Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by Author "Bezuidenhout, Johannes Andries"

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    Hypercoagulation and the structural properties of plasma proteins in rheumatoid arthritis
    (Stellenbosch : Stellenbosch University, 2022-04) Bezuidenhout, Johannes Andries; Pretorius, Etheresia; Tarr, Gareth; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Rheumatoid Arthritis (RA) is the most common chronic inflammatory joint disease globally and typically affects smaller synovial joints. The exact pathogenic cause of RA is yet to be fully determined, with the current paradigm viewing aetiology as a complex interaction between various genetic, environmental and immunological factors. The generation of self-antigens and a subsequent break in immune tolerance causes a systemic autoimmune response that affects tissues and organs other than synovial joints, including the cardiovascular system. Individuals suffering from RA have an excessive risk for early development of numerous cardiovascular diseases (CVD), such as venous and arterial thrombosis. Increased CVD morbidity and mortality risk in RA is exacerbated by heightened arthritis severity, which indicates that immunopathological mechanisms could affect cellular and soluble components responsible for regulating blood flow and perfusion. Upstream from inflammation-induced haemostatic dysregulation, posttranslational modifications of endogenous proteins that generates autoantigenicity in RA is known to target prominent clotting factors such as fibrinogen and fibrin. The extent and effect of these alterations on the normal function and structure of fibrin(ogen) within the context of coagulation has to date not been extensively considered. This study therefore aimed to identify and characterise the mechanistic and structural abnormalities of blood clot formation in RA patients. Peripheral blood samples were collected from individuals with active RA disease alongside age- and gender-matched healthy individuals that served as experimental controls. Functional and structural analysis was conducted of whole blood and fibrin clots formed from RA and control samples and compared. Clot structures were further examined to determine the possible impact of autoimmune-related citrullination and alterations of the protein secondary structure. Increased expression levels of C-reactive protein, serum amyloid A and intercellular adhesion molecule 1 measured by immunoassay was confirmed in RA patients, indicating a state of acute systemic inflammation and endothelial dysregulation. Initiation and propagation of clot formation as measured by thromboelastography occurred at a more rapid rate in RA whole blood samples compared to controls. However, maximal measured strength of formed thrombi was attenuated in RA. This observation was consistent with decreased platelet activity levels (P-selectin ELISA), alongside abnormal fibrin clot properties observed using electron microscopy, where RA clots consisted of dense fibrin fibre networks with increased fibre diameter. The identification of citrullination as a possible cause of altered fibrin clot properties in RA was accomplished by using immunofluorescence microscopy. Finally, the abrogation of fibrin clot viscoelasticity was reflected in additional microscopical and spectrochemical analysis of protein secondary structure, in which increased β-sheet generation during clot formation but similar relative β-sheet composition to that of healthy controls was indicated. These findings strongly suggests that immunopathological mechanisms intrinsic to RA may cause a thrombotic complication in which elevated fibrin deposition is offset by structural alterations that compromise clot stability and could therefore increase the risk of rupture under the influence of flow shear forces. This potential cause of thromboembolism is of important clinical relevance, and further mechanistic insights are necessary in order to identify and possibly mitigate this serious risk in the RA population.