Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Maastricht. / Auteursnaam op omslag: Jan L.M.L. van Rijn. Lit.opg. - Samenvatting in het Nederlands.

MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / Factor X deficiency is a rare bleeding disorder with an incidence of one in a million in the general population. Patients with the severe form of factor X deficiency suffer from serious bleeds occurring mainly into the joints and the muscle. In the two factor X deficient families currently looked after at the Haemophilia Comprehensive Care Centre, there are definite differences in the bleeding tendencies between and within family members. We hypothesize the differences in genetic mutations and the influence of cytokines to be responsible for these bleeding variabilities. These factors were explored in our study. The study population included a total of fourteen members of the two families with factor X deficiency. Blood for factor X measurement, cytokine studies and genetic studies was collected in the Haemophilia Comprehensive Care Centre of the Charlotte Maxeke Johannesburg Academic Hospital. Each blood was processed according to the test to be performed. Factor X activity levels were measured using the factor X assay, and the information on each patient’s bleeding episodes was obtained from the Haemophiliac Clinic database. Cytokines were analyzed in all patients using the ELISA kits from Biosource. Factor X gene was amplified using PCR and sequenced with Spectrumedix SCE 2410. iv For cytokine studies, high levels of IL-1beta and TNF-alpha were observed in frequent bleeding patients compared to infrequent bleeders. These cytokines are known to be involved in acute inflammatory process leading to cellular infiltrate and joint swelling. This results in synovitis and the creation of massive joint bleeding. The low levels of IL-1beta and TNF-alpha detected in infrequent bleeding patients appear to be related to the high levels of IL-1Ra and IL-10. These anti-inflammatory cytokines are known to inhibit the inflammatory synovitis and lessen the severity of joint bleeding. For genetic studies, differences were observed between the amino acid sequence of the three frequent bleeding patients and the consensus. In addition, a novel mutation Cys350Phe was detected in two of these patients. This mutation is characterized by very low factor X levels which sometimes are not detectable in circulation. The substituted cystine is known to cause defect in the substrate binding, leading to the lost of enzyme activity. From these findings we have concluded that the origin of the heterogeneity of bleeding in factor X deficiency is multifactorial, cytokines and genetic mutations seems to have a role in determining the clinical manifestations of the factor X deficient patients.

Alzheimer’s disease is neurodegenerative with amyloid plaque and neurofibrillary tangles as pathological hallmarks. The most abundant component in the amyloid plaque is the amyloid-β (Aβ) peptide, with presence of both isoform Aβ40 and Aβ42. In immunological methods studying the Aβ peptide a specific monoclonal antibody, 6E10, is routinly being used. In this master thesis work unspecific binding of 6E10 antibody to the blood coagulating protein factor X has been investigated. Factor X is a protein in the blood coagulation cascade where it forms protein complex that activates thrombin. Non-hemostatic functions with connections to nerves and Aβ peptide are also known. Studies with Western blot show clear binding of 6E10 to denatured factor X. Interaction studies with ELISA gives uncertain results, where binding is found but no clear binding curve is obtained. Studies with native factor X in real time measurements with SPR gave no binding at all. These results suggest binding to denatured factor X. Immunohistochemistry studies of colocalisation of factor X and Aβ peptide gave clear evidence that factor X and Aβ are found near each other in mouse brain tissue. Factor X is located outside the blood vessels and Aβ is located at the inside.

