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

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

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

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

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.

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

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.

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

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.