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

Made available in DSpace on 2015-06-17T19:34:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-29. Added 1 bitstream(s) on 2015-06-18T12:47:02Z : No. of bitstreams: 1 000831074.pdf: 615076 bytes, checksum: c97e8862ed17121a7d6c2ddd7dfb24e1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fator VIII (FVIII), o fator de von Willebrand (FvW) e o fibrinogênio são de suma importância na coagulação sanguínea, com diferentes funções fisiológicas. Por conter altas concentrações destes fatores a transfusão de crioprecipitado é uma terapia utilizada principalmente em pacientes que apresentam Doença de von Willebrand, Hemofilia A (deficiência do FVIII), ou pacientes que sofrem de hipo ou disfibrinogenemia. Este hemocomponente é um precipitado obtido após o descongelamento parcial (entre 1 e 6°C) do plasma fresco congelado, e também é conhecido como fator anti-hemofílico. Estudos têm demonstrado que o protocolo de congelamento e armazenamento do crioprecipitado afeta a qualidade do produto e a viabilidade destes fatores. Com o objetivo de avaliar a viabilidade do crioprecipitado canino em diferentes protocolos de congelamento e armazenamento foram avaliados dois grupos compostos de 10 unidades de crioprecipitado canino (n=20). Após a centrifugação das bolsas de sangue, o plasma fresco foi congelado a -80ºC (grupo I) e a -20ºC (grupo II). Vinte e quatro horas após o congelamento das bolsas, estas foram submetidas ao procedimento de extração do crioprecipitado. Os crioprecipitados das bolsas dos dois grupos foram submetidos à determinação do TP, TTPA, FVIII, FvW e fibrinogênio, no momento zero e após seis meses de estocagem. Para a realização das coletas, foram utilizadas bolsas sanguíneas triplas de plástico, com anticoagulante CPDA-1, sendo a bolsa principal com capacidade para 450 mL de sangue total (JP Indústria Farmacêutica®). Após o crioprecipitado devidamente pronto, uma alíquota de aproximadamente 5 mL da bolsa de crioprecipitado foi separada em criotubos para análise da amostra pré-estocagem e seis meses pós estocagem. As amostras obtidas em cada momento foram congeladas à -80ºC até o momento do processamento. Os resultados mostraram um decréscimo significativo dos fatores e ... / The factor VIII (FVIII), the von Willebrand factor (vWF) and the fibrinogen are extremely important to the blood clotting process, with various physiological functions. Because it contains high concentrations of these factors and fibrinogen, transfusing cryoprecipitate is a therapy mainly used in patients who have von Willebrand disease, Hemophilia A (FVIII deficiency), or who suffer from hypo/dysfibrinogenemia. This hemocomponent is a precipitate obtained after the partial thawing process (between 1 and 6ºC) of fresh frozen plasma, and which is also known as the anti-hemophilic factor. Studies have demonstrated that the cryoprecipitate freezing and storage protocol affects the product quality as well as these factors viability. In order to evaluate the canine cryoprecipitate viability in different freezing and storage protocols, two groups containing 10 units of canine cryoprecipitate (n=20) were evaluated. Following the blood centrifugation, the fresh plasma was frozen at -80ºC (group I) and at -20ºC (group II). Twenty-four hours after freezing the blood bags, they were submitted to the cryoprecipitate extraction procedure. The cryoprecipitate from both groups of blood bags were submitted to the TP, TTPA, FVIII, FvW and fibrinogen determination process, at time zero and after six months of storage. During the collections, triple plastic blood bags were used, along with the anticoagulant CPDA-1, being the main bag capacity of 450 mL of whole blood (JP Indústria Farmacêutica®). After having the cryoprecipitate properly ready, an approximately 5 mL aliquot of cryoprecipitate was separated into cryovials to be analysed pre-storage and six months after storage. However, there was no significant difference between treatments, demonstrating that the difference in initial freezing temperature did not influence the decrease of the factors after six months storage at -20°C / FAPESP: 2012/13677-6

