Le thrombus artériel, ou thrombus intra-luminal (ILT), est impliqué à des degrés divers dans la majorité des pathologies cardiovasculaires dégénératives. Actuellement, sa détection et sa caractérisation repose sur des techniques d'imagerie morphologique qui ne renseignent pas sur l'évolution possible du thrombus en relation avec son profil d'activités biologiques. Dans ce contexte, l'imagerie moléculaire du thrombus a 3 objectifs principaux : (1) la détection du thrombus initial et la recherche de localisations secondaires, (2) l'évaluation du potentiel évolutif du thrombus et de son impact sur les tissus environnants en relation avec son activité biologique, (3) l'évaluation précoce de l'efficacité thérapeutique (avant une modification morphologique). L'objectif de ce travail a été de développer des agents d'imagerie moléculaire des activités biologiques de l'ILT. En ce qui concerne l'activité proagrégante du thrombus artériel, nous avons mis en évidence le lien existant entre l'intensité du signal en Annexine A5 radiomarquée détecté in vivo et l'intensité de l'activité proagrégante dans un modèle d'endocardite infectieuse. Nous avons développé un nouveau traceur élaboré sur la base d'un ligand naturel et de haute affinité (le fucoïdan) de la P-sélectine exprimée par les plaquettes activées, puis validé sa capacité à détecter in vivo le thrombus artériel. En ce qui concerne l'activité plasminergique du thrombus artériel, nous avons utilisé l'aprotinine radiomarquée pour détecter la plasmine dans le thrombus anévrysmal humain ex vivo, puis entamé une collaboration pour optimiser son radiomarquage en utilisant un tag peptidique sans cystéine en position N-terminale. Parallèlement nous avons développé une nouvelle approche basée sur un inhibiteur peptidique conjugué à un agent chélateur bifonctionnel / Arteriel or intra-luminal thrombus (ILT) is involved to various degrees in most of the degenerative cardiovascular diseases. Its detection and characterization is currently based on morphological imaging techniques that do not provide information about its possible evolution in relation to biological activities profile. In this context, molecular imaging of thrombus has three main objectives: (1) detection of the initial thrombus and of secondary locations, (2) evaluation of the thrombus evolutive potential and its impact on surrounding tissues in relationship with its biological activity, and (3) early assessment of therapeutic efficacy (before morphological changes). The aim of this work was to develop molecular imaging agents of biological activities of ILT. Regarding the proaggregant activity, we demonstrated a relationship between annexin A5 signal intensity and vegetation proaggregant activity in a model of infective endocarditis. We also developed a novel P-selectin imaging agent based on a natural high affinity ligand (fucoidan), and validated its ability to detect ILT in vivo. Regarding the plasminergic activity of ILT, we used radiolabelled aprotinin to detect plasmin in human aneurysmal thrombus ex vivo; we also initiated a collaboration to optimize its radiolabelling using a cystein-free tag peptide in N-terminal position. In parallel we developed a new approach based on a peptide inhibitor conjugated with a bifunctional chelating agent

No description available.

