Hypercoagulation and the structural properties of plasma proteins in rheumatoid arthritis

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dc.contributor.advisorPretorius, Etheresiaen_ZA
dc.contributor.advisorTarr, Garethen_ZA
dc.contributor.authorBezuidenhout, Johannes Andries
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Physiological Sciences.en_ZA
dc.date.accessioned2022-02-07T11:04:39Z
dc.date.accessioned2022-04-29T12:51:52Z
dc.date.available2022-09-14T03:00:08Z
dc.date.issued2022-04
dc.descriptionThesis (PhD)--Stellenbosch University, 2022. en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Rumatoïede artritis (RA) is the mees algemene chroniese inflammatoriese gewrig siekte wêreldwyd en affekteer gewoonlik kleiner sinoviale gewrigte. The presiese patogeniese oorsaak van RA is tot hede nog nie ten volle bepaal nie, met die huidige etologiese siening as ‘n komplekse interaksie tussen verskeie genetiese, omgewings en immunologiese faktore. Die generering van self-antigene en ‘n daaropvolgende breek in immuun toleransie veroorsaak ‘n sistemiese outo-immuunreaksie wat weefsel en organe anders as die van sinoviale gewrigte affekteer, wat die kardiovaskulêre sisteem insluit. Individue wat ly aan RA het ‘n oortollige risiko vir vroeë ontwikkeling vand verskeie kardiovaskulêre siektes, soos veneus en arteriale trombose. Verhoogde kardiovaskulêre morbiditeit en mortaliteit in RA word vererger deur akute artritis simptome, wat aandui dat immuno-patologiese meganismes moontlik sellulêre en oplosbare komponente verantwoordelik vir die regulasie van bloedvloei en perfusie affekteer. Voorafgaande van hemostatiese wanregulasie veroorsaak deur inflammasie, is post- translasionele veranderinge van endogene proteïene wat outo-antigenisiteit in RA genereer bekend om prominente stollings faktore soos fibrinogeen en fibrien te teiken. Die omvang en effek van hierdie veranderinge op die normale funksie en struktuur van fibrinogeen en fibrien binne die konteks van koagulasie tot hede nog nie breedvoerig oorweeg nie. Hierdie studies het dus gepoog om die meganistiese end strukturele abnormaliteite van bloedstolling in RA pasiënte te indentifiseer en te karakteriseer. Perifere bloed monsters is versamel van individue met aktiewe RA tesame met ouderdom- en geslagsooreenstemende gesonde individue wat as eksperimentele kontroles gedien het. Funksionele en strukturele analises was uitgevoer op heelbloed en fibrienklonte gevorm van RA en kontrole monsters, en met mekaar vergelyk. Klontstrukture is verder bestudeer om die moontlike impak van outo-immuun verwante sitrullinasie te bepaal, tesame met veranderinge in die proteïen sekondêre struktuur. Verhoogde vlakke van C-reaktiewe proteïen, serum ameloïed A, en intersellulêre adhesie molekule 1 gemeet deur immuuntoetsing is bevestig in RA pasiënte, wat ‘n toestand van akute sistemiese inflammasie en endoteel disregulasie aandui. Die begin en aanvoering van bloedstolling gemeet deur tromboelastografie het geskied teen ‘n versnelde tempo in RA heelbloed in vergelyking met kontroles. The maksimum bepaalde vlak van trek sterkte in gevormde klonte was egter verswak in RA. Hierdie waarneming was konsekwent met verlaagde plaatjie aktiwiteit (P-selektien ELISA) tesame met abnormale fibrienklont eienskappe waargeneem met elektron mikroskopie, waar RA klonte saamgestel is deur digte fibrien netwerke met verhoogde vesel deursnee. Die wegdoening van fibrien klont viskoelastisiteit was gereflekteer in addisionele mikroskopiese en spektrochemiese analise van proteïen sekondêre struktuur, waarin verhoogde β-blad generering gedurende stolling plaasvind maar relatiewe gelyke saamstelling van β-blad inhoud bestaan met die van gesonde individue. Hierdie resultate dui baie sterk aan dat immunopatologiese meganismes intrinsiek tot RA trombotiese komplikasies mag veroorsaak. Verhoogde fibrien neerslag mag deur strukturele veranderinge teengewerk word en dus ‘n kompromie inhou vir klot stabiliteit en die risiko verhoog vir die los skeur van klonte onder invloed van vloei-skeer kragte. Die moontlike oorsaak van tromboembolisme is van uiterse belangrike kliniese belang, en verdere meganistiese insigte word benodig om sodoende hierdie ernstige risiko in die RA populasie te identifiseer en te versag.af_ZA
dc.description.versionDoctoralen_ZA
dc.embargo.terms2022-09-14
dc.format.extent196 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/125050
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshRheumatoid arthritis -- Pathogenesisen_ZA
dc.subject.lcshBlood proteins -- Propertiesen_ZA
dc.subject.lcshPlasma proteins -- Structureen_ZA
dc.subject.lcshRheumatoid arthritis -- Patientsen_ZA
dc.subject.lcshBlood -- Clottingen_ZA
dc.subject.lcshBlood -- Coagulation, Disorders ofen_ZA
dc.subject.lcshFibrinen_ZA
dc.subject.lcshImmunopathologyen_ZA
dc.subject.nameUCTDen_ZA
dc.titleHypercoagulation and the structural properties of plasma proteins in rheumatoid arthritisen_ZA
dc.typeThesisen_ZA
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