Introdução: Trombocitopenia é a redução dos níveis plaquetários ocasionada por diversas condições, como hepatopatias e a Trombocitopenia Imune (TI), por exemplo. A redução de plaquetas na hepatopatia pode ocorrer devido à deficiência medular por hipovitaminose, aumento do consumo celular e hiperesplenismo e/ou devido à coagulação intravascular disseminada. Na Trombocitopenia Imune, a redução plaquetária ocorre tanto por maior destruição destas células quanto por diminuição medular de sua produção. Portadores de trombocitopenia podem apresentar achados orais como petéquias e/ou equimoses e sangramento gengival, espontâneo ou provocado. Procedimentos cirúrgico-odontológicos nestes pacientes devem ser realizados com cautela, após avaliação do seu quadro clínico e, preferencialmente, por profissional especializado. Muita controvérsia ainda existe em relação à abordagem cirúrgico-odontológica destes pacientes tanto em relação aos níveis plaquetários, quanto em relação à reposição plaquetária prévia, não havendo ainda, um protocolo internacionalmente estabelecido. Diversos métodos hemostáticos locais auxiliam durante este tipo de abordagem, sendo o agente a base de quitosana um dos métodos que têm se mostrado efetivo em diversos estudos e apresenta-se, ainda, menos oneroso que o selante de fibrina, por exemplo, um dos métodos mais utilizados. Objetivo: Desta forma, o objetivo desta pesquisa foi observar e descrever o desempenho clínico do agente a base de quitosana em pacientes com contagem plaquetária <= a 30.000/mm3 (Grupo Quitosana) submetidos a exodontias unitárias, utilizando como padrão de comparação o grupo de pacientes trombocitopênicos com contagem plaquetária entre 31.000 e 50.000/mm3 (Grupo Controle), submetidos a exodontias unitárias, sem o uso do curativo hemostático a base de quitosana e ainda, descrever o atendimento odontológico a pacientes com contagem plaquetária inferior a 50.000/mm3 . Casuística e métodos: Pacientes trombocitopênicos com contagem abaixo de 50.000/mm3 foram submetidos a exodontias unitárias, estando divididos em Grupo Quitosana(GQ):pacientes com plaquetas abaixo de 30.000/mm3, que receberam o agente a base de quitosana após a extração e, Grupo Controle(GC): pacientes com plaquetas entre 31.000/mm3 e 50.000/mm3 e que não receberam o agente a base de quitosana. O sangramento foi observado 7 dias após a exodontia através do Índice de Sangramento Alveolar Pós- Exodontia (ISAPE) e para análise estatística foi utilizado o programa SPSS (Statistical Package for Social Sciences) 20.0; nível de significância de 5% (p=0,05), e realizado o teste de Mann-Whitney, análise de Correlação de Spearman e, teste do Chi-Quadrado e Fator de risco com intervalo de confiança (IC) de 95%. Resultados: Foram realizadas 41 exodontias unitárias entre 03/2011 e 09/2012. A média de idade dos pacientes estudados foi de 46,58 ±10,87anos, com mediana de 48,50 (variando de 20 a 64). Dentre eles, 51,20% (n=21) eram do gênero feminino e 48,79% (n=20) do masculino. 21 procedimentos foram incluídos no GQ e, 20 procedimentos, no GC. O GQ apresentou contagem plaquetária entre 5.000 e 30.000/mm3 e ISAPE médio de 0,10; sendo que 2 (9,52%) pacientes tiveram ISAPE acima de zero. Já o GC apresentou plaquetas entre 31.000 e 50.000/mm3, ISAPE médio de 0,40 e 6 (30%) pacientes com ISAPE acima de zero. Não houve significância estatística em relação ao ISAPE. Discussão: Até o presente momento não foram encontrados outros trabalhos nos moldes da presente pesquisa nas bases de dados pesquisadas (Lilacs, Medline, Bireme). Inúmeros trabalhos buscam estabelecer a contagem plaquetária mínima necessária para um paciente ser submetido à cirurgia odontológica e ainda, quando será indicada reposição plaquetária prévia. Em ambos os aspectos a literatura se mostra controvérsia, havendo autores que indicam reposição plaquetária prévia a pacientes com plaquetas em torno de 100.000/mm3, e autores que relatam que cirurgias orais simples, como as exodontias unitárias, podem ser realizadas de forma segura sem reposição plaquetária em pacientes com plaquetas em torno de 30.000/mm3. Não existe atualmente um protocolo de atendimento estabelecido internacionalmente. O ISAPE não apresentou significância estatística entre os grupos estudados, aspecto possivelmente influenciado pela heterogeneidade dos grupos e/ou ainda, pelo número reduzido da amostra. Porém, a porcentagem dos pacientes que apresentaram ISAPE maior que 0 mostra efetividade do agente hemostático em 90,48% do GQ, concordando com Belman et al.(2006), Brown et al.(2007) e Wedmore et al.