L’hémostase est un processus physiologique permettant de préserver l’intégrité du système vasculaire et de prévenir une perte de sang en réponse à une blessure. En situation pathologique, comme dans le cas de cancers, d’infections ou de maladies cardiovasculaires, il peut y avoir activation de la cascade de coagulation entraînant la formation d’un thrombus.Dans cette étude, nous avons utilisé la microscopie intravitale dans un modèle de blessure au rayon laser pour comprendre les mécanismes cellulaires et moléculaires mis en jeu dans la formation d’un thrombus plaquettaire en conditions physiologiques et pathologiques lors du développement d’un cancer. La première partie de ce travail a consisté à décrire l’implication des polynucléaires neutrophiles dans la formation d’un thrombus. Nous montrons que les neutrophiles sont les premières cellules à s’accumuler au niveau du site de blessure et représentent la principale source de facteur tissulaire menant à la génération de fibrine et à la formation du thrombus. Ces neutrophiles sont nécessaires au recrutement de cellules endothéliales progénitrices (Endothelial colony- forming cells, ECFCs), qui sont des cellules capables de jouer un rôle important dans la réparation vasculaire. Dans une seconde partie, nous avons déterminé, dans des modèles murins adaptés, l’implication de l’activation du FT et des plaquettes dans la thrombose associée au cancer. En conclusion, notre travail donne de nouvelles perspectives dans la compréhension du rôle pathophysiologique des neutrophiles, des cellules progénitrices endothéliales et des plaquettes. / Hemostasis is a physiological process to preserve the integrity of the vascular system and to prevent blood loss in response to injury. In pathological conditions, such as cancers, infections or cardiovascular diseases, the blood coagulation cascade can be activated, leading to the formation of a platelet thrombus.Using a laser-injury model coupled with a high-definition, high-speed camera, we explored the cellular and molecular mechanisms involved in thrombus formation in physiological and in pathological conditions associated with the development of a cancer. The first part of this work describes the role of polymorphonuclear neutrophils (PMNs) in thrombus formation. We show that PMNs are the first cells to accumulate at the site of injury and represent the main source of blood-borne tissue factor (TF), leading to the generation of fibrin and thrombus formation. We also show that once present at the site of injury, PMNs recruit Endothelial Progenitor Cells (endothelial colony-forming cells, ECFCs), which play a key role in vascular repair. The second part of this work we determined, in dedicated mouse models, the involvement of TF and platelet activation in thrombosis associated with cancer. Together, our findings provide new perspectives in the understanding of the pathophysiological role of polymorphonuclear neutrophils, Endothelial Progenitor Cells and platelets.

Acidentes pela serpente Bothrops jararaca (Bj) causam distúrbios hemostáticos em pacientes. Sabe-se que a fisiopatologia desses distúrbios é complexa, porém não se conhece a relevância das duas principais famílias de enzimas presentes no veneno de Bj com atividade anti-hemostática, as metaloproteinases e serinaproteases, para promover esses distúrbios. Além disso, a injúria local induzida no local da inoculação do veneno poderia estimular a liberação de fator tissular (TF) na circulação sanguínea, favorecendo a coagulopatia. Assim, o objetivo deste projeto foi investigar a contribuição das metaloproteinases e serinaproteases do veneno de Bj e a expressão de TF para a gênese dos distúrbios hemostáticos, utilizando um modelo experimental em ratos. O veneno de Bj foi previamente incubado com Na2-EDTA 13 mM ou AEBSF 4 mM para inibir as metaloproteinases e serinaproteases, respectivamente, e administrado pelas vias s.c. ou i.v. Após 3 e 6 h, os parâmetros hemostáticos e de expressão proteica de TF e isomerase de dissulfeto proteico (PDI) foram avaliados. Os níveis de veneno circulante se elevaram mais rapidamente no grupo injetado pela via i.v., e o tratamento do veneno com Na2-EDTA ou AEBSF não reduziu os níveis circulantes de veneno em comparação ao grupo controle com veneno. Em comparação com animais tratados com salina, houve uma queda abrupta na contagem plaquetária em todos os grupos e tempos administrados com veneno de Bj, sendo o grupo administrado pela via i.v. o que apresentou uma queda de maior intensidade. O pré- tratamento do veneno com o AEBSF não impediu o consumo plaquetário e somente o Na2-EDTA parcialmente reverteu a plaquetopenia. Por outro lado, o veneno não tratado causou consumo de fibrinogênio plasmático, geração de produtos de degradação de fibrinogênio e fibrina, prolongamento do tempo de protrombina (TP) e hemorragia no