Orientador: Regina Kiomi Takahira / Banca: Luiz Henrique de Araújo Machado / Banca: Simone Gonçalves Rodrigues Gomes / Resumo: O fator VIII (FVIII), o fator de von Willebrand (FvW) e o fibrinogênio são de suma importância na coagulação sanguínea, com diferentes funções fisiológicas. Por conter altas concentrações destes fatores a transfusão de crioprecipitado é uma terapia utilizada principalmente em pacientes que apresentam Doença de von Willebrand, Hemofilia A (deficiência do FVIII), ou pacientes que sofrem de hipo ou disfibrinogenemia. Este hemocomponente é um precipitado obtido após o descongelamento parcial (entre 1 e 6°C) do plasma fresco congelado, e também é conhecido como fator anti-hemofílico. Estudos têm demonstrado que o protocolo de congelamento e armazenamento do crioprecipitado afeta a qualidade do produto e a viabilidade destes fatores. Com o objetivo de avaliar a viabilidade do crioprecipitado canino em diferentes protocolos de congelamento e armazenamento foram avaliados dois grupos compostos de 10 unidades de crioprecipitado canino (n=20). Após a centrifugação das bolsas de sangue, o plasma fresco foi congelado a -80ºC (grupo I) e a -20ºC (grupo II). Vinte e quatro horas após o congelamento das bolsas, estas foram submetidas ao procedimento de extração do crioprecipitado. Os crioprecipitados das bolsas dos dois grupos foram submetidos à determinação do TP, TTPA, FVIII, FvW e fibrinogênio, no momento zero e após seis meses de estocagem. Para a realização das coletas, foram utilizadas bolsas sanguíneas triplas de plástico, com anticoagulante CPDA-1, sendo a bolsa principal com capacidade para 450 mL de sangue total (JP Indústria Farmacêutica®). Após o crioprecipitado devidamente pronto, uma alíquota de aproximadamente 5 mL da bolsa de crioprecipitado foi separada em criotubos para análise da amostra pré-estocagem e seis meses pós estocagem. As amostras obtidas em cada momento foram congeladas à -80ºC até o momento do processamento. Os resultados mostraram um decréscimo significativo dos fatores e ... / Abstract: The factor VIII (FVIII), the von Willebrand factor (vWF) and the fibrinogen are extremely important to the blood clotting process, with various physiological functions. Because it contains high concentrations of these factors and fibrinogen, transfusing cryoprecipitate is a therapy mainly used in patients who have von Willebrand disease, Hemophilia A (FVIII deficiency), or who suffer from hypo/dysfibrinogenemia. This hemocomponent is a precipitate obtained after the partial thawing process (between 1 and 6ºC) of fresh frozen plasma, and which is also known as the anti-hemophilic factor. Studies have demonstrated that the cryoprecipitate freezing and storage protocol affects the product quality as well as these factors viability. In order to evaluate the canine cryoprecipitate viability in different freezing and storage protocols, two groups containing 10 units of canine cryoprecipitate (n=20) were evaluated. Following the blood centrifugation, the fresh plasma was frozen at -80ºC (group I) and at -20ºC (group II). Twenty-four hours after freezing the blood bags, they were submitted to the cryoprecipitate extraction procedure. The cryoprecipitate from both groups of blood bags were submitted to the TP, TTPA, FVIII, FvW and fibrinogen determination process, at time zero and after six months of storage. During the collections, triple plastic blood bags were used, along with the anticoagulant CPDA-1, being the main bag capacity of 450 mL of whole blood (JP Indústria Farmacêutica®). After having the cryoprecipitate properly ready, an approximately 5 mL aliquot of cryoprecipitate was separated into cryovials to be analysed pre-storage and six months after storage. However, there was no significant difference between treatments, demonstrating that the difference in initial freezing temperature did not influence the decrease of the factors after six months storage at -20°C / Mestre