(2006) que mostram 80,0%, 79,0% e 97,0%, respectivamente. Conclusão: Não houve diferença estatística entre os grupos em relação ao desempenho clínico hemostático do curativo a base de quitosana. Foi possível descrever o protocolo de atendimento a pacientes trombocitopênicos efetivo e resolutivo, já utilizado rotineiramente na Divisão de Odontologia HC-FMUSP / Introduction: Thrombocytopenia is an abnormally low amount of platelets caused by many conditions as liver disorders and immune thrombocytopenia for example. The reduction of platele count in liver disorders may be caused by bone marrow deficiency due to hypovitaminosis, hypersplenism and disseminated intravascular coagulation while in immune thrombocytopenia may be caused by greater desctruction of these cells or its diminished production by the bone marrow. Patients with thrombocytopenia may present oral manifestations as petechiae and/or ecchymosis and provoked/spontaneous gingival bleeding. In these patients, surgical dental procedures should be performed with caution and only after a detailed medical assessment, preferably performed by a specialist. The dental management of patients with thrombocytopenia is still controversial regarding the amount of platelets and the need of platelet infusion prior to these procedures. To this date, no clinical guidelines has been developed regarding the dental management of patients with thrombocytopenia. Many hemostatic agents has been used to control post surgical bleeding. Many studies had shown thay chitosan is effective and cheaper than fibrin sealant, one of the most popular hemostatic agents. Objetive: This study was design to assess the effect of chitosan as a local haemostatic agent in patients with platelet count <= 30.000/mm3 compared to patients with thrombocytopenia and platelet count of 31.000/mm3 to 50.000/mm3 submitted to single dental extraction and to describe the protocol for dental treatment of patients with platelet count lower than 50.000/mm3 in a tertiary health science center. Methods: Patients with thrombocytopenia with platelet count lower than 50.000/mm3 were assessed. Patients with platelet count lower than 30.000/mm3 (study group) and patients with platelet count of 31.000/mm3 to 50.000/mm3 (control group) were submitted to single dental extraction. Chitosan was used only in the study group and no other type of hemostatic agent was used in the control group. Bleeding was measured according to the post dental extraction index (ISAPE) seven days after dental extraction. The results were statistically analyzed using the Statistical Package for Social Sciences (SPSS) 20.0 program. For statistical analysis, the Mann-Whitney test, Chi-square test, Spearman\'s rank correlation coeficient and confidence interval for relative risk (95%) were used. P< 0.05 was considered statistically significant. Results: Forty-one single dental extractions were performed between March/2011 and September/2012. The mean age of patients was 46,58 ±10,87 years of age. Median was 48,50 ranging from 20 to 64 years of age. Fifty-one percent (n=21) were females and 48,79% (n=20) males. Twenty-one extarctions were performe in the Chitosan group and 20 in the control group. Chitosan group presented platelet count of 5.000 to 30.000/mm3 and mean ISAPE médio of 0,10; where two patients (9.52%) had ISAPE greater than zero Control group presented with platelet count of 31.000 to 50.000/mm3, mean ISAPE of 0,40 and six patients (30%) with ISAPE greater than zero. No statistical significance was related to the ISAPE between groups. Discussion: The dental management of patients with thrombocytopenia is still controversial regarding the amount of platelets and the need of platelet infusion prior to these procedures. To this date, no clinical guidelines has been developed regarding the dental management of patients with thrombocytopenia. Many studies have tried to establish the minimal platelet count and the need of platelet infusion prior to oral surgery. Unfortunately, the scientific literature is controversial. Some authors recommend platelet infusion prior to oral surgery in patients with platelel count of 100.000/mm3 while others report that single extractions can be performed safely in patients with platelet count 30.000/mm3. In our study, the ISAPE was not stastistically diferent between groups, what may be explained by the heterogenicity of patients in both groups and by the small sample. However, the greater amount of patients (90%) who presented ISAPE greater 0 showed the effectiveness of chitosan as a hemostatic agent in agreement with previous authors as Belman et al.(2006), Brown et al.(2007) e Wedmore et al.(2006) that presented 80,0%, 79,0% e 97,0%, respectively. Conclusion: No difference was found regarding the effectiveness of chitosan as hemostatic agent between groups. A clinical guideline was developed on dental management of patients with thrombocytopenia and since then it has been used daily at the Department of Dentistry HC-FMUSP

