Conselho Nacional de Desenvolvimento Científico e Tecnológico / Apesar do desenvolvimento de novas drogas antifúngicas e da sua utilização como terapia profilática visando à prevenção de infecções fúngicas invasivas, estas ainda constituem-se num problema emergente, com elevadas taxas de mortalidade. Neste contexto, destaca-se a aspergilose invasiva, uma infecção fúngica oportunista que acomete pacientes com neutropenia profunda e prolongada, principalmente os pacientes com leucemia aguda ou submetidos a transplante de medula óssea. Aspergillus fumigatus, um fungo filamentoso, é o principal agente etiológico da aspergilose invasiva, sendo um patógeno angioinvasivo. As hifas deste fungo são capazes de causar injúria e ativação endotelial, induzindo o endotélio a um fenótipo pró-trombótico, que por sua vez, é mediado pela secreção de citocinas pró-inflamatórias, em especial, o TNF-α. O presente trabalho teve como objetivo estudar a capacidade de cepas mutantes de A. fumigatus em ativar células endoteliais, avaliando o perfil de secreção de citocinas em meio condicionado e a expressão de fator tecidual. Resumidamente, monocamadas confluentes de células endoteliais isoladas da veia umbilical humana foram incubadas com conídios e tubos germinativos de cepas selvagens (Af293 e Ku80) e mutantes (Δugm1, ΔcalA, ΔcrzA, ΔprtT) de A. fumigatus. A taxa de adesão e endocitose destas cepas às monocamadas de HUVEC foi avaliada a partir de um ensaio quantitativo de imunofluorescência diferencial. O perfil cinético de secreção de citocinas foi determinado em meio condicionado das HUVECs, por ensaio de multiplex para IL-6, IL-8 e TNF-α. A ativação endotelial, por sua vez, foi determinada pela expressão de fator tecidual por RT-PCR em tempo real. Os resultados obtidos demonstraram que a mutante para o gene ugm1, responsável por codificar a enzima UDP-galactopiranose mutase, que converte resíduos de galactopiranose a galactofuranose, apresentou um fenótipo hiperaderente às células endoteliais e um estímulo 10 vezes maior à secreção de TNF-α e 2,5 vezes maior a secreção de IL-6, quando comparada a ativação observada para as cepas selvagens. A galactofuranose é um componente importante de glicoconjugados da parede celular de A. fumigatus. Dessa forma, a ausência desse monossacarídeo na célula fúngica leva a um mecanismo compensatório caracterizado por um aumento na expressão de moléculas de galactosaminogalactana na parede celular. De maneira contrária, mutantes para os genes calA e crzA, apresentaram um fenótipo hipoaderente às HUVECs e uma perda na capacidade de induzir a secreção de citocinas e ativar o endotélio. Essas mutantes apresentam deleções que interferem na via de cálcio/calcineurina, responsável por regular a morfogênese e virulência de A. fumigatus, além de apresentarem alterações no conteúdo de beta-1-3 glucana. Já a cepa ΔprtT, mutante para o fator de transcrição prtT que regula a secreção de múltiplas proteases, apresentou um fenótipo de adesão, estímulo e ativação endotelial semelhante ao observado para as cepas selvagens. A comparação entre a capacidade de conídios e tubos germinativos em ativar células endoteliais, corroborou achados anteriores da literatura que reportam que só hifas são capazes de ativar células endoteliais, independentemente da sua viabilidade. Os dados deste estudo permitiram concluir que dentre os componentes de superfície celular de A. fumigatus, os polímeros de galactose, em especial a galactosaminogalactana, parecem ser responsáveis, pelo menos em parte, pelos mecanismos de interação e ativação endotelial. / Besides the emergency of more active and less toxic antifungal agents and the conventional use of antifungal prophylaxis, invasive mold infections have still high mortality rates, especially, invasive aspergillosis (IA). This life-threatening disease is a predominant fungal opportunistic infection for patients with long-term neutropenia, mostly HSCT recipients. Aspergillus fumigatus is the most important species causing IA and is already known as an angioinvasive fungal pathogen. Upon filamentation this fungus can damage and activate human vein endothelial cells (HUVEC) which in turn switch to a pro-thrombotic phenotype. HUVEC activation is known to be mediated by TNF-α once cell-cell contact occurs. The aim of this study was to investigate the endothelial activation ability of several mutants of A. fumigatus. Briefly, HUVECs were infected with germ tubes and conidia of A. fumigatus wild type (WT) Af293 and Ku80 and mutant (Δugm1, ΔcalA, ΔcrzA, ΔprtT) strains and a differential quantitative fluorescence assay performed to determine adhesion and internalization rates. Thus, a kinetic study of secreted pro-inflammatory cytokines and chemokines in HUVEC conditioned medium was achieved by a multiplex immuneassay. The cytokine production was assayed at three time points (4, 8 and 16 hours) using dead germ tubes, and at one time point (16 hours) using dead conidia, for an E:T ratio of 2:1. Additionally, to investigate endothelial activated phenotype, the expression of tissue factor was performed by a real time RT-PCR assay. The ugm1 mutant, which lacks the ugm1 gene encoding UDP-galactopyranose mutase, an enzyme responsible for the conversion of galactopyranose in galactofuranose, showed a hiperadherent phenotype and an increased capability to cause endothelial cell stimulation and activation. This mutant stimulated at least a 10-fold and 2.5-fold increase of TNF-alfa and IL-6 secretion, respectively and 2-fold increase of tissue factor expression by host cells, as compared to WT strains. Galactofuranose is an uncommon 5-membered ring form of galactose and a very important component of glycostructures of the A. fumigatus cell wall. The lack of this monosaccharide in the fungal cell wall leads to a compensatory mechanism characterized by an increment in the galactosaminogalactan expression. In contrast, the calA and crzA mutants, with upstream and downstream dysfunction in the calcium/calcineurin pathway, with alteration in the cell wall β-1,3-glucan content, showed a decrease capacity to adhere to HUVECs and did not induce both the secretion of pró-inflammatory cytokine and activation on endothelial cells. Furthermore, the mutant ΔprtT, witch lacks the transcriptional factor (prtT) that regulates the secretion of multiple proteases, showed the same adhesion and endothelial cell activation phenotype as observed for WT strains. As indicated in previous investigations, our data showed that conidia of A. fumigatus werent able to cause the same endothelial cell cytokine stimulation observed for germ tubes, and that endothelial cell activation is independent on fungal cell viability. Finally, its possible to conclude that the polymers of galactose in the cell wall of A. fumigatus, especially the galactosaminogalactan molecule, seem to be responsible for the endothelial cell interaction mechanisms and activation.