Orientador: Joyce Maria Annichino-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T16:00:11Z (GMT). No. of bitstreams: 1 Fonseca_BrunadeMoraesMazetto_M.pdf: 657024 bytes, checksum: 715789e040378f27c5de278879f4535b (MD5) Previous issue date: 2011 / Resumo: Níveis elevados de marcadores inflamatórios e fatores de coagulação têm sido relacionados com a patogênese do TEV. Particularmente, a relação inversa entre o FVW e a atividade da ADAMTS13 já foi previamente descrita em pacientes com trombose arterial. Níveis de FVW também mostraram-se elevados durante processos inflamatórios e portanto, poderiam desempenhar um papel de ligação entre inflamação e coagulação nos pacientes com TEV. Objetivo: Avaliar a atividade da ADAMTS13 e do FVW e sua associação com marcadores inflamatórios e evolução clínica pós-trombótica em pacientes com TEV. Pacientes e Métodos: Setenta e sete pacientes com TEV, entre sete meses e seis anos após o episódio agudo, atendidos no Hemocentro de Campinas - UNICAMP foram incluídos neste estudo e 77 indivíduos normais foram selecionados como controles, pareados por idade, gênero, etnia e grupo sanguíneo. A atividade da ADAMTS13 e do FVW foram avaliados pela ligação do FVW ao colágeno, o dímero-D por turbidimetria, a PCR por nefelometria, TNF-'alfa', IL-6, IL-8, antígeno do FVW e da ADAMTS13 foram determinados por ELISA. A presença de trombo residual foi avaliada por ultrassom com Doppler e a SPT através da escala Villalta. Resultados: Trinta pacientes (39%) tiveram TEV causado por fatores de risco transitórios, especialmente pelo uso de anticoncepcional e 47 pacientes tiveram TVE espontâneo. A atividade inflamatória estava aumentada nos pacientes em comparação aos controles, demonstrada pelo aumento significativo dos níveis séricos de TNF-'alfa' and IL-6 nos primeiros (mediana= 2,25 vs 1,59pg/mL, P?0,001; 1,16 vs 0,98pg/ml, P=0,013, respectivamente). Os níveis de IL-8 e PCR foram similares entre os 2 grupos (mediana= 18,3 vs 18,27pg/mL, P=0,47; 0.21 vs 0,17mg/dL, P=0,29, respectivamente). Trinta e dois pacientes (42,8%) foram definidos como tendo um aumento da atividade coagulante, expressa pelo dímero-D > 0,55mg/dL. Nesse grupo de pacientes todos os marcadores inflamatórios como TNF-'alfa', IL-6, IL-8 and PCR, estavam significativamente aumentados quando comparados aos pacientes com dímero-D ? 0,55 mg/L (P=0,0057; 0,001; 0,0093 e 0,0075; respectivamente). A presença de SPT e trombo residual não foram associados ao aumento da atividade coagulante. A atividade da ADAMTS13 e os níveis séricos de IL-8 estavam aumentados em pacientes com SPT quando comparados aos pacientes sem SPT. Todos os marcadores inflamatórios e parâmetros da coagulação estudados foram similares em pacientes independentemente da presença do trombo residual. Conclusão: Este estudo sugere que exista atividade inflamatória e procoagulante nos pacientes mesmo após o episódio agudo do TEV, que, entretanto, não se mostrou estar relacionada com a persistência das seqüelas clínicas e radiológicas da TVP. Além disso, o aumento do FVW nos pacientes corrobora a hipótese de ativação crônica da inflamação. Neste contexto, o aumento observado da ADAMTS 13 poderia ser compensatório frente ao aumento crônico do FVW e poderia inclusive atuar com um mecanismo protetor contra a atividade pró-trombótica observada nestes pacientes / Abstract: Introduction: Increased levels of inflammatory markers and clotting factors have been related to the pathogenesis of VTE. Particularly, the inverse relation between VWF and ADAMTS13 activity has been previously described in patients with arterial thrombosis. VWF levels are also known to be increased during inflammatory processes and therefore could play a role linking the inflammatory and coagulation systems activities in patients with VTE. Objective: To evaluate the activity of ADAMTS13 and VWF in patients with VTE and its association with inflammatory markers and clinical outcome of post-thrombotic syndrome. Patients and methods: Seventy-seven patients with VTE, 7 months to six years after the acute episode, attended at the Hemocentro of Campinas - UNICAMP, were included in this study and 77 normal subjects were selected as controls, matched by gender, age, ethnicity and ABO blood group. The activity of ADAMTS 13 was performed by VWF collagen binding, D-dímer by turbidimetry, CRP by nephelometry , and TNF-'alpha', IL-6 and IL-8, VWF and ADAMTS13 antigen by ELISA. The presence of RVO was investigated by duplex examination and PTS by Villalta scale. Results: Thirty patients (39%) had VTE caused by transient risk factors, mainly the use of oral contraceptives, and 47 patients had spontaneous VTE. Serum levels of TNF-'alpha' and IL-6 were significantly increased in patients when compared to controls (median= 2.25 vs 1.59pg/mL, P?0.001; 1.16 vs 0.98pg/ml, P=0.013, respectively) whereas levels of IL-8 and CRP were similar among the groups (median= 18.3 vs 18.27pg/mL, p=0.47; 0.21 vs 0.17mg/dL, P=0.29, respectively). Thirty-two patients (42,8%) had D-dimer > 0.55 mg/L and were defined as having increased coagulation activity. Inflammatory markers, such as TNF-'alpha', IL-6, IL-8 and CRP, were significantly higher in those patients, comparing to patients with D-dimer ? 0.55 mg/L (P=0.0057, 0.001, 0.0093 and 0.0075, respectively). The presence of PTS or RVO were not associated with increased inflammatory or coagulation activity. Only ADAMTS13-CBA and plasma levels of IL-8 were higher in patients with PTS comparing to patients without PTS. All inflammatory markers and coagulation parameters studied were similar in patients regardless the presence of RVO. Conclusion: Our findings suggest that there is an inflammatory and pro-coagulant activity in patients even after the acute episode of VTE, however, these activities were not related to the persistence of clinical and radiological sequels of DVT. Moreover, the increasing levels of VWF, observed in patients, support the hypothesis that the inflammation is chronically activated. In this context, the increasing levels of ADAMTS13, also observed in patients, could be explained as a compensatory mechanism and maybe act as a protection against pro-thrombotic activity seen in these patients / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

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

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.

