Orientador: Joyce Annichino-Bizzacchi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T06:24:29Z (GMT). No. of bitstreams: 1 Bittar_LuisFernando_D.pdf: 2765667 bytes, checksum: 034770d407c1c68eebc65646b23b7cc9 (MD5) Previous issue date: 2013 / Resumo: Introdução: Aumento dos níveis de fator VIII (FVIII) é um fator de risco prevalente e independente para tromboembolismo venoso (TEV). Síndrome pós-trombótica (SPT) é uma complicação a longo prazo da trombose venosa profunda (TVP) de membros inferiores, que está presente em 20% -50% dos pacientes, e podem ser associados a um processo inflamatório crônico. O receptor low density lipoprotein receptor-related protein 1 (LRP1) tem sido associado ao catabolismo do FVIII. Metodologia: Após uma mediana de 10 anos do primeiro episódio trombótico, avaliamos os níveis de FVIII em 68 pacientes com TEV e níveis elevados prévios deste fator, e 67 controles saudáveis. Posteriormente, foi analisada a presença de SPT em pacientes e sua relação com os níveis plasmáticos de FVIII. Além disso, foram avaliadas as regiões dos genes do FVIII e do LRP1 que codificam as regiões de afinidade entre eles, com o objetivo de verificar se essas alterações moleculares estão associadas aos níveis plasmáticos de FVIII, e com o TEV. Por último, foi avaliada a expressão proteica de LRP1 no fígado de 20 pacientes cirúrgicos. Resultados: Após 10 anos do primeiro episódio de TEV, os níveis de FVIII foram significativamente maiores em pacientes quando comparados aos controles (158,0 UI / dL vs 126,1 UI / dL, p <0,001]. Pacientes com SPT grave apresentaram níveis aumentados de FVIII (182,0 UI / dL) quando comparados aos pacientes com SPT moderada (155,5 UI / dL, p <0,001) ou sem PTS (154,0 UI / dL, p <0,001). Apesar de encontrarmos 14 alterações moleculares nos genes do FVIII e do LRP1, não foi encontrada nenhuma relação entre essas alterações moleculares e os níveis plasmáticos de FVIII ou com o TEV. Além disso, não foi observada correlação entre a expressão do LRP1 nas células hepáticas e os níveis plasmáticos de FVIII. Conclusões: Nós demonstramos um aumento persistente dos níveis de FVIII em um subgrupo de pacientes com TEV, mas de uma magnitude muito menor após 10 anos do primeiro episódio de TEV. Além disso, observamos uma associação significativa entre o aumento dos níveis plasmáticos de FVIII e SPT grave / Abstract: Introduction: Increased levels of factor VIII (FVIII) are a prevalent and independent risk factor for venous thromboembolism (VTE). Post-thrombotic syndrome (PTS) is a long-term complication of deep venous thrombosis (DVT) of the lower limbs that is present in 20%-50% of patients and can be associated to a chronic inflammatory process. The low density lipoprotein receptor-related protein 1 (LRP1) has been associated with FVIII catabolism. Methodology: After a median of 10 years of the first thrombotic episode, we evaluated FVIII coagulation levels in 68 patients with VTE and previous high levels of FVIII and in 67 healthy controls. Subsequently, we analyzed the presence of PTS in patients and its relationship with plasma levels of FVIII. Moreover, we evaluated the regions of FVIII and LRP1 genes encoding regions of affinity between these proteins, with the objective of determining whether these molecular changes are associated with plasma levels of FVIII and VTE. Finally, we evaluated the protein expression of LRP1 in the liver of 20 surgical patients. Results: After 10-years median of the first VTE episode, FVIII levels were significantly higher in patients when compared to controls (158.0 IU/dL vs. 126.1 IU/dL; p<0.001]. Patients with severe PTS showed increased levels of FVIII (182.0 IU/dL) when compared to patients with moderate PTS (155.5 IU/dL; p<0.001) or no PTS (154.0 IU/dL; p<0.001). Despite we have found 14 molecular changes in the FVIII and LRP1 genes, no relationship was found between these molecular alterations and FVIII levels or VTE. Moreover, no correlation was observed between LRP1 expression in the liver cells and plasma FVIII levels. Conclusions: We demonstrated a persistent increase of FVIII levels in a subset of patients with VTE, but in a much lower magnitude after 10 years of the first VTE episode. Moreover, we observed a significant association between increased plasma FVIII levels and severe PTS / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

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.