Os acidentes ofídicos são considerados um grande problema de saúde pública e no Estado de São Paulo a serpente Bothrops jararaca é o agente considerado mais relevante epidemiologicamente. O antiveneno é o único medicamento oficialmente aprovado para o tratamento de picadas de serpentes e, apesar de ser eficaz em diversos aspectos, mostra-se ineficaz em relação a complicações secundárias do envenenamento, como o estresse oxidativo/nitrosativo, caracterizado por um desequilíbrio no estado redox, que pode ser extremamente deletério aos pacientes. Portanto, vê-se necessária a busca por terapias complementares que possam combater o estresse oxidativo/nitrosativo. Ademais, distúrbios da hemostasia observados em envenenamentos pela B. jararaca, tais como a plaquetopenia, a hipofibrinogenemia e o aumento da atividade do fator tissular (TF) plasmático podem ter também como causa indireta o estresse oxidativo/nitrosativo, induzido por componentes do veneno de B. jararaca (BjV). Recentemente, demonstrou-se que a regulação da atividade biológica do TF, a proteína responsável pela iniciação da cascata de coagulação in vivo, pode ser controlada pela isomerase de dissulfeto proteico (PDI) e que a atividade da PDI é inibida in vivo pela quercetina-3-rutinosídeo (rutina), um antioxidante natural encontrado em plantas e alimentos. Considerando essas premissas, o presente trabalho objetivou investigar em camundongos injetados com o veneno de B. jararaca (BjV): (a) a ocorrência de distúrbios do estado redox; (b) as alterações hematológicas e hemostáticas (em plasma e tecidos) associadas ao desequilíbrio do estado redox; (c) a atividade da rutina como agente modulador sobre essas alterações. Para isso, camundongos Swiss (30-35 g) foram divididos em 4 grupos experimentais: controle salina (salina, controle negativo), controle rutina (rutina+salina), BjV+salina (BjV+salina, controle positivo) e BjV+rutina (BjV+rutina, tratamento). Após 3, 6 e 24 h da administração dos tratamentos (via s.c.), foram coletados sangue e fragmentos de tecidos para posteriores análises. O envenenamento induziu um aumento de espécies reativas, diminuição da capacidade antioxidante total, alterações hematológicas (plaquetopenia, neutrofilia e diminuição de eritrócitos), distúrbios hemostáticos (hipofibrinogenemia, prolongamento do tempo de protrombina e aumento da atividade de TF no plasma), além de diminuir a expressão proteica de PDI no coração. A rutina não inibiu in vitro a atividade biológica de proteínas presentes no BjV (metaloproteinases, serinaproteases, fosfolipases A2 e L-aminoácido oxidases) e nem mesmo a agregação plaquetária induzida pelo veneno. No entanto, a administração do veneno incubado com rutina foi capaz de reduzir os níveis de espécies reativas, impedir a queda de eritrócitos, normalizar os níveis de fibrinogênio e o tempo de protrombina e alterar a atividade de TF e expressão proteica de TF e PDI em tecidos. Desse modo, concluímos que a rutina foi capaz de favoravelmente modular importantes alterações hemostáticas e de estado redox no envenenamento por B. jararaca, o que indica seu grande potencial como agente terapêutico complementar em envenenamentos / Snakebites are a major public health issue and in São Paulo State, and Bothrops jararaca snakes are considered the most important agents epidemiologically. Antivenom is the only officially approved treatment for snakebites and although effective in many aspects, it is ineffective in combating secondary complications induced by envenomation, e.g. oxidative/nitrosative stress (ONS), which can be extremely deleterious to patients. Therefore, it is necessary to search for new complementary therapies that could attenuate ONS. Furthermore, the hemostatic disturbances in B. jararaca envenomation - characterized by the presence of thrombocytopenia, hypofibrinogenemia and increased tissue factor (TF) activity in plasma - could be indirectly evoked by ONS induced by B. jararaca venom (BjV) components. Recently, the biological activity of TF - the protein responsible for initiating the extrinsic pathway of the coagulation cascade - was demonstrated to be regulated by protein disulfide isomerase (PDI), which in turn, can be inhibited in vivo by quercetin-3-rutinose (rutin), a natural antioxidant found in plants and diet. Based on that, the present study aimed to investigate in mice injected with BjV: (a) the alterations in redox status in plasma and tissues; (b) the hematological/hemostatic disturbances (in