local de inoculação do veneno. No entanto, o Na2-EDTA, e não o AEBSF, bloqueou completamente esses parâmetros. Não houve redução dos níveis de fator VII ao longo do envenenamento, e seus níveis apresentou um notável aumento nos animais envenenados no grupo 6 h s.c. Ratos envenenados apresentaram notável aumento dos níveis do TF plasmáticos, que foi inibido pelo pré-tratamento com o Na2-EDTA. Houve também aumento da expressão de TF no pulmão e pele. Nos grupos injetados com veneno de Bj em 6 h ocorreu uma redução da expressão de PDI. Os resultados mostram que as metaloproteinases são componentes essenciais para o desencadeamento da coagulopatia do envenenamento. No entanto, as metaloproteinases e serinaproteases não estão diretamente envolvidas na gênese da plaquetopenia induzida pelo veneno de Bj e outros mecanismos/toxinas parecem estar envolvidos. Os resultados também demonstraram o aumento dos níveis de TF plasmático durante o envenenamento, similar àquele observado na coagulação intravascular disseminada, o que sugere a importância da geração de TF como um mecanismo para promover distúrbios sistêmicos da coagulação / Bites by Bothrops jararaca (Bj) snakes evoke hemostatic disturbances in patients. The pathophysiology of such disturbances is complex, but the importance of the major enzyme families with anti-hemostatic activity, found in Bj venom. i.e., metalloproteinases and serine proteinases, to promote them is not known. Moreover, the local injury induced at the site of venom inoculation might also stimulate tissue factor (TF) release into bloodstream, favoring the coagulopathy. The aim of this study was to investigate the contribution of metalloproteinases and serine proteinases of Bj venom, as well the TF expression to the genesis of hemostatic disturbances, using an experimental model in rats. Crude Bj venom was previously incubated with 13 mM Na2-EDTA or 4 mM AEBSF to inhibit metalloproteinases and serine proteinases, respectively, and administered s.c. or i.v. into rats. After 3 and 6 h, hemostatic parameters and TF and protein disulfide isomerase (PDI) expression were evaluated in plasma and samples of skin and lungs. Circulating venom levels increased more rapidly in i.v. group, and neither Na2-EDTA nor AEBSF treatment reduced the circulating venom levels in comparison with the control group. Platelet counts showed a marked decrease in all groups administered with Bj venom in comparison with saline-treated rats; the fall in platelet counts was more intense in animals administered i.v. with Bj venom. The pre-treatment of venom with AEBSF failed to block the fall in platelets count, and only Na2-EDTA minimally reversed thrombocytopenia. Nonetheless, non-treated venom provoked plasma fibrinogen consumption, generation of fibrin(ogen) degration products, prolongation of prothrombin time (TP), and hemorrhage at the site of venom inoculation. However, Na2-EDTA, but not AEBSF, completely blocked these parameters. Factor VII levels were not reduced during envenomation, and they showed a marked increase in envenomed rats at 6 h in the s.c. group. Envenomed rats showed a marked increase in plasma TF levels, which was also blocked by Na2-EDTA. In addition, TF expression was increased in the lung and skin samples. PDI expression in skin was reduced at 6 h in all groups treated with venom. These findings demonstrate that metalloproteinases are essential venom components involved in the Bj-induced coagulopathy. Nonetheless, metalloproteinases and serine proteases had no direct involvement in the genesis of Bj-induced thrombocytopenia and other venom mechanisms/toxins seem to be associated therein. High levels of TF in plasma may occur during snake envenomation, so that the etiopathogenesis of coagulopathy in snake envenomation resembled that of true disseminated intravascular coagulation syndrome

O curto período de armazenamento dos concentrados de plaquetas (CP), de 5 a 7 dias, torna esse hemocomponente crítico para os serviços de hemoterapia. A criopreservação se apresenta então como uma possibilidade para a manutenção dos estoques por período mais prolongado. Essa prática exige a adição de substância crioprotetora e a adequação do volume em relação à concentração de plaquetas. O CP obtido por aférese (CPAF) é um hemocomponente proveniente de um único doador, coletado em sistema automatizado, que equivale a 6-8 unidades de CP obtidas da coleta de doadores convencional. Objetivo: Avaliar o efeito da criopreservação de CPAF no quinto dia do armazenamento, por meio de análises imunofenotípicas e funcionais das plaquetas, utilizando o crioprotetor dimetilsulfóxido (DMSO) a 5%, em freezer a -80°C e descongelamento em banho maria a 37°C, sem a remoção do crioprotetor. Material e Métodos: Foram analisadas 20 unidades de CPAF em quatro fases diferentes do processo: Fase I (pré-redução de