Motivation In the black population of South Africa, cardiovascular disease (CVD) is rapidly increasing due to urbanisation. Stroke is usually accompanied by a prothrombotic haemostatic profile. Changing lifestyle factors that accompany the urbanisation process could have a negative impact on the haemostatic profile of black South Africans. Elevated levels of pro-coagulant factors, von Willebrand factor (vWF), fibrinogen and fibrin D-dimer have been reported in the black population, which could increase the black population’s susceptibility to CVD. However, low levels of plasminogen activator inhibitor-1 (PAI-1) previously reported in the black population could contribute towards a pro-fibrinolytic state, which may counteract the hypercoagulant state. This may have a beneficial effect on the haemostatic profile of the black population. More investigation into the haemostatic profile of black South Africans is therefore needed to determine if an altered haemostatic profile exists in this group, and if so, to what extent these alterations may relate to cardiovascular dysfunction. This study included markers of both the coagulation (vWF, fibrinogen, fibrin D-dimer) and fibrinolytic (PAI-1, fibrin D-dimer and fibrinolytic potential) systems in an attempt to investigate the haemostatic profile of the black population of South Africa, and for comparison purposes that of the white population as well. The relationship of these markers’ with selected markers of cardiovascular function was also examined to determine if they could possibly contribute to an increase in cardiovascular risk, especially in the black population. Aims The aims of this study were to first compare coagulation and fibrinolysis markers in the black and white populations of South Africa. Furthermore, to determine if associations exist between the selected components of the haemostatic system and markers of cardiovascular function, especially in the black population of South Africa, who tends to be at a higher cardiovascular risk due to altered metabolic and haemostatic profiles. Methodology The Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study was a prospective cohort study that consisted of 409 participants at baseline (2008-2009) that were equally distributed according to both ethnicity (200 black; 209 white) and gender (black, 101 men, 99 women; white, 101 men, 108 women). At follow-up (2011/2012) the cohort totalled 359 participants (170 black, 88 men and 82 women; 189 white, 93 men and 96 women). Data from baseline measurements were used for the first two manuscripts (chapters 2 and 3), while followup data was used for the third manuscript (chapter 4). vWF, fibrinogen, PAI-1, fibrin D-dimer, CLT, serum peroxides, glutathione, glutathione peroxidase and reductase activity were determined, and ambulatory blood pressure and the retinal vessel calibres were measured. The groups were stratified by ethnicity as specified by statistical interaction terms. T-tests and chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between the components of the haemostatic system and cardiovascular function markers. This was followed by multiple linear regression analyses to investigate whether independent associations exist between the variables in both ethnic groups. P-values ≤0.050 were deemed significant. Results and conclusion of each manuscript The first manuscript (chapter 2) compares the haemostatic profiles of the black and white population to determine whether ambulatory blood pressure is related to components of the haemostatic system. The black participants displayed a prothrombotic profile with significantly higher vWF, fibrinogen, PAI-1, fibrin D-dimer and a longer CLT than their white counterparts. Furthermore, partial and multiple linear regression analyses showed a positive association of systolic and diastolic blood pressure with fibrin D-dimer in the black population, while a negative association existed between ambulatory blood pressure and CLT in the white population. These associations suggest that fibrin D-dimer may contribute, at least in part, to the high prevalence of hypertension in the black population. The second manuscript (chapter 3) determined associations between markers of the haemostatic and oxidant-antioxidant systems in the black and white populations. In addition to the prothrombotic profile that exists in the black population, this group also had significantly higher serum peroxides (oxidative stress) and lower glutathione peroxidase activity (antioxidant) levels. Multiple linear regression analyses indicated positive associations between fibrinogen and serum peroxides in both populations. In the white population, an additional positive association was found between serum peroxide and CLT. In the black population, vWF and CLT were negatively associated with GPx activity. The results suggest that there are ethnic-specific relationships between the haemostatic