plasma and tissues) associated with the imbalance in redox status; (c) the activity of rutin as a modulatory agent on those alterations. Swiss mice (30-35 g) were divided in four experimental groups: saline control (saline, negative control), rutin control (rutin+saline), BjV+saline (BjV+saline, positive control) and BjV+rutin (BjV+rutin, treatment) and after 3, 6 and 24 h of the injection of treatments (s.c. route), blood and tissues were collected to further analyses. Envenomation induced an increase in reactive species, a decrease in total antioxidant capacity, hematological alterations (thrombocytopenia, neutrophilia and a decrease in red blood cell counts), hemostatic disturbances (hypofibrinogenemia, prolonged prothrombin time and an increase in the TF activity in plasma), and a decrease in protein expression of PDI in the heart. In vitro, rutin failed to inhibit the biological activity of the main BjV enzymes (metalloproteinases, serine proteases, phospholipases A2 and L-amino acid oxidases) and BjV-induced platelet aggregation. However, when the venom was incubated with rutin, there were reduced reactive species levels, less intense red blood cell drops, recovery of fibrinogen levels and prothrombin time, and alteration of TF activity and protein expression of TF and PDI in tissues. Therefore, we conclude that rutin favorably modulated important hemostatic and redox status alterations in B. jararaca envenomation, indicating its great potential as a complementary therapeutic agent for snakebites

Des résultats d’expériences cliniques de reconstruction de l’os maxillaire faites à partir de la greffe d’une "pâte osseuse" gélifiée par l’ajout de facteur tissulaire ont été le primum movens de ce travail. Cette "pâte osseuse", faite d’os en poudre et de plasma enrichi en plaquette (PRP) à laquelle on ajoute du facteur tissulaire, est un modèle à la fois de la coagulation et de la régénération osseuse.<p>Pour analyser des effets de la coagulation, nous avons utilisé un modèle connu :la culture primaire de cellules endothéliales (HUVEC). Les effets in vitro des facteurs de coagulation, dénommés protéases de la coagulation, pris séparément, ont été bien étudiés dans ces cellules, néanmoins aucune information sur l’effet combiné de ces protéases ou du plasma en coagulation n’était connue. Nous avons mesuré la "signalisation calcium" comme réponse cellulaire aux différents agents et ces mesures de la signalisation calcium ont été complétées par la mesure d’une autre réponse biologique, à savoir la sécrétion de cytokines pro-inflammatoires (IL-6 et IL-8). Pour l’étude de la régénération osseuse, la signalisation calcium a été mesurée sur une lignée d’ostéosarcomes humains (SaOS-2), stimulée par des protéases de la voie extrinsèque de la coagulation (facteur VIIa, facteur Xa et thrombine). Comme réponse biologique complémentaire, nous avons évalué l’effet des protéases d’intérêt sur l’apoptose induite par l’absence de sérum dans le milieu de culture.<p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Venous thromboembolism (VTE), defined as deep vein thrombosis and/or pulmonary embolism is the second leading cause of mortality in cancer patients, second only to cancer itself. A number of reports suggest that circulating extracellular vesicles (EVs) may be increased in cancer patients with VTE. The aim of this study was to examine circulating EVs in high-risk ambulatory cancer patients, determine if levels are associated with hematological outcomes (VTE, major bleeding event), and to assess the impact of prophylactic antithrombotic therapy (Apixaban). We hypothesized that elevated levels of circulating large EVs will be predictive of cancer associated VTE and/or bleeding events and that treatment with Apixaban will reduce EV levels and incidence of cancer VTE. Plasma samples from patients at baseline, and 90-days follow-up from the Apixaban for the Prevention of Venous Thromboembolism in High-Risk Ambulatory Cancer patients (AVERT) trial were investigated. Total EVs were quantified by their pro-coagulant activity using the Zymuphen MP-Activity kit. Platelet, endothelial and tissue-factor EV levels were quantified by flow cytometry. We observed that circulating EVs exhibited significant associations with sex, age, and cancer type, however we did not observe any relationships with clinical outcomes. Thus, it appears that circulating EVs may not have a role in risk stratification for VTE in in high-risk ambulatory cancer patients.