volume), Fase II (pós-repouso, agitação e adição do DMSO), Fase III (pós-descongelamento) e Fase IV (após duas horas de descongelamento e agitação). Todas as bolsas foram avaliadas quanto à presença do \"swirling\" plaquetário, e análise microbiológica, contagem de plaquetas e leucócitos, determinação do volume plaquetário médio (VPM), análise do pH, dosagem de desidrogenase lática (DHL) e glicose, a ativação plaquetária por meio de citometria de fluxo com os marcadores CD61, CD62P e anexina V e para a avaliação funcional, as técnicas de microagregação plaquetária e retração do coágulo. Os resultados de cada fase foram analisados e comparados, considerando resultados p< 0,05 de significância estatística, avaliada pelos testes de Wilcoxon e Teste-T pareado. Resultados: Todos os CPs criopreservados apresentaram na inspeção visual presença de \"swirling\" e ausência de grumos. O pH manteve-se com mediana de 7,185 (6,076-7,528) pra fase III, análise microbiológica foi negativa em todas as unidades criopreservadas, o número mediano de plaquetas caiu de 3,04x1011/U na Fase II para 2,27x1011/U na fase III (redução de 25,32%). A ativação plaquetária na fase II foi de 23% CD62P+ para 44% CD62P+ na fase III (p=0,067). O marcador anexina V estava expresso em 13% das plaquetas na fase II e em 11% na fase III, (p=0,33). A LDH aumentou de 747 U/L (4-3079) para 1.428 U/L (662-2303) da fase II para a fase III, respectivamente (p=0,055). A glicose diminuiu em todas as fases (p<0,0001). Os testes de função plaquetária revelaram que a plaquetas descongeladas mantêm sua função. Conclusão: Os resultados obtidos mostraram que, embora tenham ocorrido ativação e redução significativa do número de plaquetas, o produto final conservou quantidade suficiente de plaquetas, cujas funções foram mantidas, o que torna viável a utilização do hemocomponente CP criopreservado. Sugere ainda que os CPAF possam ser criopreservados no quinto dia de armazenamento, com o uso do DMSO a 5%, ideal para que não se faça necessário sua remoção pós-descongelamento. / The concentration time of the platelet concentrates (CP), from 5 to 7 days, makes this blood component critical for hemotherapy services. Cryopreservation presents itself as a possibility of maintaining stocks for a longer period. This practice requires an additional cryoprotectant and a suitability of volume in relation to platelet concentration. The CP collected by the same (CPAF) is a blood component of a single dosage, being collected in an automated system, which is equivalent to 6-8 CP units from the collection of conventional donors. Objective: To evaluate the effect of CPAF cryopreservation on the fifth day of storage, by means of immunophenotypic and functional platelet analysis using the 5% DMSO cryoprotectant in a freezer at -80 ° C and thawing in a Maria bath at 37 ° C, without removal of the cryoprotectant. Material and Methods: 20 units of CPAF were analyzed in four different phases of the process: Phase I (pre-reduction of volume), Phase II (post-rest, agitation and DMSO addition), Phase III (post-thaw) and Phase IV (after two hours of thawing and shaking). All units were evaluated for platelet swirling and microbiological analysis, platelet and leukocyte counts, determination of mean platelet volume (MVP), pH analysis, lactate dehydrogenase (LDH) and glucose, platelet activation by means of flow cytometry with the markers CD61, CD62P and annexin V and for the functional evaluation, platelet microaggregation and clot retraction techniques. The results of each phase were analyzed and compared, considering results p <0.05 of statistical significance, evaluated by the Wilcoxon and Paired T-test. Results: All cryopreserved CPs presented visual presence of \"swirling\" and absence of lumps. The pH was maintained at a median of 7.185 (6.076- 7.528) for phase III, microbiological analysis was negative in all cryopreserved units, the median number of platelets fell from 3.04x1011/U in Phase II to 2.27x1011/U in phase III (reduction of 25.32%). Phase II platelet activation was 23% CD62P + to 44% CD62P + in phase III (p=0.067). The annexin V marker was expressed in 13% of platelets in phase II and 11% in phase III (p=0.33). LDH increased from 747 U/L (4-3,079) to 1,428 U / L (662-2303) from phase II to phase III, respectively (p<0,0055). Glucose decreased in all phases (p <0.0001). Platelet function tests have revealed that thawed platelets maintain their function. Conclusion: The results showed that, although activation and significant reduction of platelet count occurred, the end product preserved sufficient quantity of platelets, whose functions were maintained, which makes the use of the cryopreserved CP blood component viable. It also suggests that CPAFs can be cryopreserved on the fifth day of storage using 5% DMSO, so that their post-thaw removal is not required.