and oxidant-antioxidant systems. The third manuscript (chapter 4) investigated the relationships between the retinal vessel calibres and components of the haemostatic system in the black and white population. The investigation focussed specifically on arteriolar diameters in the lower median, since a narrow arteriolar diameter is known to be associated with elevated blood pressure. In both ethnic groups, a narrower arteriolar calibre was accompanied by narrower venular calibres. Independent positive associations were found between the central retinal vein equivalent (CRVE) and fibrinogen in the black population, as well as vWF and CLT in the white population. In addition, independent negative associations were found between the central retinal artery equivalent and CLT in the black population and with vWF in the white population. The results suggest that haemostatic alterations are linked to early vascular changes that may differ between ethnicities. General conclusion Ethnic-specific relationships between the components of the haemostatic system and measures of cardiovascular function are evident. The prothrombotic profile that is observed in the black population, together with the adverse associations of the haemostatic components with blood pressure, a compromised oxidant-antioxidant profile, and retinal vessel calibres may contribute, at least in part, to the high cardiovascular and cerebrovascular risk evident in this population group. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Motivation In the black population of South Africa, cardiovascular disease (CVD) is rapidly increasing due to urbanisation. Stroke is usually accompanied by a prothrombotic haemostatic profile. Changing lifestyle factors that accompany the urbanisation process could have a negative impact on the haemostatic profile of black South Africans. Elevated levels of pro-coagulant factors, von Willebrand factor (vWF), fibrinogen and fibrin D-dimer have been reported in the black population, which could increase the black population’s susceptibility to CVD. However, low levels of plasminogen activator inhibitor-1 (PAI-1) previously reported in the black population could contribute towards a pro-fibrinolytic state, which may counteract the hypercoagulant state. This may have a beneficial effect on the haemostatic profile of the black population. More investigation into the haemostatic profile of black South Africans is therefore needed to determine if an altered haemostatic profile exists in this group, and if so, to what extent these alterations may relate to cardiovascular dysfunction. This study included markers of both the coagulation (vWF, fibrinogen, fibrin D-dimer) and fibrinolytic (PAI-1, fibrin D-dimer and fibrinolytic potential) systems in an attempt to investigate the haemostatic profile of the black population of South Africa, and for comparison purposes that of the white population as well. The relationship of these markers’ with selected markers of cardiovascular function was also examined to determine if they could possibly contribute to an increase in cardiovascular risk, especially in the black population. Aims The aims of this study were to first compare coagulation and fibrinolysis markers in the black and white populations of South Africa. Furthermore, to determine if associations exist between the selected components of the haemostatic system and markers of cardiovascular function, especially in the black population of South Africa, who tends to be at a higher cardiovascular risk due to altered metabolic and haemostatic profiles. Methodology The Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study was a prospective cohort study that consisted of 409 participants at baseline (2008-2009) that were equally distributed according to both ethnicity (200 black; 209 white) and gender (black, 101 men, 99 women; white, 101 men, 108 women). At follow-up (2011/2012) the cohort totalled 359 participants (170 black, 88 men and 82 women; 189 white, 93 men and 96 women). Data from baseline measurements were used for the first two manuscripts (chapters 2 and 3), while followup data was used for the third manuscript (chapter 4). vWF, fibrinogen, PAI-1, fibrin D-dimer, CLT, serum peroxides, glutathione, glutathione peroxidase and reductase activity were determined, and ambulatory blood pressure and the retinal vessel calibres were measured. The groups were stratified by ethnicity as specified by statistical interaction terms. T-tests and chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between the components of the haemostatic system and cardiovascular function markers. This was followed by multiple linear regression analyses to investigate whether independent associations exist between the variables in both ethnic groups. P-values ≤0.050 were deemed significant. Results and conclusion of each manuscript The first manuscript (chapter 2) compares the haemostatic profiles