L'inhibiteur de la Voie du Facteur Tissulaire (TFPI) est une protéine régulatrice de la coagulation plasmatique intervenant à la phase initiale de la cascade. Il inhibe en présence de la protéine S (PS) le facteur Xa et ce complexe TFPI-Xa inactive ensuite le complexe FT-VIIa. Nous avons recherché une résistance à l'activité anticoagulante du TFPI. La sensibilité du plasma à une quantité fixe de TFPI a été évaluée sur la base d'un temps de thromboplastine diluée (TTD) réalisé avec et sans TFPI : - chez des patients ayant présenté une thrombose veineuse profonde inexpliquée ; cette résistance suspectée sur une 1ère étude n'a pas été confirmée sur la 2ème. - chez des patientes enceintes ; une résistance au TFPI acquise a été montrée et rapportée au déficit acquis en PS ; cependant le degré de résistance au TFPI ne peut pas être utilisé comme marqueur de risque de pathologie vasculaire placentaire. Chez des patients obèses l'effet inhibiteur des taux élevés de Lp(a) sur l'activité TFPI décrit in vitro n'a pas été retrouvé in vivo pas plus que l'effet de l'aspirine sur la normalisation des taux de Lp(a). Le TFPI joue un rôle dans les manifestations hémorragiques des hémophiles. Nous avons montré que les hémophiles B ont comparativement aux A des taux moindres de TFPI ce qui pourrait expliquer leur différence en terme de manifestations hémorragiques. Les taux de TFPI libre sont bien corrélés aux paramètres de la génération de thrombine surtout au temps de latence. En présence d’un anti TFPI humain la génération de thrombine est corrigée chez l'hémophile. Cette correction dépend de la concentration d'anti TFPI, est saturable et doit être étudiée sur du plasma riche en plaquettes / TFPI is a multivalent Kunitz-type proteinase inhibitor that directly inhibits FXa and produces FXa-dependent feedback inhibition of the FVIIa–TF complex. It was recently demonstrated that Protein S (PS) plays the role of TFPI cofactor by enhancing the TFPI inhibition of factor Xa in vivo. Approximately 80% of plasma TFPI circulates as a complex with plasma lipoproteins, about 5–20% circulating as free TFPI. Under quiescent conditions, approximately 50–80% of intravascular TFPI is stored in association with the endothelium. Full-length TFPI α carried in platelets constitutes 8-10% of the total amount of TFPI in the blood, corresponding to a quantity comparable to that of soluble full-length TFPI α in the plasma. We searched for resistance to TFPI activity in patients who presented idiopathic venous thrombosis at a young age. Plasma sensitivity to TFPI was evaluated on the basis of diluted prothrombin time (dPT) measured in patients and in control plasma in the presence (W) and absence (Wo) of exogenous TFPI. At the same time, dPT was measured on a reference plasma to establish a normalized ratio termed TFPI NR and defined as (dPT wTFPI/ dPT Wo TFPI) patient or control / (dPT wTFPI/ dPT Wo TFPI) reference plasma. In an initial study, we found that TFPI resistance could be considered as a new coagulation abnormality that could be related to unexplained thrombosis. In a second study, we failed to demonstrate a role of TFPI resistance in patients with venous thrombosis, abnormal TFPI NR being more likely related to the non-respect of preanalytical conditions rather than to an inherited trait. However, in another study, we showed that inherited or acquired PS deficiency was responsible for a TFPI resistance, providing an ex vivo demonstration that PS is the cofactor of TFPI activity. We showed that this TFPI resistance existed throughout pregnancy and that it disappeared when PS returned to normal values after delivery. We evaluated this TFPI resistance as a possible marker of the risk of a gestational vascular complication (GVC) in 72 patients at risk of developing a GVC. TFPI NR did not differ between GVC+ patients (n =15) and GVC– patients (n = 57). High levels of Lipoprotein(a) (Lp(a) have been shown to be an independent risk factor for cardiovascular disease, lowering of these levels not being achievable by any treatment except possibly aspirin. An in vitro study showed that TFPI activity could be inhibited by Lp(a). We did not confirm this TFPI inhibition in vivo in 20 obese patients with coronary insufficiency who had either normal Lp(a) levels (≤ 0.3 g/L; n = 15) or high Lp(a) levels (≥ 0.3 g/L; n = 5) . Moreover, we found no effect of aspirin treatment on Lp(a) whatever the initial level of Lp(a). Haemophilia B patients bleed less than haemophilia A patients. We showed that this difference in bleeding profile could be explained by lower free TFPI levels in haemophilia B patients compared to haemophilia A patients. In an ongoing study, we showed that in haemophilia A patients there was a strong correlation between the different parameters of thrombin generation (TG) and free TFPI. We also showed, in a TG assay performed in platelet-rich plasma (PRP) with a low TF concentration, that LT was sensitive to free TFPI levels whatever the type of haemophilia and whatever theseverity of the disease. We demonstrated that blocking TFPI by an anti-TFPI Antibody (Ab) allows complete correction of the TG profile in PRP. We showed that it is of major importance to perform a TG assay in PRP in order to evaluate the efficacy of anti-TFPI Ab in correcting TG parameters in haemophilia patients

Distúrbios de hemostasia são uma das principais manifestações clínicas observadas nos acidentes por serpentes do gênero Bothrops. Tendo em vista a importância da ativação de fatores da cascata de coagulação no desenvolvimento da patologia no envenenamento, o presente trabalho descreve o isolamento e a caracterização bioquímica e funcional de uma metaloprotease capaz de induzir a ativação de fatores de coagulação, a partir da peçonha de Bothrops moojeni. A metaloprotease foi isolada por três etapas cromatográficas utilizando colunas de exclusão molecular (Sephacryl S-200), interação hidrofóbica (Phenyl Sepharose) e troca aniônica (ES 502N). A protease isolada, denominada moojenactivase, é uma glicoproteína com massa molecular de aproximadamente 89 kDa e ponto isoelétrico de 4,92, sendo composta por três cadeias com massas de 66; 17 e 14 kDa, ligadas por pontes dissulfeto. A determinação da sequência de aminoácidos por espectrometria de massas evidenciou grande identidade sequencial com