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.

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.

Von Willebrand disease (vWD) is one of the most common inherited bleeding diseases, caused by a qualitative or quantitative deficiency of the von Willebrand factor (vWF). vWF is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells and it is present in the subendothelial matrix, blood plasma, platelets and endothelium. This glycoprotein represents an important role in thrombus formation by initiating platelet adhesion to sites of injury as well as platelet aggregation. The objective of this study was determine the activities of NTPDase (CD39), 5 -nucleotidase (EC 3.1.3.5, CD73) and Ectonucleotide Pyrophosphatase/Phosphodiesterase (E-NPP) enzymes in platelets patients from von Willebrand disease and healthy patients, as well as ristocetin-induced platelet aggregation (RIPA) and polymorphisms of the α2 gene of the α2β1 integrin. The following groups were studied: 14 patients diagnosed with vWD and 14 healthy patients for control group. For ristocetin-induced platelet aggregation was used a Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP), using a final concentration of ristocetin in 1.25mg/mL. The polymorphisms of the α2 gene was analyzed through the Polymerase chain reaction (PCR), used for digestion of the PCR product of the Bgl II restriction site. NTPDase and E-NPP were decreased in platelets of patients with vWD compared to the group control. Moreover, the activity of the enzyme 5'- nucleotidase was not statistically significant. The results of the RIPA were significantly reduced in patients with vWD compared with the control. The allelic frequencies among vWD patients were found to be 78.57% for 807C and 21.43% for 807T. Our results indicate an decreased NTPDase and E-NPP activities in platelets, may be related to the low adhesiveness of platelets in patients with vWD. The allelic frequency 807C predominant suggests, in agreement with other studies, this polymorphism and factor characteristic of hemorrhagic manifestations in patients with DvW. / A doença de von Willebrand (DvW) é uma das mais comuns entre as doenças hemorrágicas, e é provocada por uma deficiência qualitativa ou quantitativa do fator de von Willebrand (FvW). O FvW é uma glicoproteína multimérica sintetizada por megacariócitos e células endoteliais e está presente no matriz subendotelial, no plasma sanguíneo, nas plaquetas e no endotélio. Esta glicoproteína desempenha um papel importante na formação do trombo plaquetário, iniciando a adesão plaquetária ao local do dano vascular, bem como, a agregação plaquetária. O objetivo deste estudo foi determinar a atividade das enzimas NTPDase (EC 3.6.1.5, CD39), 5'-nucleotidase (EC 3.1.3.5, CD73) e Ectonucleotideo pirofasfatase/fosfodiesterase (E-NPP) em plaquetas de pacientes com a DvW e em plaquetas de pacientes saudáveis, bem como agregação plaquetária induzida pela ristocetina (RIPA) e o polimorfismo do gene α2 da integrina α2β1 da superfície de plaquetas. Os grupos foram divididos da seguinte forma: 14 pacientes diagnosticados com DvW e 14 pacientes saudáveis, para o grupo controle. Para a RIPA foram utilizados um Plasma Rico em Plaquetas (PRP) e um Plasma Pobre em Plaquetas (PPP), utilizando-se uma concentração final de ristocetina de 1.25mg/mL. O polimorfismo do gene α2 foi analisado através da reação em cadeia de polimerase (PCR), utilizando para a digestão do produto da PCR a enzima de restrição Bgl II. Constatou-se que a atividade das enzimas NTPDase e E-NPP foram reduzidas em plaquetas de pacientes com DvW comparadas ao grupo controle. Por outro lado, a atividade da enzima 5'-nucleotidase não foi estatisticamente significativa. Os resultados para os RIPA foram significativamente reduzidos em pacientes com DvW comparado com o controle. A freqüência alélica encontrada entre os pacientes com DvW foi de 78,57% para o alelo 807C e de 21,43% para o alelo 807T. Nossos resultados indicam que a redução da atividade da NTPDase e da E-NPP em plaquetas pode estar relacionada à baixa adesividade das plaquetas em pacientes com DvW. A freqüência alélica 807C predominante sugere, de acordo com outros estudos, que este polimorfismo é fator característico das manifestações hemorrágicas em pacientes portadores da DvW.