Bielefeld, Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Bielefeld, Universiẗat, Diss., 2001.

Antithrombin (AT) is a plasma serine protease inhibitor that regulates thrombin and other activated clotting factors in the intrinsic and common pathways of coagulation. As the most abundant coagulation pathway inhibitor, AT serves to maintain balance in the coagulation system by inducing an anticoagulant effect. The importance of AT is evident in cases where deficiencies of AT lead to increased risk of venous thromboembolic disease. Since an AT antagonist would be considered a procoagulant, inactivating AT could provide a novel approach to restoring hemostasis in patients with inherited or acquired bleeding disorders. In vitro selection is a powerful tool used to screen large combinatorial oligonucleotide libraries against a target molecule or protein. By employing this technique in combination with high-throughput screening, we identified novel RNA aptamer candidates capable of binding AT with high affinity and inhibiting AT’s inactivation of its main target protease, thrombin. Kinetic characterization of the most abundant aptamer candidates showed a reduction in AT-mediated thrombin inhibition of 50-60%. The most inhibitory aptamer, R6_15, mediated a decrease of AT’s second-order rate constant from 2.37 ± 0.06 x 104 to 1.57 ± 0.02 x 104 M-1 s-1 (mean ± SD) The interaction between the aptamers and AT was also measured in human plasma. In a clotting assay, aptamer R6_15 accelerated clotting time by approximately 7 seconds (from 44.3 ± 0.8 to 37.3 ± 0.7 seconds). This difference in clotting time was the greatest noticed among all the other aptamer candidates. By measuring the change in AT’s fluorescence intensity, we were able to determine the aptamers’ binding capacity. The binding affinities (kd) of aptamers R6_15 and R6_19 were 65.3 ± 8.7 and 67.5 ± 14.5 nM, respectively. Truncation of R6_15 on either its 5’ or 3’ end did not increase its inhibitory activity or binding affinity towards AT. By pairing the selection data with dynamic molecular modelling, the interface of aptamer R6_15 to AT was predicted to be at the site of heparin binding, specifically at residue K114. Although these computer-generated results are not conclusive, they provide a testable hypothesis for future experimentation. Ultimately, this work provides evidence that the application of in vitro evolution has yielded a novel anti-serpin aptamer. With some modifications, the selection protocol employed in this study could be revisited to identify tighter binders and more potent inhibitors of AT. Either aptamer R6_15 or a future higher affinity AT-binding aptamer could be tested for its efficacy in reducing bleeding in vivo using mouse models of acquired hemophilia or traumatic bleeding. / Thesis / Master of Science (MSc)

Thesis (doctoral)--Universität Hamburg, 1979.

One of the most fundamental aspects of extracorporeal membrane oxygenation (ECMO) is maintaining proper anticoagulation management in order to prevent hemorrhagic or thrombotic events. Anticoagulation on ECMO is most commonly achieved with the use of unfractionated heparin to maintain a minimum anticoagulation level as monitored by activated clotting time (ACT). Heparin's main effect is exerted by binding to and potentiating antithrombin III. Many factors may contribute to a sub-therapeutic ATIII level that may decrease the effectiveness of heparin. A retrospective record review was performed on all adult ECMO patients at the University of Arizona Medical Center between 2008 and 2011, in order to determine optimal ATIII levels for maintaining proper anticoagulation. In addition, we investigated correlations between ATIII levels and hemorrhagic and/or thrombotic events. Variables measured include, ACTs, heparin dose, ATIII dose, ATIII levels, blood product use, and adverse events. Thirty-five patients received ATIII over the course of the ECMO run. Six patients did not receive ATIII and they were found to have used significantly more blood products than those who did receive ATIII. Also, heparin dose dropped significantly 24h after the first dose of ATIII. There is a significant positive correlation between the amount of ATIII given per day and the amount of packed red blood cells transfused per day. The results suggest an ideal therapeutic range of ATIII dosing, where lack of or too much ATIII administration can lead to excessive bleeding.

Zugl.: Jena, Univ., Diss., 2006 u.d.T.: Hagel, Stefan: Protein-C- und Antithrombin-III-Aktivität / Hergestellt on demand