outras metaloproteases, indicando a presença dos domínios metaloprotease, desintegrina-like e lectinas-like e classificando-a como uma protease da classe PIIId. Funcionalmente, a moojenactivase foi capaz de induzir a cogulação de plasma humano pela ativação dos fatores II (protrombina) e X da cascata de coagulação, gerando -trombina e fator X ativado, respectivamente. A protease apresentou atividade fibrinogenolítica, especialmente sobre a cadeia da molécula de fibrinogênio, porém não foi capaz de induzir a formação do coágulo de fibrina pela ativação deste. A moojenactivase foi parcialmente inibida quando incubada em condições de pH entre 3,5 e 5,0 e em pH 9,0, além de temperaturas acima de 60ºC, bem como na presença de ions Cu2+, além dos inibidores EDTA, SDS, DTT e soro anti-ofídico crotalico/botrópico. A protease induziu agregação plaquetária e não apresentou atividades fibrinolítica e hemorrágica. Células mononucleares de sangue periférico (PBMC) tratadas com a protease foram capazes de produzir TNF- assim como expressar fator tecidual (Fator III da coagulação) na forma ativa, fazendo com que essas células apresentassem caráter procoagulante. Com o objetivo avaliar os efeitos nos parâmetros hematológicos in vivo, a moojenactivase foi administrada em ratos (3g/Kg) onde foi observado que a protease foi capaz de prolongar o tempo de sangramento dos animais e induzir a diminuição do número de plaquetas sanguíneas, caracterizando um quadro de trombocitopenia. Ainda, o plasma dos animais administrados com a moojenactivase apresentaram valores elevados do tempo de protrombina e tempo de tromboplastina parcialmente ativada, assim como redução na concentração de fibrinogênio. Na análise dos parâmetros da série branca, foi observado aumento leucocitário na circulação, com predominância de neutrófilos até 3h após a administração, indicando a instalação de um quadro inflamatório. Com relação à análise da série vermelha, a moojenactivase não foi capaz de alterar nenhum dos parâmetros estudados. Os resultados obtidos no presente trabalho mostram, pela primeira vez, o isolamento de uma metaloprotease da classe P-IIId da peçonha de Bothrops moojeni capaz de atuar sobre diferentes ii eventos do processo hemostático, sendo essa ação prócoagulante responsável pelo quadro de incoagulabilidade sanguínea em animais. Os dados gerados podem auxiliar no entendimento dos distúrbios de coagulação em pacientes envolvidos em acidentes por serpentes da espécie Bothrops moojeni, levando ao melhor direcionamento na terapia anti-ofídica. Ainda, a função da moojenactivase sobre componentes biológicos credencia a molécula para uma possível aplicação biotecnológica em processos que envolvem o sistema hemostático. / Haemostasis disorders are a major clinical manifestation induced by Bothrops snake envenomations. Considering the relevance of the activation of coagulation factors during the envenomation pathophysiology, the present work describes, for the first time, the isolation and functional and biochemical characterization of a coagulation factor activator metalloprotease from Bothrops moojeni snake venom. The protease was purified by three chromatographic procedures using size exclusion (Sephacryl S-200), hydrophobic interaction (Phenyl Sepharose) and anion exchange (ES 502N) chromatographies. The isolated protease, named moojenactivase, is a glycoprotein with molecular mass of approximately 89 kDa by SDS-PAGE, and composed of 66 kDa, 17 kDa and 14 kDa disulfide linked chains, with pI of 4,92. The amino acid sequence determination of tryptic peptides from moojenactivase by mass spectrometry presented fragments with high identity to snake venom metalloproteases, confirming the presence of the metalloprotease, disintegrin-like and lectin-like domains, which allowed its classification as a PIIId class snake venom metalloprotease. Regarding its functional properties, the protease was capable to induce human plasma coagulation by inducing activation of coagulation factors II and X, forming-thrombin and factor X activated, respectively. Also, moojenactivase presented fibrinogenolitic activity, by cleaving preferentially -chain of fibrinogen, however was not capable to induce the formation of fibrin clot from fibrinogen. The enzyme stability was assessed and showed that moojenactivase presented a reduced functional activity when preincubated in pH values ranging from 3,5 to 5,0 and at pH 9,0, and in temperature conditions over 60ºC. Cu2+ ions and inhibitors such as EDTA, SDS, DTT and crotalic/bothropic antiophidian serum reduced the protease activity. Moojenactivase induced platelet aggregation, but no fibrinolytic and haemorrhage activities. In order to evaluate the stimulation of peripheral blood mononuclear cells (PBMC), cells were treated with the protease and we observed the release of proinflammatory cytokine TNF- and expression of active Tissue Factor (coagulant factor III), inducing a procoagulant state on PBMC. In order to evaluate in vivo haematological effects, the protease (3 g/Kg) was administered in rat (i.v.) and was observed that moojenactivase induced a prolonged bleeding time and reduced platelet counting (indicating a thrombocytopenia state). Moreover, the evaluation of the hemostasis parameters was assessed by the the prothrombin time and activated partial thromboplastin time assays and showed a prolonged clot time on both tests, and also a decrease in fibrinogen plasma levels. The leukogram analysis showed an increase in the circulating leukocyte number up to 3 hours after moojenactivase administration, composed predominantly of neutrophils. However, parameters envolving red cells shows that the protease do not affect. The results obtained in the present work show, for the first time, the isolation of a PIIId class metalloprotease from Bothrops moojeni snake venom involved on the activation of several hemostatic events, inducing a pro coagulant activity and leading to blood unclottable state in experimental animals. These data can assit in understanding coagulation disturbs in iv patients involved in Bothrops moojeni envenomation and leading to a better anti ophidic therapy guidance. Moreover, moojenactivase functional activities accredits this protease as a possible molecular instrument applied on biotechnological prospect related to the hemostasis.