In einem präoperativen Screening mit Hilfe eines standardisierten Blutungsanamnesebogens und dem PFA-100 sind primäre Hämostasestörungen häufig, sekundäre (plasmatische) selten. Nach einer positiven Blutungsanamnese können mit dem PFA-100 über 90% der Hämostasestörungen erfaßt werden. In unserer Untersuchung wären ohne den PFA-100 27% der Patienten mit Hämostasestörungen nicht erkannt worden. Wenn die standardisierte Blutungsanamnese eindeutig negativ ist, kann auf ein präoperatives Screening mit den sogenannten Routinetests (aPTT u. Quick-Wert) verzichtet werden. Wenn der Quick-Wert, die aPTT, die Thrombozytenzahl und der PFA-100: Kollagen-Epinephrin nur in den indizierten Fällen mit positiver Blutungsanamnese (11.2%) durchgeführt würden, bedeutet das in Deutschland pro Jahr eine Einsparung von 14.2 Millionen Euro. In einer eigenen ersten Untersuchung wurde die Mikroangiopathie im Bereich der nutritiven Hautkapillaren (Fingernagelfalz) erstmal beim von Willebrand-Jürgens-Syndrom systematisch mit der intravitalen Videokapillarmikroskopie diagnostiziert. Die Nachfolgeuntersuchung an über 500 Patienten mit Hämostasestörungen im Vergleich zu einer Kontrollgruppe an Gesunden (n=100) bestätigte die Meßergebnisse für das von Willebrand-Jürgens-Syndrom (Typ 1 und 2a) und zeigte eine hohen positiven predictive value von 98%. Insbesondere die einfach und schnell durchgeführte Begutachtung der Kapillarmorphologie mit Veränderungen in Form von Kapillardilatationen, Extravasaten (frische oder alte Kapillarblutungen oder Blutungssäume) und gleichzeitig vorliegender Torquierung (Schlängelung) der Kapillaren ist bereits hoch prädiktiv (98%) für das Vorliegen eines von-Willebrand-Jürgens-Syndroms (Typ 1 und Typ 2a). In dem präoperativen therapeutischen Stufenkonzept zum Management primärer Hämostasestörungen zeigt sich die gute Wirksamkeit von DDAVP. Transfusionspflichtige Blutungen können bei Patienten mit primären Hämostasestörungen durch dieses präoperative Therapieregime verhindert werden. In der Patientengruppe mit primären Hämostasestörungen ohne präoperatives blutstillendes Management steigen die durchschnittlichen Kosten für die Transfusion von Blutkomponenten um das 5-fache auf ungefähr 1700 Euro. Hierbei sind Patientengruppen aller operativen Disziplinen berücksichtigt. / In a preoperative screening using a standardized questionaire of bleeding history and the PFA-100 platelet analyzer, primary hemostatic disorders are frequently found, whereas secondary disorders are rare. In cases with a positive bleeding history more than 90% of hemostatic disorders can be identified by PFA-100 platelet analyzer. In our investigation 27% of patients with impaired hemostasis would have been missed without using the PFA-100 platelet analyzer. In cases where the standardized bleeding history is clearly negative, the "so-called" routine preoperative coagulation tests (aPTT and prothrombin time) can be left out. Assuming that PT, aPTT, platelet count and PFA-100:collagen-epinephrine would only be performed in indicated cases with positive bleeding history (11.2%), this would lead to reduction of costs of 14.2 million Euro. Our investigations started with a systematic examination of the micrangiopathy in the nutritive nailfold capillaries of patient with von Willebrand Disease (vWD) using the intravital video capillary microscopy. In a follow-up investigation microangiopathy in the nutritive nailfold capillaries was examined in more than 500 patients with hemostatic disorders and 100 healthy volunteers. The results confirmed the findings for the vWD (type 1 and type 2a) and showed a high positive predictive value of 98%. In the preoperative phased therapy plan approach in the management of primary hemostatic disorders, DDAVP shows a good drug action. In these patients, the PFA-100 platelet analyzer is able to determinate the hemostatic effect of drugs, such as DDAVP, tranexamic acid, aprotinin, conjugated estrogens and platelet transfusion. In the patient group with primary hemostatic disorders without preoperative management, the average costs for blood transfusions increases 5-fold to roughly 1700 Euro. In this consideration patients of all operative disciplines are included.