of the black and white population to determine whether ambulatory blood pressure is related to components of the haemostatic system. The black participants displayed a prothrombotic profile with significantly higher vWF, fibrinogen, PAI-1, fibrin D-dimer and a longer CLT than their white counterparts. Furthermore, partial and multiple linear regression analyses showed a positive association of systolic and diastolic blood pressure with fibrin D-dimer in the black population, while a negative association existed between ambulatory blood pressure and CLT in the white population. These associations suggest that fibrin D-dimer may contribute, at least in part, to the high prevalence of hypertension in the black population. The second manuscript (chapter 3) determined associations between markers of the haemostatic and oxidant-antioxidant systems in the black and white populations. In addition to the prothrombotic profile that exists in the black population, this group also had significantly higher serum peroxides (oxidative stress) and lower glutathione peroxidase activity (antioxidant) levels. Multiple linear regression analyses indicated positive associations between fibrinogen and serum peroxides in both populations. In the white population, an additional positive association was found between serum peroxide and CLT. In the black population, vWF and CLT were negatively associated with GPx activity. The results suggest that there are ethnic-specific relationships between the haemostatic and oxidant-antioxidant systems. The third manuscript (chapter 4) investigated the relationships between the retinal vessel calibres and components of the haemostatic system in the black and white population. The investigation focussed specifically on arteriolar diameters in the lower median, since a narrow arteriolar diameter is known to be associated with elevated blood pressure. In both ethnic groups, a narrower arteriolar calibre was accompanied by narrower venular calibres. Independent positive associations were found between the central retinal vein equivalent (CRVE) and fibrinogen in the black population, as well as vWF and CLT in the white population. In addition, independent negative associations were found between the central retinal artery equivalent and CLT in the black population and with vWF in the white population. The results suggest that haemostatic alterations are linked to early vascular changes that may differ between ethnicities. General conclusion Ethnic-specific relationships between the components of the haemostatic system and measures of cardiovascular function are evident. The prothrombotic profile that is observed in the black population, together with the adverse associations of the haemostatic components with blood pressure, a compromised oxidant-antioxidant profile, and retinal vessel calibres may contribute, at least in part, to the high cardiovascular and cerebrovascular risk evident in this population group. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Drs Dandachli and Arzamendi contributed equally to this work. / L’adhésion, l’activation et l’agrégation des plaquettes représentent les étapes initiales dans la formation du thrombus aux sites des lésions vasculaires. Malgré l’utilisation des médicaments antiplaquettaires comme l’Aspirine, le Plavix et les inhibiteurs de la glycoprotéine IIb/IIIa (GPIIb/IIIa), l’incidence de thrombus dans la maladie coronarienne aigue reste élevée. Le dommage aux artères coronaires induit l’exposition du collagène de la matrice sous-endothéliale et sa liaison au facteur Von-Willebrand (vWF). Ceci contribue au recrutement et à l’adhésion des plaquettes via la liaison du domaine A1 du vWF à la GPIb des plaquettes. Nous avons postulé que l’inhibition de la liaison vWF/GPIb pourra représenter une stratégie efficace pour inhiber l’adhésion initiale des plaquettes et ainsi réduire la propagation du thrombus. L’objectif de notre étude était de déterminer le potentiel anti-thrombotique d’un inhibiteur du vWF. L’aptamère dirigé contre le domaine A1 du vWF (ARC1779) a été développé et fourni par la compagnie Archemix. Son effet et celui du Reopro (abciximab, inhibiteur de la GPIIb/IIIa comme témoin positif) ont été testés en utilisant du sang provenant de 5 volontaires sains et de 27 patients coronariens traités avec l’Aspirine (inhibiteur du cyclo-oxygénase ou COX) et le Plavix (anti-récepteur de l’adénosine diphosphate ou ADP), en accord avec le comité d’éthique de l’ICM. Les plaquettes ont été marquées avec l’Indium-111 afin de pouvoir quantifier leur adhésion dans le sang complet sur des surfaces artérielles porcines dénudées. L’adhésion des plaquettes a été effectuée dans des chambres de perfusion sous des forces de cisaillement de 6974/sec pendant 15 minutes à 37 °C. L’activation plaquettaire, suite à l’étude de l’adhésion, a été évaluée par l’expression de la P-sélectine et du vWF par la cytométrie en