Die Schädigung des Organs durch Ischämie-Reperfusion (IR) im Rahmen der kadaverischen Organtransplantation hat bedeutenden Anteil an der Pathogenese verzögert einsetzender Organfunktion und Auswirkungen auf das Langzeitüberleben des Transplantats. In der vorliegenden Studie sollte der Einfluss unspezifischer Schädigung durch IR verglichen mit spezifischen Alloantigen-abhängigen Mechanismen während der Frühphase nach der Transplantation sowie die Auswirkungen prolongierter Aufbewahrung auf Schädigung und Immunogenität des Organs ermittelt werden. Nach vorausgegangener vierstündiger kalter Ischämiezeit wurden Organe aus syngen (Lew/Lew) und allogen (F344/Lew) transplantierten Ratten an 8 aufeinander folgenden Zeitpunkten innerhalb der ersten 10 Tage zu funktionellen, immunhistochemischen und morphologischen Veränderungen untersucht. In weiteren Gruppen wurden syngen transplantierte Organe 24 Stunden nach der Transplantation untersucht, die zuvor ansteigenden kalten Ischämiezeiten zwischen 2 und 48 Stunden ausgesetzt wurden. Im zeitlichen Verlauf zeigten sich bis 7 Tage nach der Transplantation keine wesentlichen Unterschiede zu Nierenfunktion, Morphologie, Zellinfiltration und Expression von Adhesionsmolekülen zwischen allogenen und isogenen Gruppen. Die zunächst eintretende Verschlechterung der Nierenfunktion war begleitet von einem Einstrom neutrophiler und monozytärer Zellen und morphologischen Veränderungen im Sinne von akuter Tubulusnekrose (ATN). Unter zunehmender Infiltration von Monozyten/Makrophagen kam es funktionell und morphologisch zur Regeneration. Neutrophile traten vornehmlich über Interaktion von ICAM-1/LFA-1 und Monozyten/Makrophagen über VCAM-1/VLA-4 aus dem Gefäßsystem aus. Gabe von Cyclosporin A führte zu signifikanter Reduktion ED-1-positiver Makrophagen nach 10 Tagen, ohne jedoch den Anteil des aktivierten Makrophagensubtyps ED-2 zu beeinflussen. Ansteigende kalte Aufbewahrung des Organs führte zu größerer vaskulärer Schädigung, die sich durch abnehmende Intensität und lückenhaftere Verteilung von PECAM-1 auf dem Endothel äußerte. Die Zunahme der Intensität von Tissue Factor auf Endothel und infiltrierenden Leukozyten deutete neben gesteigerter Thrombogenese auf alternative Adhäsionsmechanismen hin. Diese Ergebnisse zeigen, dass innerhalb der ersten 10 Tage nach der Transplantation wichtige Phasen der Gewebeschädigung und Regeneration ausgelöst durch die Schädigung nach IR und weitestgehend ohne Beteiligung Alloantigen-abhängiger Faktoren ablaufen. Eine bedeutende Rolle als Mediatoren während dieser Phasen kommt dabei den Monozyten/Makrophagen zu. / Organ damage due to long cold preservation is associated with delayed graft function and has important effects on graft survival. Aim of this study was to determine the impact of ischemia-reperfusion (IR) injury compared to antigen-specific mechanisms and the effect of prolonged cold ischemia on intragraft injury and antigenicity during the early phase post transplantation. Rat renal grafts were four-hours cold-preserved, transplanted to syngeneic (Lew/Lew) or allogeneic recipients (F344/Lew) and harvested at 8 different time points after transplantation for further investigation of functional, immunhistochemical and histologic changes. In five additional syngen groups organs were cold preserved from 2 hours to 48 hours and harvested after 24 hours post transplantation. No significant differences in renal function, morphologic changes, cellular infiltration and expression of adhesion molecules occurred between syngeneic and allogeneic groups within the first 7 days. Initial functional impairment was accompanied by the influx of neutrophils and monocytes/macrophages together with morphologic changes reflecting acute tubular necrosis (ATN). Increasing infiltration of monocytes/macrophages paralleled functional and morphologic regeneration. Extravasation of neutrophils was mediated mainly by interaction of ICAM-1/LFA-1 and infiltration of monocytes/macrophages by VCAM-1/VLA-4. Treatment with the standard dose of Cyclosporin A (CsA) lead to a significant decrease of ED1-positive macrophage infiltration 10 days post NTx but the portion of ED2-positive macrophage subtype was not affected. Prolonged cold organ preservation lead to more severe vascular damage indicated by decreased color intensity and continuity of PECAM-1 staining on endothelial cells. Higher staining intensity for Tissue Factor (TF) on endothelium and infiltrating leukocytes implicated enhanced intragraft procoagulant capacity and alternative adhesion mechanisms. These results show that within the first 10 days post transplantation phases of tissue injury and repair after ischemia-reperfusion are largely independent of the immunologic background and monocytes/macrophages play an important role as mediators during these processes.

