Factor XI (FXI) is the zymogen of the coagulation protease factor XIa (FXIa) that contributes to thrombin generation through the intrinsic pathway. FXI is activated by the contact pathway protease, factor XIIa (FXIIa), in a high molecular weight kininogen-dependent manner. It is also known to be activated by thrombin in a positive feedback reaction, however, the mechanism of this activation is not yet completely understood. Therefore, our objectives were to identify the role of polyanions in the thrombin mediated FXI activation, and the role of the thrombin exosites in the activation. To study this activation, we assessed the activation of FXI by thrombin in the presence and absence of the polyanions, dextran sulfate and polyphosphate (polyP). We utilized surface plasmon resonance to determine whether FXI and thrombin bind to the polyanions, and how the exosites effect thrombin’s ability to bind using thrombin exosite 1 and 2 variants. To investigate the role of the exosites in FXI activation, we analyzed the activation of FXI by the thrombin variants. In addition, we explored how inhibiting the thrombin exosites using DNA aptamers affects thrombin’s ability to bind to polyanions and activate FXI. We found that polyanions are required as a cofactor for the activation of FXI by thrombin, and stimulate the activation in a concentration dependent fashion, suggesting a template mechanism. Our findings also show that exosite 1 and 2 are required for thrombin to bind to polyanions, however, exosite 2 plays the predominant role in FXI activation. Our aptamer data showed that either exosite can be targeted to inhibit FXI activation. These findings enrich our understanding of the mechanism of FXI activation by thrombin and provides further insight on how to attenuate the activation for potential antithrombotic therapies. / Thesis / Master of Science (MSc)

A disponibilidade de hemoderivados é um parâmetro importante para medir a qualidade da saúde em um país. Dentre os produtos hemoderivados, imunoglobulinas tem alto valor agregado. O Instituto Butantan tem por objetivo o estabelecimento de uma planta industrial para fracionamento de plasma, com um processo produtivo baseado principalmente em cromatografias. Eventos tromboembólicos a partir de infusões de imunoglobulinas por via intravenosa (IgIV) foram relacionados com presença de Fator XI de coagulação (FXI) como contaminantes nas preparações de IgIVs. Com objetivo de detectar o FXI nas frações, o processo cromatográfico foi testado em escala piloto, bancada, e em cromatografia direta e o FXI foi dosado nas frações iniciais e no produto final IgIV. Foram estabelecidos os métodos de dosagem de atividade de FXI por tempo de coagulação e ensaio cromogênico. Concluímos que o FXI acompanha a IgG nas etapas iniciais dos processos cromatográficos e verificamos que ocorre a presença de FXI nos produtos finais. Este trabalho contribui para o desenvolvimento do conjunto de testes de controle de qualidade de biofármacos derivados de plasma humano. / The availability of hemoderivatives is an important parameter to measure a countrys health quality. Among hemoderivatives products, immunoglobulin have high value.. Instituto Butantan, aims the establishment of an industrial plant for plasma fractionation with a process drawn, based mainly in chromatographies. Thromboembolic events from infusions of intravenous immunoglobulins (IVIg) have been related with the presence of coagulation Factor XI (FXI) as contaminant in the IVIG preparations. With an objective of tracking FXI in the chromatographic fractions, the process was tested in pilot and bench scales and in direct chromatography and the FXI was measured in the initial fractions and in the final product IVIg. The methods of measurement of FXI activity thru coagulation time and chromogenic assay were established. We have concluded that FXI accompanies IgG in the early stages of the chromatographic processes and verified that the presence occurs in the final products, This work contributes to the development of the set of quality control tests of biopharmaceuticals derivatives from human plasma.

