and nine unaffected (GII) to vWD. Animals were evaluated during the pregnancy, parturition, lactation, and during various reproductive cycle stages in non-pregnant bitches. vWF concentration did not change during reproductive cycle stages in non-pregnant bitches... (Complete abstract click electronic access below) / Orientador: Regina Kiomi Takahira / Coorientador: João Pessoa Araujo Junior / Banca: Raimundo Souza Lopes / Banca: Luiz Henrique de Araújo Machado / Banca: Mere Erika Saito / Banca: Paulo Ricardo de Oliveira Paes / Doutor

The hamster estrous cycle lasts four days and is considered to be a physiological model for angiogenesis. Angiogenesis is the formation of new capillaries from preexisting vessels, and it occurs extensively during corpus luteum formation in the estrous cycle. Von Willebrand Factor (vWF) is a glycoprotein that is secreted uniquely in endothelial cells and megakaryocytes. It is frequently used as an endothelial cell marker and it is able to detect vessels within tissues when it is used in immunohistochemical staining techniques. This study explores von Willebrand Factor expression within Golden Hamster ovarian tissue. In particular, this study uses cage control and antiorthostatic cage suspension tissue. Antiorthostatic cage suspension is a model developed to mimic and study the physiological effects caused by microgravity, such as that experienced in space flight. It is hypothesized that simulated microgravity caused by antiorthostatic cage suspension would result in lower levels of vasculature and expression of vWF within ovarian tissue. Due to financial considerations, conclusive data was not obtained due to a lack of statistics. However, our study indicates that vasculature and vWF expression may be increased in antiorthostatic cage suspension tissue.

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.

The force-induced activation of adhesive proteins such as von Willebrand Factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