Thirty thousand colonies of a bovine liver cDNA library were screened with a mixture of synthetic oligodeoxyribonucleotides coding for bovine factor X. Five positive colonies were identified, and plasmid DNA was isolated. Cleavage with restriction endonucleases showed that these plasmids (designated pBXl-5) contained inserts of 1530 bp, 770 bp, 700 bp, 1100 bp and 930 bp. DNA sequence analysis of the plasmid with the largest insert (pBXl) confirmed that bovine factor X cDNAs had been cloned. The cDNA sequence predicts that factor X is synthesized as a single chain precursor in which the light and heavy chains of plasma factor X are linked by the dipeptide Arg-Arg. The cDNA sequence also predicts that factor X is synthesized with a preproleader peptide. It is proposed that at least five specific proteolytic events occur during the conversion of preprofactor X to plasma factor Xa. A human liver cDNA library was screened by colony hybridization with a bovine factor X cDNA probe. Three of the positive plasmids contained overlapping DNA that coded for most of human factor X mRNA. A second human liver cDNA library was screened by in situ hybridization with 32P-labeled human factor X cDNA clones obtained from the first screen. Several clones were isolated that contained longer inserts. DNA sequence analysis of these clones allowed the prediction of the amino acid sequence of the precursor form of human plasma factor X. From these studies, it is predicted that human factor X is synthesized as a single polypeptide chain precursor in which the light and heavy chains of plasma factor X are linked by the tripeptide Arg-Lys-Arg. The cDNA sequence also predicts that human factor X is synthesized as a preproprotein having an aminoterminal leader peptide of 40 amino acid residues. A comparison of the amino acid sequences of human and bovine factor X shows high sequence identity around the calcium- binding regions and catalytic regions but low sequence identity around the nonfunctional regions. A human genomic phage library was screened with a human factor X cDNA as a hybridization probe. Thirty-two overlapping phage clones were isolated. Characterization of six of these clones indicates that over 32 Kbp of contiguous sequence is represented. DNA sequence and restriction map analysis shows that the factor X gene is comprised of at least 8 exons and 7 introns. No clones representing the 5' untranslated region and the prepeptide of the leader sequence were identified. Two further genomic phage libraries and two libraries specific for the 5' region of the factor X gene were screened, but no 5' end clones were obtained. Restriction enzyme mapping and Southern blot analysis indicate that thus far, the human factor X gene maps to 24 Kbp of the human genome. Comparison of the factor X gene with other vitamin K-dependent blood coagulation factor genes reveals homologous exon organization. Within the blood coagulation serine proteases factor X, factor IX, factor VII, and protein C form a closely related gene family. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Les adénovirus (Ad) sont parmi les vecteurs les plus utilisés en thérapie génique. Cependant, les données d’essais pré-Cliniques et cliniques ont montré qu’ils induisaient une forte toxicité hépatique consécutive à leur tropisme hépatique, une réponse inflammatoire et une forte thrombocytopénie. Différents travaux avaient montré que l’interaction de l’Ad avec le facteur de la coagulation X (FX).était responsable de la transduction in vivo des hépatocytes après administration systémique des vecteurs Ad. Cependant, des résultats précédents du laboratoire avaient montré également que le pseudotypage d’une autre protéine de capside, la fibre, permettait de réduire la transduction hépatique. Dans le but de mieux comprendre le rôle de la fibre dans le tropisme et la toxicité des Ad, nous avons comparé des Ad recombinants pseudotypés pour tout (tige et tête de la fibre : AdF3) ou partie (tige : AdS3K5) de la fibre Ad3 avec un Ad5 à capside non modifiée (Adwt). Après administration systémique chez la souris, l’AdF3 et l’AdS3K5 induisent une plus faible expression du transgène dans le foie et la rate comparativement à l’Ad5wt. Cette réduction ne résulte ni d’un défaut de capture de ces vecteurs dans le foie ni de leur incapacité à utiliser le FX. Cependant, nos résultats ont révélé que les Ad pseudotypés par la fibre Ad3 étaient capturés de façon plus importante par les cellules de Kupffer. Nous avons montré que cette capture était une propriété intrinsèque de la fibre Ad3 puisqu’elle était observée également après administration systémique d’un Ad de sérotype 3. De façon intéressante, les Ad pseudotypés par la fibre Ad3 restent capables de transférer des gènes dans les tumeurs aussi efficacement que l’Adwt.Dans la deuxième partie de nos travaux, nous avons cherché à mieux comprendre les mécanismes de la thrombocytopénie consécutive à l’administration d’Ad. Nous avons défini