Embora pacientes em terapia dupla antiplaquetária, composta de aspirina e um antagonista do receptor purinérgico P2Y12, tenham melhor prognóstico, eles ainda sofrem eventos trombóticos. Frequentemente, relaciona-se o nível de inibição plaquetária destes pacientes à ineficácia do tratamento. A reatividade plaquetária destes pacientes é uma função do nível do bloqueio do receptor P2Y12 e dos níveis de óxido nítrico e prostaciclina, importantes mediadores endoteliais, os quais agem em sinergia tripla aumentando o nível dos nucleotídeos cíclicos intraplaquetários mantendo a plaqueta em estado quiescente. Pacientes com disfunção endotelial têm menor disponibilidade destes mediadores e, consequentemente, menor inibição plaquetária in vivo. Propõe-se uma nova estratégia terapêutica composta de um inibidor do receptor purinérgico P2Y12, um ativador da enzima guanilato ciclase e um inibidor da fosfodiesterase, visando aumento dos nucleotídeos cíclicos, resultando em um forte efeito antiplaquetário localizado e tendo aplicação direta à pacientes com disfunção endotelial. / Disorders on the cardiovascular (CV) system are responsible for 31% of deaths worldwide. When inappropriately activated, platelets can cause myocardial infarction and stroke. Dual anti-platelet therapy (DAPT), composed of acetylsalicylic acid and a P2Y12 receptor blocker (clopidogrel, prasugrel, etc) is recommended for the prevention of recurrent CV events. Whilst DAPT is associated with an improvement in patient outcomes, thrombotic events do still occur. Platelet reactivity of patients under DAPT is a function of the level of P2Y12 receptor blockade and the levels of nitric oxide (NO) and prostacyclin (PGI2), two important endothelium-derived autacoids. Therefore, a new antithrombotic therapy is proposed using soluble guanylate cyclase (sGC) allosteric modulators independent of NO and phosphodiesterase (PDE) inhibitors. Based on the results observed, the combination of a sGC activator and a PDE inhibitor given at low doses, in the presence of P2Y12 receptor blocker, could produce enhanced and localized platelet inhibition.

L'aspirine est l'anti-plaquettaire le plus largement prescrit à la phase aiguë de l'accident vasculaire cérébral (AVC) ischémique. Cependant, la survenue de récidives, malgré cette prescription, est fréquente. La description de l'effet de l'aspirine sur l'activité plaquettaire durant cette phase n'a jamais été réalisée. Elle pourrait mettre en évidence une moindre réponse plaquettaire et aider à établir de nouvelles stratégies thérapeutiques. Cinquante patients, ont reçu par voie orale 300 mg d'aspirine, suite à un AVC ischémique. Ensuite, des prélèvements sanguins ont été réalisés : entre 2 et 3 heures (T1), entre 23 et 24 heures (T2) après la prise d'aspirine et, pour des patients déjà traités quotidiennement par une dose inférieure, avant la prise d'aspirine (T0). Les concentrations sériques de thromboxane (TX) B2 ont été mesurées, ainsi que les agrégations induites par l'acide arachidonique, par le collagène à la concentration de 2µg/L (Col2) et 20 µg/L (Col20). Afin de diminuer l'effet des variations de condition d'expérience, les résultats pour Col2 ont été rapportés à ceux pour Col20 (Col2/20). Tous les patients ont présenté une réponse à l?aspirine visible à T1 avec de plus, des concentrations de TXB2 abaissées en comparaison à T0. Il existe une récupération de l'activité plaquettaire à T2 comparée à T1, montrée par les concentrations de TXB2 et le rapport Col2/20. La dose orale de 300 mg d'aspirine, donnée à la phase aiguë de l'AVC, entraîne une inhibition plaquettaire, mais avec une récupération visible sur 24 heures. Pour les patients déjà traités quotidiennement par une dose inférieure, elle permet de compléter l'inhibition de la voie TXA2 dépendante / Aspirin is the most commonly used antiplatelet treatment during the acute phase of cerebral ischemic events. But, despite this protection, early ischemic recurrences are frequent, and considered as clinical failures of this therapy. We studied laboratory parameters of the first 300 mg oral dose of aspirin given, within 48 hours, after ischemic cerebral event. Fifty patients were included. For all patients, two blood sampling were performed, the first, during the third hour after aspirin intake (T1) and the second during the twenty-fourth hour (T2). For patients already treated with a daily dose of aspirin, a supplementary withdrawn was done before aspirin intake (T0). Platelet reactivity was studied on the basis of serum thromboxane (TX) B2 levels and light transmission aggregometry after stimulation of platelet-rich plasma by acid arachidonic and collagen 2µg/mL reported to results with collagen 20 µg/mL (ratio Col2/20). Inhibition of platelet activity was observed, at T1, for all patients. There is a significant increase of TXB2 values, and of relative values of the ratio Col2/20, at T2 as compared to T1. For already aspirin treated patients, there is a significant decrease of TXB2 levels at T1 as compared to T0. There is a platelet reactivity recovery within 24 hours, following the first 300 mg oral dose of aspirin, during the acute phase of a cerebral ischemic event, and demonstrated by TXB2 levels and ratio Col2/20. This fact would favour early ischemic recurrences. However, this dose is able to complete the inhibition of the TXA2 pathway for already aspirin treated patients