Introdução: Trombocitopenia é a redução dos níveis plaquetários ocasionada por diversas condições, como hepatopatias e a Trombocitopenia Imune (TI), por exemplo. A redução de plaquetas na hepatopatia pode ocorrer devido à deficiência medular por hipovitaminose, aumento do consumo celular e hiperesplenismo e/ou devido à coagulação intravascular disseminada. Na Trombocitopenia Imune, a redução plaquetária ocorre tanto por maior destruição destas células quanto por diminuição medular de sua produção. Portadores de trombocitopenia podem apresentar achados orais como petéquias e/ou equimoses e sangramento gengival, espontâneo ou provocado. Procedimentos cirúrgico-odontológicos nestes pacientes devem ser realizados com cautela, após avaliação do seu quadro clínico e, preferencialmente, por profissional especializado. Muita controvérsia ainda existe em relação à abordagem cirúrgico-odontológica destes pacientes tanto em relação aos níveis plaquetários, quanto em relação à reposição plaquetária prévia, não havendo ainda, um protocolo internacionalmente estabelecido. Diversos métodos hemostáticos locais auxiliam durante este tipo de abordagem, sendo o agente a base de quitosana um dos métodos que têm se mostrado efetivo em diversos estudos e apresenta-se, ainda, menos oneroso que o selante de fibrina, por exemplo, um dos métodos mais utilizados. Objetivo: Desta forma, o objetivo desta pesquisa foi observar e descrever o desempenho clínico do agente a base de quitosana em pacientes com contagem plaquetária <= a 30.000/mm3 (Grupo Quitosana) submetidos a exodontias unitárias, utilizando como padrão de comparação o grupo de pacientes trombocitopênicos com contagem plaquetária entre 31.000 e 50.000/mm3 (Grupo Controle), submetidos a exodontias unitárias, sem o uso do curativo hemostático a base de quitosana e ainda, descrever o atendimento odontológico a pacientes com contagem plaquetária inferior a 50.000/mm3 . Casuística e métodos: Pacientes trombocitopênicos com contagem abaixo de 50.000/mm3 foram submetidos a exodontias unitárias, estando divididos em Grupo Quitosana(GQ):pacientes com plaquetas abaixo de 30.000/mm3, que receberam o agente a base de quitosana após a extração e, Grupo Controle(GC): pacientes com plaquetas entre 31.000/mm3 e 50.000/mm3 e que não receberam o agente a base de quitosana. O sangramento foi observado 7 dias após a exodontia através do Índice de Sangramento Alveolar Pós- Exodontia (ISAPE) e para análise estatística foi utilizado o programa SPSS (Statistical Package for Social Sciences) 20.0; nível de significância de 5% (p=0,05), e realizado o teste de Mann-Whitney, análise de Correlação de Spearman e, teste do Chi-Quadrado e Fator de risco com intervalo de confiança (IC) de 95%. Resultados: Foram realizadas 41 exodontias unitárias entre 03/2011 e 09/2012. A média de idade dos pacientes estudados foi de 46,58 ±10,87anos, com mediana de 48,50 (variando de 20 a 64). Dentre eles, 51,20% (n=21) eram do gênero feminino e 48,79% (n=20) do masculino. 21 procedimentos foram incluídos no GQ e, 20 procedimentos, no GC. O GQ apresentou contagem plaquetária entre 5.000 e 30.000/mm3 e ISAPE médio de 0,10; sendo que 2 (9,52%) pacientes tiveram ISAPE acima de zero. Já o GC apresentou plaquetas entre 31.000 e 50.000/mm3, ISAPE médio de 0,40 e 6 (30%) pacientes com ISAPE acima de zero. Não houve significância estatística em relação ao ISAPE. Discussão: Até o presente momento não foram encontrados outros trabalhos nos moldes da presente pesquisa nas bases de dados pesquisadas (Lilacs, Medline, Bireme). Inúmeros trabalhos buscam estabelecer a contagem plaquetária mínima necessária para um paciente ser submetido à cirurgia odontológica e ainda, quando será indicada reposição plaquetária prévia. Em ambos os aspectos a literatura se mostra controvérsia, havendo autores que indicam reposição plaquetária prévia a pacientes com plaquetas em torno de 100.000/mm3, e autores que relatam que cirurgias orais simples, como as exodontias unitárias, podem ser realizadas de forma segura sem reposição plaquetária em pacientes com plaquetas em torno de 30.000/mm3. Não existe atualmente um protocolo de atendimento estabelecido internacionalmente. O ISAPE não apresentou significância estatística entre os grupos estudados, aspecto possivelmente influenciado pela heterogeneidade dos grupos e/ou ainda, pelo número reduzido da amostra. Porém, a porcentagem dos pacientes que apresentaram ISAPE maior que 0 mostra efetividade do agente hemostático em 90,48% do GQ, concordando com Belman et al.