A disponibilidade de hemoderivados é um parâmetro importante para medir a qualidade da saúde em um país. Dentre os produtos hemoderivados, imunoglobulinas tem alto valor agregado. O Instituto Butantan tem por objetivo o estabelecimento de uma planta industrial para fracionamento de plasma, com um processo produtivo baseado principalmente em cromatografias. Eventos tromboembólicos a partir de infusões de imunoglobulinas por via intravenosa (IgIV) foram relacionados com presença de Fator XI de coagulação (FXI) como contaminantes nas preparações de IgIVs. Com objetivo de detectar o FXI nas frações, o processo cromatográfico foi testado em escala piloto, bancada, e em cromatografia direta e o FXI foi dosado nas frações iniciais e no produto final IgIV. Foram estabelecidos os métodos de dosagem de atividade de FXI por tempo de coagulação e ensaio cromogênico. Concluímos que o FXI acompanha a IgG nas etapas iniciais dos processos cromatográficos e verificamos que ocorre a presença de FXI nos produtos finais. Este trabalho contribui para o desenvolvimento do conjunto de testes de controle de qualidade de biofármacos derivados de plasma humano. / The availability of hemoderivatives is an important parameter to measure a countrys health quality. Among hemoderivatives products, immunoglobulin have high value.. Instituto Butantan, aims the establishment of an industrial plant for plasma fractionation with a process drawn, based mainly in chromatographies. Thromboembolic events from infusions of intravenous immunoglobulins (IVIg) have been related with the presence of coagulation Factor XI (FXI) as contaminant in the IVIG preparations. With an objective of tracking FXI in the chromatographic fractions, the process was tested in pilot and bench scales and in direct chromatography and the FXI was measured in the initial fractions and in the final product IVIg. The methods of measurement of FXI activity thru coagulation time and chromogenic assay were established. We have concluded that FXI accompanies IgG in the early stages of the chromatographic processes and verified that the presence occurs in the final products, This work contributes to the development of the set of quality control tests of biopharmaceuticals derivatives from human plasma.

The clinical management of Factor XI (FXI) deficiency is problematic due to the marked phenotypic heterogeneity between individuals with this disorder and the lack of a reliable test to predict bleeding risk. FXI-deficient individuals are currently at risk of being over- or under treated, with associated risks of transfusion-related complications or haemorrhage respectively. The improvement of care of FXI-deficient patients requires the development of measures that can predict bleeding phenotype and enable the identification of individuals who need treatment at times of haemostatic challenge. In addition, for those requiring treatment, there is a need for development of tests which can determine the optimal type and dose of FXI replacement on an individually tailored basis, as well as assays which can accurately monitor the effect of treatment and guide clinicians in the requirement for further perioperative treatment. This thesis addresses these objectives by studying global haemostasis assays and bleeding scores as tools to predict bleeding tendency and by studying the utility of global haemostasis assays as potential tests by which FXI replacement treatment can be determined and monitored. For prediction of bleeding tendency, this research demonstrated that the thrombin generation assay (TGA) was able to differentiate bleeding tendency provided the sample conditions used in the assay were optimised to assess FXI involved coagulation pathways thought to be of relevance in vivo: using platelet rich plasma with inhibition of in vitro contact activation and a low tissue factor trigger. Thromboelastometry measured using the same sample type was similarly able to distinguish bleeding phenotype. However, when the potential clinical utility of the assays was compared using receiver operating characteristic curve analysis, thromboelastometry was inferior to TGA as an identifier of bleeding tendency. When the thromboelastometry sample type used was whole blood, or where assays were performed in the presence of tissue plasminogen activator the assays did not differentiate bleeding phenotype. For purposes of treatment planning, the potential of the TGA to determine the optimal dose of FXI replacement was assessed by in vitro spiking experiments using two commercially available FXI concentrates and samples from individuals with major FXI deficiency. Each concentrate improved thrombin generation, but dose response curves were found to differ, suggesting different properties for the two products. The clinical utility of the approach was then demonstrated with comparable TGA results obtained in ex vivo samples from patients treated with FXI concentrate and baseline samples spiked in vitro with equivalent amounts of the same FXI concentrate. The utility of global haemostasis assays to monitor the effect of FXI replacement in FXI-deficient individuals undergoing surgery was also tested. Improvement in assay parameters after treatment with solvent-detergent fresh frozen plasma or FXI concentrate was demonstrated suggesting assay value in FXI replacement monitoring. Finally the use of recently developed bleeding assessment tools and bleeding scores as descriptive, diagnostic or predictive measures was tested along with correlation with FXI:C levels and TGA parameters. This analysis confirmed that bleeding scores have a limited value in the clinical assessment of FXI deficiency.