flux. L’effet de l’ARC1779 a été également déterminé sur l’agrégation plaquettaire, dans le sang complet par impédance, induite par l’acide arachidonique (AA), l’ADP, la Ristocétine et le peptide agoniste du récepteur de la thrombine-1 (TRAP-1). L’adhésion des plaquettes a été également observée par microscopie électronique à balayage. Dans un premier temps, nous avons trouvé que l’adhésion des plaquettes des volontaires sains à l’artère endommagée était élevée (80 x 106/cm2). Cette adhésion a été réduite significativement de plus que 90% par l’abciximab (100 nM) et d’une façon dose dépendante avec l’ARC1779 (25-250 nM). La perfusion du sang avec ou sans ARC1779 n’entraine pas une activation plaquettaire, telle que déterminée par l’expression de la P-sélectine et du vWF à la surface des plaquettes. Suite à ces résultats, l’étude avec le sang des patients a été poursuivie avec des doses de 25, 83 et 250 nM d’ARC1779. L’agrégation plaquettaire du sang des patients a été complètement inhibée en réponse à l’AA et à l’ADP, ce qui confirme que ces patients étaient bien traités avec l’Aspirine et le Plavix. L’adhésion des plaquettes aux surfaces artérielles endommagées a été réduite, chez les volontaires sains et les patients, d’une manière dépendante de la dose d’ARC1779, lorsqu’il était incubé avant la perfusion. Cependant, l’ARC1779 et aussi l’abciximab étaient sans effets significatifs sur l’adhésion plaquettaire, lorsqu’ils étaient ajoutés 10 minutes après la perfusion. L’inhibition de l’adhésion avec 25 nM d’ARC1779 était comparable à celle obtenue avec l’abciximab. Cependant, l’agrégation plaquettaire en réponse au TRAP-1 n’était pas affectée par l’ARC1779, alors qu’elle était complètement inhibée par l’abciximab. L’ARC1779 est un inhibiteur spécifique de la liaison du vWF au GPIb des plaquettes. Il inhibe l’adhésion plaquettaire aux surfaces artérielles endommagées sans affecter l’agrégation plaquettaire et confère une protection anti-thrombotique similaire à l’abciximab. L’ARC1779 pourra être considéré comme un nouvel antiplaquettaire qui possède des propriétés anti-thrombotiques plus intéressantes que l’abciximab. / Anti-platelet therapy in coronary artery disease (CAD) patients reduces recurrent athero-thrombosis, but at the cost of increased risk of bleeding. Because von Willebrand factor (vWF) functions predominantly in a high-shear environment, the vWF-specific aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein Ib, may deliver a site-specific anti-thrombotic effect while minimizing bleeding risk. We investigated the efficiency of ARC1779 on platelet activation, adhesion, and aggregation in CAD patients on double anti-platelet therapy. Blood from 27 patients taking aspirin and clopidogrel and 5 normal volunteers was labeled with 111In-autologous platelets and perfused over denuded porcine arteries at high shear rate for 15 minutes. Blood was treated with either 25, 83 and 250 nM ARC1779; 100nM abciximab or placebo, 5 min before (upstream therapy) or 10 min after (downstream therapy) beginning the perfusion. Under upstream, but not downstream therapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nM and by abciximab vs. placebo (4.8, 3.8 and 2.9 vs. 7.3 platelets x 106/cm2, p <0.05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation in response to thrombin receptor activating peptide-1, arachidonic acid and adenosine diphosphate. In addition, ARC1779 was without any effect on P-selectin expression and platelet-leukocyte binding. In conclusion, ARC1779 has comparable anti-thrombotic efficacy to abciximab among CAD patients receiving aspirin and clopidogrel, but with lesser systemic effects on platelet activation and aggregation. These important proof-of-concept data form the framework for randomized clinical investigations of this novel anti-platelet therapy among CAD patients.

No description available.

L’adhésion, l’activation et l’agrégation des plaquettes représentent les étapes initiales dans la formation du thrombus aux sites des lésions vasculaires. Malgré l’utilisation des médicaments antiplaquettaires comme l’Aspirine, le Plavix et les inhibiteurs de la glycoprotéine IIb/IIIa (GPIIb/IIIa), l’incidence de thrombus dans la maladie coronarienne aigue reste élevée. Le dommage aux artères coronaires induit l’exposition du collagène de la matrice sous-endothéliale et sa liaison au facteur Von-Willebrand (vWF). Ceci contribue au recrutement et à l’adhésion des plaquettes via la liaison du domaine A1 du vWF à la GPIb des plaquettes. Nous avons postulé que l’inhibition de la liaison vWF/GPIb pourra représenter une stratégie efficace pour inhiber l’adhésion initiale des plaquettes et ainsi réduire la propagation du thrombus. L’objectif de notre étude était de déterminer le potentiel anti-thrombotique d’un inhibiteur du vWF. L’aptamère dirigé contre le domaine A1 du vWF (ARC1779) a été développé et fourni par la compagnie Archemix. Son