A doença de von Willebrand (DvW) é um defeito qualitativo/quantitativo do fator de von Willebrand (FvW), uma glicoproteína que desempenha um papel essencial na adesão e agregação plaquetária. Cães acometidos por esta diátese hemorrágica hereditária podem ser assintomáticos, ou apresentar sinais clínicos de um problema hemostático primário, especialmente hemorragias em superfícies mucosas. O diagnóstico da DvW baseia-se na quantificação do FvW plasmático, este sendo atualmente realizado por ELISA – antígeno FvW (Ag:FvW). O presente trabalho desenvolveu um ensaio para quantificação do Ag:FvW em amostras caninas e aplicou o teste em uma população de cães. Também determinou intervalos de referência para o ensaio e para tempos de coagulação. O ensaio apresentou R² médio de 0,9810, com coeficientes de variação intra-teste de 1,83 a 4,54% e entre-teste de 9,02 a 17,75%. Valores de referência para Ag:FvW, Tempo de Protrombina e Tempo de Tromboplastina Parcial Ativada obtidos foram de 24,87 a 224,5%, 6,0 a 9,3 segundos e 15,2 a 24,5 segundos, respectivamente. Completando, foi feita uma revisão da literatura sobre a doença em cães. Em conclusão, a determinação do Ag:FvW é um teste essencial para pacientes com histórico de diátese hemorrágica sem coagulopatia e/ou trombocitopenia e pode ser conclusivo para o diagnóstico de DvW. Obter valores de referência para a população local e padronizar os reagentes e instrumentos utilizados são extremamente importantes para um diagnóstico acurado dos distúrbios de hemostasia. Finalmente, um caso clínico de um cão com DvW também foi descrito. / The von Willebrand disease (vWD) is a von Willebrand factor (vWF) quantitative/qualitative defect. This glycoprotein plays a crucial role on platelet adhesion and aggregation. Dogs affected by the hemorrhagic diathesis may be asymptomatics, or show clinical signs of a primary hemostatic disturbance, such as bleeding from mucosal surfaces. The diagnosis is based on vWF quantification by ELISA techniques – vWF antigen (vWF:Ag). The current study developed an assay for vWF:Ag quantification on canine samples, and the test was conducted on a dog population. Also, reference intervals were determined for this assay and for clotting times. The assay achieved a mean R² of 0.9810, with coefficient of variation for intra-assay of 1.83 to 4.54% and for inter-assay of 9.02 to 17.75%. The vWF:Ag assay, Prothrombin Time and activated Partial Thromboplastin Time reference intervals were 24.87 to 224.5%, 6.0 to 9.3 seconds and 15.2 to 24.5 seconds, respectively. In addition, a literature review about the disease in dogs was done. In conclusion, vWF:Ag determination is an essential assay for patients with hemorrhagic diathesis history without coagulopathy and/or thrombocytopenia, and might be conclusive for the disease diagnosis. Reference values for local population and standard protocols are extremely important for an accurate diagnosis of disorders of hemostasis. Finally, a case report of a dog with vWD was also described.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-25Bitstream added on 2014-06-13T18:50:22Z : No. of bitstreams: 1 utsunomia_ek_me_botfm.pdf: 415505 bytes, checksum: 904bb5b0f94628384d4ffd205f0db712 (MD5) / A doença de von Willebrand (DvW) é decorrente da deficiência qualitativa ou quantitativa do Fator de von Willebrand (FvW) e é considerada a coagulopatia hereditária mais comum na população com uma prevalência de 1,1% na população geral. Para o seu diagnóstico são utilizados diversos métodos como o teste do cofator da ristocetina, que é amplamente utilizado na rotina laboratorial por ser considerado padrão-ouro na avaliação da função do fator de von Willebrand e por estar diminuído em todos os casos da doença. O kit comercial de plaquetas liofilizadas é importado, de elevado custo e possui uma validade de 30 dias após sua reconstituição se conservado de 2C a 8C. Devido à baixa prevalência da DvW na população, muitas vezes não há uma grande quantidade de exames a serem realizados, fazendo com que os plasmas desses pacientes sejam congelados e armazenados até o momento onde apareça um número mínimo de pacientes. O estudo teve como objetivo padronizar uma técnica como alternativa na utilização de plaquetas fixadas em formalina e o seu teste em indivíduos saudáveis e com a suspeita da doença, onde foram avaliados a porcentagem de atividade expressa considerando o montante de aglutinação induzida pela ristocetina, que se relaciona proporcionalmente com a concentração do FvW e o percentual de atividade normal medido através do agregômetro. O estudo possui um caráter preliminar e apresentou-se viável à rotina laboratorial, e se faz necessário não só a coleta de um número amostral maior, mas também da análise de diversas variáveis (grupo sanguíneo, idade, hormônios, estresse, uso de medicamentos) para se estabelecer nessa população um valor de referência para essa técnica / The von Willebrand disease (VWD) is caused by a qualitative or quantitative deficiency of von Willebrand Factor (vWF) and is considered the most common hereditary bleeding disorder in the population with a prevalence of 1.1% in the general population. For its diagnosis, several methods are used to test the ristocetin cofactor, which is widely used in laboratories because it is considered the gold standard in assessing the function of von Willebrand factor and to be decreased in all cases of the disease. The imported commercial kit of lyophilized platelets is costly and expires 30 days after reconstitution if stored from 2C to 8C. Due to the low prevalence of v WD in the population, usually there are not enough tests to be done, making it necessary to freeze and store the plasma of these patients until there is a minimum number of patients for testing. This study aimed to standardize a technique as an alternative to the use of formalin-fixed platelets and to test it in healthy subjects with suspected disease. Thus, the percentage of expressed activity was evaluated considering the amount of agglutination induced by ristocetin proportionally related to the concentration of vWF and the percentage of normal activity measured by an aggregometer. The study has a preliminary character and showed to be feasible for laboratory routine, and it is necessary not only to collect a larger sample size, but also to analyze several variables (blood group, age, hormones, stress, use of drugs) to establish a reference value for this technique in the population