la cinétique de la thrombocytopénie ainsi que l’effet de la dose virale. Nous avons montré que certains facteurs de l’hôte comme les facteurs de la coagulation ou la rate n’étaient pas impliqués dans la thrombocytopénie. De façon intéressante, nous avons montré que la fibre Ad5 jouait un rôle dans l’induction de la baisse plaquettaire puisque l’administration des virus à fibre Ad3 n’induisait plus de forte baisse plaquettaire. Parallèlement, nous avons observé un profil inflammatoire associé à l’administration des Ad à fibre modifiée beaucoup plus réduit que celui de l’Adwt. Nos travaux en cours évaluent l’existence possible d’une corrélation entre la production de cytokines/chimiokines et la thrombocytopénie.L’ensemble de ces résultats montre que le pseudotypage des Ad5 par la fibre de l’Ad3 permet de réduire leur toxicité et de limiter la réponse inflammatoire tout en conservant un transfert de gènes efficace dans les tumeurs. L’introduction de ce type de modification de capside dans les Ad oncolytiques devrait permettre de conserver leur capacité à se répliquer dans les tumeurs tout en limitant les toxicités liées à leur dissémination par voie systémique. / To date adenoviruses (Ad) are the most used vectors in gene therapy. However, Ad use is hampered by a strong liver tropism that leads to hepatotoxicity, a strong inflammatory response and the induction of thrombocytopenia. Binding of Ad hexon to coagulation factor X (FX) is responsible for hepatocyte transduction in vivo. As a consequence, mutation of hexon protein abrogates Ad interaction with FX and reduces liver transduction. However, previous results of our lab have demonstrated that Ad5 pseudotyping with fiber Ad3 also resulted in significant reduction of liver transduction. To understand how fiber modification affects in vivo Ad tropism, we used two pseudotyped viruses with whole (AdF3) or only the shaft (AdS3K5) of Ad3 fiber.Following systemic delivery of fiber-Modified Ads, a reduced transduction was observed 2 days p.i. in liver and spleen. This reduction was not due to the impairment of fiber-Modified Ads liver entry or FX use in vivo. Remarkably, after Kupffer cells depletion, a restored transgene expression level was observed, suggesting that fiber-Modified Ads are strongly uptaken by Kupffer cells. We have demonstrated that this strong uptake is an Ad3 intrinsic property since Ad3 was also strongly uptaken by Kupffer cells. Interestingly, fiber-Modified Ads transduce tumours as efficiently as Ad5. In the second part of this work, we aimed to better understand the mechanism of Ad-Induced thrombocytopenia. We first defined the kinetic and dose-Dependence of Ad-Induced thrombocytopenia. Then, we have shown that factors of the host such as the coagulation factors and the spleen were not involved in the thrombocytopenia development. Interestingly, we demonstrated o role for Ad5 in this platelet count reduction since fiber-Modified Ad induced only a modest thrombocytopenia. In parallel, we have observed a reduced production of inflammatory cytokine and chemokine following fiber-Modified Ad administration. Experiments are ongoing to investigate a possible correlation between inflammatory responses and thrombocytopenia. Altogether, our findings demonstrate that Ad5 pseudotyping with Ad3 fiber allows à reduced toxicity and inflammatory response while tumour transduction efficacy is remained. Therfore, oncolytic Ad pseudotyped with Ad3 fiber might be potent tool in tumor virotherapy while limiting risk of toxicity.

Proteins containing Kunitz domain are mostly inhibitors of serine proteases. Their general characteristic is the presence of three disulfide bonds and small sizes around 6-10 kDa, although sometimes they consist of several Kunitz domains or they are part of more complex proteins. Their function is usually related to the regulation of physiological and proteolytic processes, but also to an interaction with pathogens or other defense mechanisms, such as being part of the sea anemone mucus or the venom of snakes and other invertebrates. We focused on Kunitz proteins in Eudiplozoon nipponicum, a helminth of the class Monogenea parasiting on gills of common carp (Cyprinus carpio). In the transcriptome of this parasite, several sequences with Kunitz domain have been identified based on similarities with the one already described Kunitz protein, EnKT1, suggesting that this parasite, like other bloodfeeding parasites, uses a whole set of these serine protease inhibitors with other specific functions. Several sequences with the Kunitz domain found in the transcriptome were verified by PCR and optionally supplemented by RACE-PCR. One protein, called EnKC1, was subsequently produced by recombinant expression in E. coli cells of SHuffleTM and Rosetta Gami B strains. Recombinant protein with the Kunitz domain...

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.

No description available.

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.