effet et celui du Reopro (abciximab, inhibiteur de la GPIIb/IIIa comme témoin positif) ont été testés en utilisant du sang provenant de 5 volontaires sains et de 27 patients coronariens traités avec l’Aspirine (inhibiteur du cyclo-oxygénase ou COX) et le Plavix (anti-récepteur de l’adénosine diphosphate ou ADP), en accord avec le comité d’éthique de l’ICM. Les plaquettes ont été marquées avec l’Indium-111 afin de pouvoir quantifier leur adhésion dans le sang complet sur des surfaces artérielles porcines dénudées. L’adhésion des plaquettes a été effectuée dans des chambres de perfusion sous des forces de cisaillement de 6974/sec pendant 15 minutes à 37 °C. L’activation plaquettaire, suite à l’étude de l’adhésion, a été évaluée par l’expression de la P-sélectine et du vWF par la cytométrie en flux. L’effet de l’ARC1779 a été également déterminé sur l’agrégation plaquettaire, dans le sang complet par impédance, induite par l’acide arachidonique (AA), l’ADP, la Ristocétine et le peptide agoniste du récepteur de la thrombine-1 (TRAP-1). L’adhésion des plaquettes a été également observée par microscopie électronique à balayage. Dans un premier temps, nous avons trouvé que l’adhésion des plaquettes des volontaires sains à l’artère endommagée était élevée (80 x 106/cm2). Cette adhésion a été réduite significativement de plus que 90% par l’abciximab (100 nM) et d’une façon dose dépendante avec l’ARC1779 (25-250 nM). La perfusion du sang avec ou sans ARC1779 n’entraine pas une activation plaquettaire, telle que déterminée par l’expression de la P-sélectine et du vWF à la surface des plaquettes. Suite à ces résultats, l’étude avec le sang des patients a été poursuivie avec des doses de 25, 83 et 250 nM d’ARC1779. L’agrégation plaquettaire du sang des patients a été complètement inhibée en réponse à l’AA et à l’ADP, ce qui confirme que ces patients étaient bien traités avec l’Aspirine et le Plavix. L’adhésion des plaquettes aux surfaces artérielles endommagées a été réduite, chez les volontaires sains et les patients, d’une manière dépendante de la dose d’ARC1779, lorsqu’il était incubé avant la perfusion. Cependant, l’ARC1779 et aussi l’abciximab étaient sans effets significatifs sur l’adhésion plaquettaire, lorsqu’ils étaient ajoutés 10 minutes après la perfusion. L’inhibition de l’adhésion avec 25 nM d’ARC1779 était comparable à celle obtenue avec l’abciximab. Cependant, l’agrégation plaquettaire en réponse au TRAP-1 n’était pas affectée par l’ARC1779, alors qu’elle était complètement inhibée par l’abciximab. L’ARC1779 est un inhibiteur spécifique de la liaison du vWF au GPIb des plaquettes. Il inhibe l’adhésion plaquettaire aux surfaces artérielles endommagées sans affecter l’agrégation plaquettaire et confère une protection anti-thrombotique similaire à l’abciximab. L’ARC1779 pourra être considéré comme un nouvel antiplaquettaire qui possède des propriétés anti-thrombotiques plus intéressantes que l’abciximab. / Anti-platelet therapy in coronary artery disease (CAD) patients reduces recurrent athero-thrombosis, but at the cost of increased risk of bleeding. Because von Willebrand factor (vWF) functions predominantly in a high-shear environment, the vWF-specific aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein Ib, may deliver a site-specific anti-thrombotic effect while minimizing bleeding risk. We investigated the efficiency of ARC1779 on platelet activation, adhesion, and aggregation in CAD patients on double anti-platelet therapy. Blood from 27 patients taking aspirin and clopidogrel and 5 normal volunteers was labeled with 111In-autologous platelets and perfused over denuded porcine arteries at high shear rate for 15 minutes. Blood was treated with either 25, 83 and 250 nM ARC1779; 100nM abciximab or placebo, 5 min before (upstream therapy) or 10 min after (downstream therapy) beginning the perfusion. Under upstream, but not downstream therapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nM and by abciximab vs. placebo (4.8, 3.8 and 2.9 vs. 7.3 platelets x 106/cm2, p <0.05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation in response to thrombin receptor activating peptide-1, arachidonic acid and adenosine diphosphate. In addition, ARC1779 was without any effect on P-selectin expression and platelet-leukocyte binding. In conclusion, ARC1779 has comparable anti-thrombotic efficacy to abciximab among CAD patients receiving aspirin and clopidogrel, but with lesser systemic effects on platelet activation and aggregation. These important proof-of-concept data form the framework for randomized clinical investigations of this novel anti-platelet therapy among CAD patients. / Drs Dandachli and Arzamendi contributed equally to this work.

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. / <p>Härtill 6 uppsatser</p> / digitalisering@umu

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

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