Coherent diffractive imaging (CDI) has become a very popular technique over the past two decades. CDI is a "lensless" imaging method which replaces the objective lens of a conventional microscope by a computational image reconstruction procedure. Its increase in popularity came together with the development of X-ray free-electron lasers (XFELs) which produce extremely bright and coherent X-rays. By facilitating these unique properties, CDI enables structure determination of non-crystalline samples at nanometre resolution and has many applications in structural biology, material science and X-ray optics among others. This work focuses on two specific CDI techniques, flash X-ray diffractive imaging (FXI) on biological samples and X-ray ptychography. While the first FXI demonstrations using soft X-rays have been quite promising, they also revealed remaining technical challenges. FXI becomes even more demanding when approaching shorter wavelengths to allow subnanometre resolution imaging. We described one of the first FXI experiments using hard X-rays and characterized the most critical components of such an experiment, namely the properties of X-ray focus, sample delivery and detectors. Based on our findings, we discussed experimental and computational strategies for FXI to overcome its current difficulties and reach its full potential. We deposited the data in the Coherent X-ray Database (CXIDB) and made our data analysis code available in a public repository. We developed algorithms targeted towards the needs of FXI experiments and implemented a software package which enables the analysis of diffraction data in real time. X-ray ptychography has developed into a very useful tool for quantitative imaging of complex materials and has found applications in many areas. However, it involves a computational reconstruction step which can be slow. Therefore, we developed a fast GPU-based ptychographic solver and combined it with a framework for real-time data processing which already starts the ptychographic reconstruction process while data is still being collected. This provides immediate feedback to the user and allows high-throughput ptychographic imaging. Finally, we have used ptychographic imaging as a method to study the wavefront of a focused XFEL beam under typical FXI conditions.  We are convinced that this work on developing strategies and algorithms for FXI and ptychography is a valuable contribution to the development of coherent diffractive imaging.

The newly emerging technology of X-ray free-electron lasers (XFELs) has the potential to revolutionise molecular imaging. XFELs generate very intense X-ray pulses and predictions suggest that they may be used for structure determination to atomic resolution even for single molecules. XFELs produce femtosecond pulses that outrun processes of radiation damage and permit the study of structures at room temperature and of structural dynamics. While the first demonstrations of flash X-ray diffractive imaging (FXI) on biological particles were encouraging, they also revealed technical challenges. In this work we demonstrated how some of these challenges can be overcome. We exemplified, with heterogeneous cell organelles, how tens of thousands of FXI diffraction patterns can be collected, sorted, and analysed in an automatic data processing pipeline. We improved  image resolution and reduced problems with missing data. We validated, described, and deposited the experimental data in the Coherent X-ray Imaging Data Bank. We demonstrated that aerosol injection can be used to collect FXI data at high hit ratios and with low background. We reduced problems with non-volatile sample contaminants by decreasing aerosol droplet sizes from ~1000 nm to ~150 nm. We achieved this by adapting an electrospray aerosoliser to the Uppsala sample injector. Mie scattering imaging was used as a diagnostic tool to measure positions, sizes, and velocities of individual injected particles. XFEL experiments generate large amounts of data at high rates. Preparation, execution, and data analysis of these experiments benefits from specialised software. In this work we present new open-source software tools that facilitates prediction, online-monitoring, display, and pre-processing of XFEL diffraction data. We hope that this work is a valuable contribution in the quest of transitioning FXI from its first experimental demonstration into a technique that fulfills its potentials.