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. TF is over-expressed on many malignant cells and apart from increasing the risk of thrombosis, the presence of TF/FVIIa also promotes the progression of cancer and metastasis by intracellular signaling. TF expressing microparticles (MP) are, moreover, often found in the circulation of cancer patients. Aim: The aim of this thesis was to study different aspects of TF activity, e.g. the importance of procoagulant MP and TF-induced intracellular signaling pathways, with focus on cell migration (chemotaxis) and apoptosis. Results: The TF signaling complexes were shown to prevent apoptosis induced by serum starvation and TRAIL in cancer cells by reduced activation of caspase-8 in a PI3k/AKT-dependent manner. FVIIa also decreased transcription of pro-apoptotic genes in cancer cells treated with TRAIL. Simvastatin triggered apoptosis by transcriptional reduction of BCL-2 due to cytosolic retention of NFκB. Simvastatin also inactivated the PI3k/AKT pathway and reduced the production of the MP-like prostasomes which, respectively, impaired the anti-apoptotic signaling by TF and reduced the procoagulant activity in the vicinity of prostate cancer cells. Intracellular events conducted by the TF/FVIIa complex selectively enhanced PDGF-BB induced chemotaxis which was partly explained by the TF/FVIIa-induced transactivation of the PDGFβ-receptor. This was dependent on Src-family members and engagement of PAR2. Conclusions: The results presented in this thesis extend the current knowledge of TF-mediated signaling. We report the TF complexes to govern the extrinsic pathway of apoptosis, present data on FVIIa-dependent regulation of apoptosis-related genes, and exclude known surface proteins as transmitters of the anti-apoptotic signals. We moreover describe TF/FVIIa to transactivate the PDGFβ-receptor and play a decisive role in the potentiated chemotaxis toward PDGF-BB in a number of cell types. Finally, we explain the mechanism behind simvastatin-induced apoptosis in cancer cells and how statins interfere with TF-dependent signaling and coagulation.

Distúrbios de hemostasia são uma das principais manifestações clínicas observadas nos acidentes por serpentes do gênero Bothrops. Tendo em vista a importância da ativação de fatores da cascata de coagulação no desenvolvimento da patologia no envenenamento, o presente trabalho descreve o isolamento e a caracterização bioquímica e funcional de uma metaloprotease capaz de induzir a ativação de fatores de coagulação, a partir da peçonha de Bothrops moojeni. A metaloprotease foi isolada por três etapas cromatográficas utilizando colunas de exclusão molecular (Sephacryl S-200), interação hidrofóbica (Phenyl Sepharose) e troca aniônica (ES 502N). A protease isolada, denominada moojenactivase, é uma glicoproteína com massa molecular de aproximadamente 89 kDa e ponto isoelétrico de 4,92, sendo composta por três cadeias com massas de 66; 17 e 14 kDa, ligadas por pontes dissulfeto. A determinação da sequência de aminoácidos por espectrometria de massas evidenciou grande identidade sequencial com outras metaloproteases, indicando a presença dos domínios metaloprotease, desintegrina-like e lectinas-like e classificando-a como uma protease da classe PIIId. Funcionalmente, a moojenactivase foi capaz de induzir a cogulação de plasma humano pela ativação dos fatores II (protrombina) e X da cascata de coagulação, gerando -trombina e fator X ativado, respectivamente. A protease apresentou atividade fibrinogenolítica, especialmente sobre a cadeia da molécula de fibrinogênio, porém não foi capaz de induzir a formação do coágulo de fibrina pela ativação deste. A moojenactivase foi parcialmente inibida quando incubada em condições de pH entre 3,5 e 5,0 e em pH 9,0, além de temperaturas acima de 60ºC, bem como na presença de ions Cu2+, além dos inibidores EDTA, SDS, DTT e soro anti-ofídico crotalico/botrópico. A protease induziu agregação plaquetária e não apresentou atividades fibrinolítica e hemorrágica. Células mononucleares de sangue periférico (PBMC) tratadas com a protease foram capazes de produzir TNF- assim como expressar fator tecidual (Fator III da coagulação) na forma ativa, fazendo com que essas células apresentassem caráter procoagulante. Com o objetivo avaliar os efeitos nos parâmetros hematológicos in vivo, a moojenactivase foi administrada em ratos (3g/Kg) onde foi observado que a protease foi capaz de prolongar o tempo de sangramento dos animais e induzir a diminuição do número de plaquetas sanguíneas, caracterizando um quadro de trombocitopenia. Ainda, o plasma dos animais administrados com a moojenactivase apresentaram valores elevados do tempo de protrombina e tempo de tromboplastina parcialmente ativada, assim como redução na concentração de fibrinogênio. Na análise dos parâmetros da série branca, foi observado aumento leucocitário na circulação, com predominância de neutrófilos até 3h após a administração, indicando a instalação de um quadro inflamatório. Com relação à análise da série vermelha, a moojenactivase não foi capaz de alterar nenhum dos parâmetros estudados. Os resultados obtidos no presente trabalho mostram, pela primeira vez, o isolamento de uma metaloprotease da classe P-IIId da peçonha de Bothrops moojeni capaz de atuar sobre diferentes ii eventos do processo hemostático, sendo essa ação prócoagulante responsável pelo quadro de incoagulabilidade sanguínea em animais. Os dados gerados podem auxiliar no entendimento dos distúrbios de coagulação em pacientes envolvidos em acidentes por serpentes da espécie Bothrops moojeni, levando ao melhor