La Sphingosine 1-phosphate (S1P) est un lipide bioactif qui joue un rôle majeur dans de nombreux processus tels que le développement vasculaire, la circulation des cellules immunitaires et la régulation de l’intégrité vasculaire. L’absence de S1P circulante engendre une augmentation constitutive de la perméabilité vasculaire ainsi qu’une sensibilité accrue à l’anaphylaxie systémique passive, cependant l’implication respective des différentes sources de la S1P au cours de ces processus, demeure méconnue. Ceci est plus particulièrement vrai pour les plaquettes qui constituent une réserve importante de S1P. La S1P d’origine plaquettaire ne contribue pas au pool de S1P plasmatique, il n’est libéré que lors de l’activation plaquettaire. Nous avons étudié le rôle de la S1P d’origine plaquettaire au sein des vaisseaux sanguins, au cours du développement embryonnaire et chez des souris adultes, en présence ou en absence d’autres sources de S1P circulante, notamment des globules rouges. Par délétion des gènes codant pour les sphingosine kinases 1&2 au sein des megakaryocytes, nous avons généré des souris dont les plaquettes sont incapables de synthétiser et donc de sécréter la S1P. Ces plaquettes sont par ailleurs incapables, in vitro, de promouvoir le maintien de la barrière endothéliale. Néanmoins, une déficience en S1P d’origine plaquettaire n’augmente pas le risque d’hémorragie au cours du développement embryonnaire, dans un contexte d’inflammation, d’irradiation, ou de traumatisme ; cela suggère que la S1P d’origine plaquettaire n’est pas indispensable au cours de ces processus. Cependant, nous avons découvert que la résistance au choc anaphylactique est compromise en l’absence de S1P d’origine plaquettaire. L’aspirine bloquant la sécrétion de S1P par les plaquettes, un traitement à l’aspirine induit également une sensibilisation au choc anaphylactique de façon similaire à celle observée dans le cas d’une déficience en S1P d’origine plaquettaire. Il semblerait que dans ce contexte, la S1P d’origine plaquettaire joue plutôt un rôle pour promouvoir le tonus vasculaire via S1P2, que de maintenir l’intégrité vasculaire. / Sphingosine 1-phosphate (S1P) is a bioactive lipid that plays key roles in vascular development, immune cell trafficking and regulation of vascular integrity. Lack of circulating S1P leads to constitutive vascular leak and sensitivity to passive systemic anaphylaxis, but the relative roles of different sources of S1P in these processes is unclear. This is especially true for platelets, which carry large amounts of S1P but do not contribute S1P to plasma unless activated. We have addressed roles of platelet S1P in developing and mature murine vessels in the presence or absence of other circulating S1P sources, notably red blood cells. By deletion of the genes encoding sphingosine kinases 1&2 in megakaryocytes, we generated mice with platelets that were greatly impaired in their ability to secrete S1P. These platelets were also unable to promote endothelial barrier function in vitro. Nevertheless, platelet S1P deficiency did not sensitize to bleeding during embryonic development, or in association with inflammation, irradiation or traumatic injury, suggesting that platelet S1P is dispensable for these processes. Instead, we find that recovery from anaphylactic shock is impaired in the absence of platelet-derived S1P. Consistent with the ability of aspirin to block S1P release from platelets, it impaired recovery from anaphylactic shock to a similar degree as platelet S1P deficiency. Instead of protecting vascular integrity, platelet-derived S1P appeared to act via the promotion of vascular tone through S1P2 in this context. Collectively, we find that S1P is necessary for the capacity of platelets to promote endothelial barrier function in vitro, but that this capacity becomes redundant in the presence of red blood cell-derived S1P in vivo. Nevertheless, during anaphylactic shock, both sources of S1P are necessary for complete recovery. Blocking S1P release with aspirin, an over-the-counter anti-inflammatory drug, may impair the protective functions of platelet S1P.