(2006), Brown et al.(2007) e Wedmore et al.(2006) que mostram 80,0%, 79,0% e 97,0%, respectivamente. Conclusão: Não houve diferença estatística entre os grupos em relação ao desempenho clínico hemostático do curativo a base de quitosana. Foi possível descrever o protocolo de atendimento a pacientes trombocitopênicos efetivo e resolutivo, já utilizado rotineiramente na Divisão de Odontologia HC-FMUSP / Introduction: Thrombocytopenia is an abnormally low amount of platelets caused by many conditions as liver disorders and immune thrombocytopenia for example. The reduction of platele count in liver disorders may be caused by bone marrow deficiency due to hypovitaminosis, hypersplenism and disseminated intravascular coagulation while in immune thrombocytopenia may be caused by greater desctruction of these cells or its diminished production by the bone marrow. Patients with thrombocytopenia may present oral manifestations as petechiae and/or ecchymosis and provoked/spontaneous gingival bleeding. In these patients, surgical dental procedures should be performed with caution and only after a detailed medical assessment, preferably performed by a specialist. The dental management of patients with thrombocytopenia is still controversial regarding the amount of platelets and the need of platelet infusion prior to these procedures. To this date, no clinical guidelines has been developed regarding the dental management of patients with thrombocytopenia. Many hemostatic agents has been used to control post surgical bleeding. Many studies had shown thay chitosan is effective and cheaper than fibrin sealant, one of the most popular hemostatic agents. Objetive: This study was design to assess the effect of chitosan as a local haemostatic agent in patients with platelet count <= 30.000/mm3 compared to patients with thrombocytopenia and platelet count of 31.000/mm3 to 50.000/mm3 submitted to single dental extraction and to describe the protocol for dental treatment of patients with platelet count lower than 50.000/mm3 in a tertiary health science center. Methods: Patients with thrombocytopenia with platelet count lower than 50.000/mm3 were assessed. Patients with platelet count lower than 30.000/mm3 (study group) and patients with platelet count of 31.000/mm3 to 50.000/mm3 (control group) were submitted to single dental extraction. Chitosan was used only in the study group and no other type of hemostatic agent was used in the control group. Bleeding was measured according to the post dental extraction index (ISAPE) seven days after dental extraction. The results were statistically analyzed using the Statistical Package for Social Sciences (SPSS) 20.0 program. For statistical analysis, the Mann-Whitney test, Chi-square test, Spearman\'s rank correlation coeficient and confidence interval for relative risk (95%) were used. P< 0.05 was considered statistically significant. Results: Forty-one single dental extractions were performed between March/2011 and September/2012. The mean age of patients was 46,58 ±10,87 years of age. Median was 48,50 ranging from 20 to 64 years of age. Fifty-one percent (n=21) were females and 48,79% (n=20) males. Twenty-one extarctions were performe in the Chitosan group and 20 in the control group. Chitosan group presented platelet count of 5.000 to 30.000/mm3 and mean ISAPE médio of 0,10; where two patients (9.52%) had ISAPE greater than zero Control group presented with platelet count of 31.000 to 50.000/mm3, mean ISAPE of 0,40 and six patients (30%) with ISAPE greater than zero. No statistical significance