A antitrombina de B. jararaca foi isolada por meio de cromatografia de afinidade em coluna HiTrap Heparin HP (GE Healthcare). A análise da interação da antitrombina humana ou de B. jararaca com a heparina em sistema BIAcoreT200 demonstrou que a antitrombina de B. jararaca apresenta maior afinidade pela heparina que a antitrombina humana. Com relação à sua atividade anti-inflamatória, os resultados obtidos evidenciaram o efeito anti-inflamatório do pré e do pós-tratamento com a antitrombina de B. jararaca na resposta inflamatória aguda. A análise proteômica do exsudato inflamatório de camundongos identificou algumas proteínas possivelmente relacionadas ao mecanismo de inibição da antitrombina, como a enzima C3 do sistema complemento, a sorotransferrina, a a1-antitripsina, a apolipoproteína AI, o fibrinogênio, o cininogênio e a albumina. O processo de clonagem permitiu a obtenção da sequência completa da antitrombina de B. jararaca e apesar da longa distância evolutiva entre serpentes e humanos, diversas características da antitrombina encontram-se conservadas. / B. jararaca antithrombin was isolated by affinity chromatography using HiTrap Heparin HP column (GE Healthcare). The interaction analysis of human or B. jararaca antithrombin with heparin using a BIAcoreT200 system (GE Healthcare) demonstrated that the affinity of B. jararaca antithrombin for heparin is higher than human antithrombin. Regarding the anti-inflammatory activity of B. jararaca antithrombin, the results showed the anti-inflammatory effect of pre- and post-treatment with this molecule in acute inflammation. The proteomic analysis of inflammatory exudate of mice identified some proteins possibly related to the mechanism of inhibition of antithrombin, such as C3 complement, serum transferrin, a1-antitrypsin, apolipoprotein AI, fibrinogen, albumin and kininogen. The molecular cloning process allowed the determination of the complete sequence of B. jararaca antithrombin and despite the evolutionary distance between snakes and human, a number of characteristics are preserved in antithrombin molecule.

O presente trabalho teve como objetivo caracterizar o desenvolvimento da semente de Araucaria angustifolia através da proteômica comparativa, buscando compreender as alterações fisiológicas e metabólicas que ocorrem durante esse processo. Inicialmente, foram avaliados três diferentes metodologias de extração de proteínas. A metodologia composta por solução de extração contendo 7 M de uréia, 2 M de tiouréia, 1% de ditiotreitol, 2% de Triton-100, 1 mM de fluoreto de fenilmetilsulfonil e 5 µM de pepstatina, seguido de precipitação em 20% de ácido tricloroacético apresentou géis de maior resolução e reprodutibilidade, tendo sido escolhida como metodologia de extração protéica para o estudo das alterações no proteoma da semente de A. angustifolia. Uma dificuldade associada ao estudo do proteoma de espécies não sequenciadas é a baixa representatividade nos bancos de dados protéicos, resultando em identificações baseadas em homologia. Estratégias proteômicas baseadas em fracionamento em gel resultam em grandes contaminações por fragmentos de queratina. Sendo assim, foi desenvolvido um programa de remoção de espectros de baixa qualidade para utilização em proteômica baseada em homologia. As análises mostraram que o programa reduz o tempo de busca, melhora a qualidade dos alinhamentos e não resulta em perda de identificações positivas. Finalmente, utilizando as metodologias descritas, foram estudadas as alterações no proteoma durante o desenvolvimento da semente de A. angustifolia. Noventa e seis proteínas foram identificadas e agrupadas de acordo com sua função biológica e padrão de detecção. Os resultados obtidos permitiram o estabelecimento de marcadores protéicos no início e final do desenvolvimento embrionário. A análise das proteínas abundantes no início da embriogênese indica um maior controle no metabolismo oxidativo em relação aos estádios finais. Contrariamente, o final da embriogênese é caracterizado por um alto metabolismo de assimilação de carbono e acúmulo de proteínas de reserva. As implicações dos resultados obtidos no controle e melhoramento de sistemas de embriogênese somática na espécie também foram discutidas / The aim of the present work was to characterize the seed development of Araucaria angustifolia through proteomics in order to understand the physiological and biochemical changes during this process. For that, initially, three different protein extraction methods were evaluated. The extraction based on protein solubilization in 7 M urea, 2 M thiourea, 1% dithiothreitol, 2% Triton-100, 1 mM phenylmethylsulphonyl fluoride, 5 µM pepstatin, followed by 20% trichloroacetic acid precipitation showed the highest gel resolution and reprodutivity and, thus, was chosen to be used in the analysis of the proteome of A. angustifolia seeds. One aspect that hampers the proteome study of unsequenced species is the low protein representativity in databases. So, protein identification is usually carried out through homology. Strategies based on 2-DE result in high keratin contamination. In the present work a spectra filtering software was developed and evaluated for use in homology driven proteomics. The software reduced the time of search, improved alignment quality and did not result in lost of positive identifications. Finally, using the described strategies, the changes in the proteome of A. angustifolia seeds were studied. Ninety six proteins were identified and classified according to their biological functions and expression profiles during seed development. The identified proteins may be used as protein markers of early and late embryogenesis. Proteins involved in the control of oxidative metabolism were highly expressed during the early stages of seed development; while, carbon metabolism and storage proteins were highly expressed in late stages. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed.

Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.

<p>Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.</p>