A fosfolipase A2 (PLA2) é uma enzima chave no metabolismo dos fosfolípides de membrana e é um dos principais componentes envolvidos na sinalização celular. Alterações da atividade da PLA2 tem sido descritas no cérebro e no sangue (soro, plasma e plaquetas) de pacientes com diversas doenças neuropsiquiátricas. Neste estudo foi desenvolvido um ensaio radioenzimático para detectar em plaquetas, a atividade dos três principais grupos de PLA2, que são PLA2 secretórias ou PLA2 extracelular dependente de Ca 2+ (sPLA2); PLA2 citósólicas dependentes de Ca 2+ (cPLA2) e as PLA2 intracelulares independentes de Ca 2+ (iPLA2). Para confirmar a presença desses grupos da enzima em plaquetas, algumas variáveis foram testadas, como as diferenças de preferência ao ácido graxo como substrato, o requerimento de cálcio e a inibição seletiva com os inibidores Bromoenol lactone (BEL) e o Methyl Arachidonyl Fluorophosphonate (MAFP). Os resultados obtidos demonstram a presença dos três principais grupos de PLA2 (sPLA2, cPLA2, and iPLA2) em plaquetas. Estes achados sugerem o uso de plaquetas, uma amostra biológica de fácil acesso, como possível modelo periférico de neurônios para o estudo do metabolismo de fosfolípides. / Phospholipase A2 (PLA2) is a key-enzyme in the metabolism of membrane phospholipids and is one of the major components involved in cell signaling. Alterations of PLA2 activity have been reported in brains and blood cells in several neuropsychiatric diseases. In this study we developed a radio-enzymatic assay to detect in platelets the activity of the three main groups of PLA2, which are secretory PLA2 or extracellular calcium dependent PLA2 (sPLA2), cytosolic calcium dependent PLA2 (cPLA2) and intracellular calcium independent PLA2 (iPLA2). To confirm the presence of these PLA2 groups some variables were tested, such as differences in the preferred fatty acid substrate, calcium dependence, and selective inhibition with Bromoenol lactone (BEL) and Methyl Arachidonyl Fluorophosphonate (MAFP). Our findings demonstrate the presence of the three main groups of PLA2 (sPLA2, cPLA2, and iPLA2) in platelets. In addition, this study is in line with others suggesting that platelets, a typical biological sample, can be used as a peripheral model for neurons.

Acidentes pela serpente Bothrops jararaca (Bj) causam distúrbios hemostáticos em pacientes. Sabe-se que a fisiopatologia desses distúrbios é complexa, porém não se conhece a relevância das duas principais famílias de enzimas presentes no veneno de Bj com atividade anti-hemostática, as metaloproteinases e serinaproteases, para promover esses distúrbios. Além disso, a injúria local induzida no local da inoculação do veneno poderia estimular a liberação de fator tissular (TF) na circulação sanguínea, favorecendo a coagulopatia. Assim, o objetivo deste projeto foi investigar a contribuição das metaloproteinases e serinaproteases do veneno de Bj e a expressão de TF para a gênese dos distúrbios hemostáticos, utilizando um modelo experimental em ratos. O veneno de Bj foi previamente incubado com Na2-EDTA 13 mM ou AEBSF 4 mM para inibir as metaloproteinases e serinaproteases, respectivamente, e administrado pelas vias s.c. ou i.v. Após 3 e 6 h, os parâmetros hemostáticos e de expressão proteica de TF e isomerase de dissulfeto proteico (PDI) foram avaliados. Os níveis de veneno circulante se elevaram mais rapidamente no grupo injetado pela via i.v., e o tratamento do veneno com Na2-EDTA ou AEBSF não reduziu os níveis circulantes de veneno em comparação ao grupo controle com veneno. Em comparação com animais tratados com salina, houve uma queda abrupta na contagem plaquetária em todos os grupos e tempos administrados com veneno de Bj, sendo o grupo administrado pela via i.v. o que apresentou uma queda de maior intensidade. O pré- tratamento do veneno com o AEBSF não impediu o consumo plaquetário e somente o Na2-EDTA parcialmente reverteu a plaquetopenia. Por outro lado, o veneno não tratado causou consumo de