was related to the ISAPE between groups. Discussion: The dental management of patients with thrombocytopenia is still controversial regarding the amount of platelets and the need of platelet infusion prior to these procedures. To this date, no clinical guidelines has been developed regarding the dental management of patients with thrombocytopenia. Many studies have tried to establish the minimal platelet count and the need of platelet infusion prior to oral surgery. Unfortunately, the scientific literature is controversial. Some authors recommend platelet infusion prior to oral surgery in patients with platelel count of 100.000/mm3 while others report that single extractions can be performed safely in patients with platelet count 30.000/mm3. In our study, the ISAPE was not stastistically diferent between groups, what may be explained by the heterogenicity of patients in both groups and by the small sample. However, the greater amount of patients (90%) who presented ISAPE greater 0 showed the effectiveness of chitosan as a hemostatic agent in agreement with previous authors as Belman et al.(2006), Brown et al.(2007) e Wedmore et al.(2006) that presented 80,0%, 79,0% e 97,0%, respectively. Conclusion: No difference was found regarding the effectiveness of chitosan as hemostatic agent between groups. A clinical guideline was developed on dental management of patients with thrombocytopenia and since then it has been used daily at the Department of Dentistry HC-FMUSP

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The epidemiology, pathogenesis, clinical and pathological aspects of the spontaneous and experimental poisoning of cattle by bracken fern (Pteridium aquilinum) were studied. Two scientific papers that stemmed from these studies are presented and discussed here. Initially, 6,256 necropsy reports from cattle necropsied during a de 43-year-period (1964-2006) were reviewed. Of those, 15 cases were consistent with acute poisoning caused by the ingestion of P. aquilinum and they occurred in cattle from small farms in the Central region of the State of Rio Grande do Sul, Brazil. In 40% of the farms the disease occurred in small outbreaks affecting several cattle per farm and in 60% only one bovine was affected in each farm. Morbidity and mortality were 17.9% and lethality was virtually 100%. The poisoning was experimentally produced in four calves; it was concluded that exclusively the events of the primary hemostasis due to thrombocytopenia are responsible for the hemorrhages. Blood culture from three affected calves yield the growth of Klebsiella oxytoca, Staphylococcus hyicus and S. aureus, indicating that septicemia, facilitated by neutropenia could have a role in the death of cattle acutely poisoned due to the ingestion of P. aquilinum. / Foram estudados a epidemiologia, a patogênese, os aspectos clínicos e patológicos da intoxicação aguda, espontânea e experimental, por samambaia (Pteridium aquilinum) em bovinos. Dois trabalhos científicos que resultaram desse estudo são aqui apresentados e discutidos. Inicialmente, foram revisados 6.256 laudos de necropsia de bovinos num período de 43 anos (1964-2006). Desses, 15 casos corresponderam a quadros de intoxicação aguda causada pela ingestão de P. aquilinum e os casos ocorreram em bovinos de pequenas propriedades rurais da Região Central do Rio Grande do Sul. Em 40% das propriedades a doença ocorreu em pequenos surtos e em 60% delas apenas um bovino era afetado por propriedade. As taxas médias de morbidade e mortalidade foram de 17,9% e a letalidade foi virtualmente 100%. A intoxicação foi produzida experimentalmente em quatro bovinos e foi demonstrado que apenas eventos da hemostasia primária devidos a trombocitopenia são responsáveis pelas hemorragias. A hemocultura de três dos bovinos intoxicados produziu crescimentos de Klebsiella oxytoca, Staphylococcus hyicus e S. aureus, indicando que a septicemia, facilitada pela neutropenia, pode ter participação na causa da morte de bovinos na intoxicação aguda pela ingestão de P. aquilinum.