direcionamento na terapia anti-ofídica. Ainda, a função da moojenactivase sobre componentes biológicos credencia a molécula para uma possível aplicação biotecnológica em processos que envolvem o sistema hemostático. / Haemostasis disorders are a major clinical manifestation induced by Bothrops snake envenomations. Considering the relevance of the activation of coagulation factors during the envenomation pathophysiology, the present work describes, for the first time, the isolation and functional and biochemical characterization of a coagulation factor activator metalloprotease from Bothrops moojeni snake venom. The protease was purified by three chromatographic procedures using size exclusion (Sephacryl S-200), hydrophobic interaction (Phenyl Sepharose) and anion exchange (ES 502N) chromatographies. The isolated protease, named moojenactivase, is a glycoprotein with molecular mass of approximately 89 kDa by SDS-PAGE, and composed of 66 kDa, 17 kDa and 14 kDa disulfide linked chains, with pI of 4,92. The amino acid sequence determination of tryptic peptides from moojenactivase by mass spectrometry presented fragments with high identity to snake venom metalloproteases, confirming the presence of the metalloprotease, disintegrin-like and lectin-like domains, which allowed its classification as a PIIId class snake venom metalloprotease. Regarding its functional properties, the protease was capable to induce human plasma coagulation by inducing activation of coagulation factors II and X, forming-thrombin and factor X activated, respectively. Also, moojenactivase presented fibrinogenolitic activity, by cleaving preferentially -chain of fibrinogen, however was not capable to induce the formation of fibrin clot from fibrinogen. The enzyme stability was assessed and showed that moojenactivase presented a reduced functional activity when preincubated in pH values ranging from 3,5 to 5,0 and at pH 9,0, and in temperature conditions over 60ºC. Cu2+ ions and inhibitors such as EDTA, SDS, DTT and crotalic/bothropic antiophidian serum reduced the protease activity. Moojenactivase induced platelet aggregation, but no fibrinolytic and haemorrhage activities. In order to evaluate the stimulation of peripheral blood mononuclear cells (PBMC), cells were treated with the protease and we observed the release of proinflammatory cytokine TNF- and expression of active Tissue Factor (coagulant factor III), inducing a procoagulant state on PBMC. In order to evaluate in vivo haematological effects, the protease (3 g/Kg) was administered in rat (i.v.) and was observed that moojenactivase induced a prolonged bleeding time and reduced platelet counting (indicating a thrombocytopenia state). Moreover, the evaluation of the hemostasis parameters was assessed by the the prothrombin time and activated partial thromboplastin time assays and showed a prolonged clot time on both tests, and also a decrease in fibrinogen plasma levels. The leukogram analysis showed an increase in the circulating leukocyte number up to 3 hours after moojenactivase administration, composed predominantly of neutrophils. However, parameters envolving red cells shows that the protease do not affect. The results obtained in the present work show, for the first time, the isolation of a PIIId class metalloprotease from Bothrops moojeni snake venom involved on the activation of several hemostatic events, inducing a pro coagulant activity and leading to blood unclottable state in experimental animals. These data can assit in understanding coagulation disturbs in iv patients involved in Bothrops moojeni envenomation and leading to a better anti ophidic therapy guidance. Moreover, moojenactivase functional activities accredits this protease as a possible molecular instrument applied on biotechnological prospect related to the hemostasis.

La greffe d’îlots pancréatiques est caractérisée par une réponse inflammatoire et procoagulante précoce, connue sous le nom d’IBMIR (Instant Blood Mediated Inflammatory Reaction). Les microparticules (MPs) porteuses de facteur tissulaire (TF) sont le témoin d’un important remodelage membranaire et constituent des acteurs centraux dans la dissémination du stress de l’IBMIR. Nous avons exploré l’effet d’un stress inflammatoire sur la survie et la fonction de la cellule β dans un modèle de communication cellulaire médiée par les MPs. La modulation pharmacologique par les analogues du GLP-1 a été évaluée, la par la mesure de la sécrétion d’insuline, de l’activité TF et l'analyse du remodelage de la membrane plasmique. Nos résultats décrivent les MPs comme des effecteurs autocrines et indiquent que les MPs sont des cibles potentielles pour les analogues du GLP-1 au cours de l'IBMIR. Les données apportent de nouvelles pistes sur les mécanismes cellulaires mis en jeu lors des phénomènes d’ischémie reperfusion durant l’IBMIR. / Pancreatic islets graft is characterized by early inflammatory and procoagulant events known as Instant Blood Mediated Inflammatory Reaction (IBMIR). Tissue factor (TF) bearing microparticles (MPs) are surrogates of important membrane remodeling and key players in the systemic and local dissemination of such stress.We investigated the effect of inflammatory stress on β cell survival and function in a MP-mediated cell crosstalk model. Pharmacological modulation by GLP-1 analogues was evaluated by measurement of insulin secretion, TF activity and assessment of plasma membrane remodeling. Our data evidenced MPs as autocrine effectors and possible new target for GLP-1 analogues. They bring new hints on the cellular mechanisms prompted by ischemia reperfusion during IBMIR.