fibrinogênio plasmático, geração de produtos de degradação de fibrinogênio e fibrina, prolongamento do tempo de protrombina (TP) e hemorragia no local de inoculação do veneno. No entanto, o Na2-EDTA, e não o AEBSF, bloqueou completamente esses parâmetros. Não houve redução dos níveis de fator VII ao longo do envenenamento, e seus níveis apresentou um notável aumento nos animais envenenados no grupo 6 h s.c. Ratos envenenados apresentaram notável aumento dos níveis do TF plasmáticos, que foi inibido pelo pré-tratamento com o Na2-EDTA. Houve também aumento da expressão de TF no pulmão e pele. Nos grupos injetados com veneno de Bj em 6 h ocorreu uma redução da expressão de PDI. Os resultados mostram que as metaloproteinases são componentes essenciais para o desencadeamento da coagulopatia do envenenamento. No entanto, as metaloproteinases e serinaproteases não estão diretamente envolvidas na gênese da plaquetopenia induzida pelo veneno de Bj e outros mecanismos/toxinas parecem estar envolvidos. Os resultados também demonstraram o aumento dos níveis de TF plasmático durante o envenenamento, similar àquele observado na coagulação intravascular disseminada, o que sugere a importância da geração de TF como um mecanismo para promover distúrbios sistêmicos da coagulação / Bites by Bothrops jararaca (Bj) snakes evoke hemostatic disturbances in patients. The pathophysiology of such disturbances is complex, but the importance of the major enzyme families with anti-hemostatic activity, found in Bj venom. i.e., metalloproteinases and serine proteinases, to promote them is not known. Moreover, the local injury induced at the site of venom inoculation might also stimulate tissue factor (TF) release into bloodstream, favoring the coagulopathy. The aim of this study was to investigate the contribution of metalloproteinases and serine proteinases of Bj venom, as well the TF expression to the genesis of hemostatic disturbances, using an experimental model in rats. Crude Bj venom was previously incubated with 13 mM Na2-EDTA or 4 mM AEBSF to inhibit metalloproteinases and serine proteinases, respectively, and administered s.c. or i.v. into rats. After 3 and 6 h, hemostatic parameters and TF and protein disulfide isomerase (PDI) expression were evaluated in plasma and samples of skin and lungs. Circulating venom levels increased more rapidly in i.v. group, and neither Na2-EDTA nor AEBSF treatment reduced the circulating venom levels in comparison with the control group. Platelet counts showed a marked decrease in all groups administered with Bj venom in comparison with saline-treated rats; the fall in platelet counts was more intense in animals administered i.v. with Bj venom. The pre-treatment of venom with AEBSF failed to block the fall in platelets count, and only Na2-EDTA minimally reversed thrombocytopenia. Nonetheless, non-treated venom provoked plasma fibrinogen consumption, generation of fibrin(ogen) degration products, prolongation of prothrombin time (TP), and hemorrhage at the site of venom inoculation. However, Na2-EDTA, but not AEBSF, completely blocked these parameters. Factor VII levels were not reduced during envenomation, and they showed a marked increase in envenomed rats at 6 h in the s.c. group. Envenomed rats showed a marked increase in plasma TF levels, which was also blocked by Na2-EDTA. In addition, TF expression was increased in the lung and skin samples. PDI expression in skin was reduced at 6 h in all groups treated with venom. These findings demonstrate that metalloproteinases are essential venom components involved in the Bj-induced coagulopathy. Nonetheless, metalloproteinases and serine proteases had no direct involvement in the genesis of Bj-induced thrombocytopenia and other venom mechanisms/toxins seem to be associated therein. High levels of TF in plasma may occur during snake envenomation, so that the etiopathogenesis of coagulopathy in snake envenomation resembled that of true disseminated intravascular coagulation syndrome