L’hémostase primaire est un processus permettant la formation d’un clou plaquettaire qui sera stabilisé par un réseau de fibrine. Ce caillot est également consolidé grâce à des phases tardives de l’hémostase primaire résultant des fonctions plaquettaires ; il s’agit principalement de la rétraction qui diminue la taille du caillot afin de le stabiliser. Cette phase est déclenchée par une signalisation « outside-in », consécutive à l’activation de l’intégrine αIIbβ3 et à l’agrégation plaquettaire, et est dépendante d’une réorganisation du cytosquelette. Le premier objectif de ce travail a été d’étudier la signalisation impliquée dans la rétraction, et en particulier l’implication des protéines ROCK, MLCK, Rac-1 et de l’actine dans l’activité de la chaine légère de la myosine (MLC) . MLC est en effet une protéine clé de la réorganisation du cytosquelette. Nous avons mis en évidence une phosphorylation biphasique de MLC dont le deuxième pic, corrélé à la rétraction, est dépendant de Rac1 et de la polymérisation de l’actine. Cette étude a été appliquée à une pathologie, le syndrome de Lowe. Il s’agit d’une maladie génétique rare, également appelée OCRL (Oculo cérébro rénal de Lowe) en référence aux organes majoritairement touchés. Suite à l’observation d’événements hémorragiques per et postopératoires suggérant une instabilité du caillot et l’observation dans une étude précédente d’un temps d’occlusion allongé au PFA100®, nous avons mis en place une étude sur 15 patients et 15 témoins pour lesquels nous avons étudié les différentes phases de l’hémostase primaire. Outre une anomalie et un retard de maturation des mégacaryocytes, nous avons mis en évidence pour la première fois chez ces patients un défaut de la voie « outside-in » responsable d’une anomalie de l’étalement plaquettaire et de la rétraction du caillot. Ce défaut de rétraction, dû à un défaut d’activation de MLC, pourrait être en partie responsable des événements hémorragiques observés chez ces patients. / Primary hemostasis is a mechanism allowing platelet clot formation that is thereafter stabilized by a fibrin network. Fibrin clot is also consolidated following post occupancy events, mainly clot retraction that decrease clot size and thus strengthen it. This phase is triggered by « outside-in » signaling. It is consecutive to αIIbβ3 integrin activation and platelet aggregation, dependent on cytoskeleton organization. Our first objective was to investigate signaling events underlying retraction, and particularly the involvement of ROCK, MLCK, Rac-1, and actin in MLC (Myosin Light Chain) phosphorylation. Indeed, MLC, involved in cytoskeleton rearrangement, is a key protein of this mechanism. We described a MLC biphasic phosphorylation profile, which second peak was dependent of Rac1 and actin polymerization. In a second part, we studied clot retraction signaling in patients with the Lowe syndrome. It is a rare genetic disease, caused by absence of OCRL (oculo cerebro renal of Lowe) protein in reference to the majority of affected organs. The rationale of this study was a previous observation of hemorrhagic events during and after surgeries, suggesting clot instability. A thrombopathy was suggested by a closure time lengthening in the PFA-100 system. The study enrolled 15 patients and 15 controls. Besides a defect of megakaryocyte maturation, we described a defect of « outside-in » signaling responsible for spreading and clot retraction abnormality. This retraction defect, caused by a MLC activity defect, could be partly responsible for hemorrhagic events reported in these patients.

No description available.

La drépanocytose est associée à un état d’hypercoagulabilité qui se manifeste sur le plan clinique par un risque augmenté de thromboses artérielles et veineuses. L’exploration de la coagulation chez les patients drépanocytaires montrait surtout une activation de la coagulation et des altérations des acteurs pro- et anticoagulants du système hémostatique. Les tests de coagulation globale de routine tels que l’aPTT et le PT sont peu sensibles aux états d’hypercoagulabilité. La fonction hémostatique globale des patients drépanocytaires était donc peu connue. Le test de génération de thrombine est un test de coagulation globale, sensible aux états d’hypo- et d’hypercoagulabilité, facile à réaliser de nos jours avec une bonne reproductibilité. Nous l’avons utilisé pour démontrer que la coagulation globale des enfants drépanocytaires était caractérisée par une accélération des réactions de formation de la thrombine et par une augmentation du potentiel de thrombine endogène. Nous avons par la suite montré que les taux élevés de microparticules pro-coagulantes et du facteur VIII chez les enfants drépanocytaires seraient les facteurs déterminant l’accélération des réactions de formation de thrombine tandis que la réduction de l’activité anticoagulante du système protéine C / protéine S serait le facteur déterminant l’augmentation du potentiel de thrombine endogène. Les marqueurs de l’hémolyse corrélaient significativement avec ces facteurs ainsi qu’avec les paramètres de génération de thrombine, suggérant que l’hémolyse serait le mécanisme pathologique à la base de l’augmentation du potentiel de génération de thrombine chez les enfants drépanocytaires. Les paramètres de génération de thrombine n’étaient pas significativement différents entre l’état de stabilité clinique et l’état de crise vaso-occlusive. Chez les enfants hétérozygotes composites, ces paramètres avaient des valeurs intermédiaires entre celles des enfants contrôles et celles des enfants drépanocytaires homozygotes. Près de 40 % des enfants drépanocytaires homozygotes avaient un potentiel hémostatique supérieur à la moyenne + 2DS des enfants contrôles du même âge. Ces enfants présentant une génération de thrombine élevée se distinguaient des autres par leur plus jeune âge, une plus grande intensité de l’hémolyse, une plus courte durée de traitement par l’hydroxyurée et des vélocités du flux sanguin au doppler transcrânien plus élevées. Ces données suggèrent davantage un lien entre le potentiel de génération de thrombine et la vasculopathie cérébrale chez les enfants drépanocytaires. L’analyse de 4 enfants ayant reçu une greffe de cellules souches hématopoïétiques montrait une tendance à la réduction du potentiel de génération de thrombine et des autres altérations de la coagulation trois mois après la greffe. Le test de génération de thrombine permet une meilleure exploration de la coagulation globale des enfants drépanocytaires. Sa réalisation sur sang total permettrait une analyse plus globale en intégrant la participation des éléments figurés du sang particulièrement les globules rouges./<p>Sickle cell disease is associated with a hypercoagulable state that express clinically by an increased risk of arterial and venous thrombosis. The exploration of coagulation in sickle cell patients showed mainly activation of coagulation and alterations pro-and anticoagulants actors of the hemostatic system. Routine global testing of coagulation such as the prothrombin time and the activated partial thromboplastin time are insensitive to hypercoagulable states. The overall hemostatic function in sickle cell disease was so little known. The thrombin generation test is a test of overall coagulation. It is sensitive to both hypo- and hypercoagulable states. It is easy to perform nowadays with good reproducibility. We used it to demonstrate that the overall coagulation of sickle cell disease children was characterized by an acceleration of the reactions of thrombin formation and an increase of the endogenous thrombin potential. We have subsequently shown that high levels of procoagulant microparticles and high levels of factor VIII in children with sickle cell disease are the factors determining the acceleration of reactions leading to thrombin formation. Our results also showed that the reduced activity of the protein C / S anticoagulant pathway is a determining factor of the increased endogenous thrombin potential in sickle cell children. Markers of hemolysis correlated significantly with these factors as well as the parameters of thrombin generation, suggesting that hemolysis is probably the pathological mechanism underlying the increased potential for thrombin generation in children with sickle cell disease. Nearly 40% of children with homozygous sickle cell disease had their hemostatic potential above the mean + 2SD that of controls children of the same age. These children with high thrombin generation differed from others by their younger age, greater intensity of hemolysis, a shorter duration of treatment with hydroxyurea. They also had higher velocity of blood flow using transcranial Doppler. These data further suggest a potential link between thrombin generation and cerebral vasculopathy in children with sickle cell disease. Analysis of four children who received hematopoietic stem cells transplantation showed a tendency towards a reversibility of thrombin generation and other alterations of coagulation three months after the transplant. Thrombin generation assay allows a better exploration of the global coagulation of sickle cell disease children. Its realization on whole blood would be a more comprehensive analysis as it would integrate the participation of blood cells particularly red blood cells. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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