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Extracorporeal membrane oxygenation (ECMO) is a medical treatment that aims to support patients' respiratory and circulatory systems by oxygenation of blood outside of the patient. The therapy exposes blood to an artificial environment, which increases the risk of clot formations in the blood. This thesis proposes a noninvasive method to detect the development of thrombi in the ECMO circuit (which may cause patient complications) by measuring the blood pump motor effect and blood flow. To show the feasibility of this approach, a code that calculates pump efficiency changes due to adjustments of flow resistance shall be written and tested with a mock-up of an extracorporeal life support (ECLS) circuit. Results indicate there exist different flow efficiency relations. Efficiency seems to be influenced by design; certain rotation speeds have higher efficiency than others. As flow increases, so do efficiency (for our values, 3-5 Litres per minute, LPM). For 3 LPM, the highest efficiency was achieved at around 2800 RPM; 4 and 5 LPM start with higher efficiency but decreases as RPM increases. It was concluded that it is possible to differentiate between various flow restrictions using power consumption assessments. Low resistance changes, reduction of cross-section area for flow by 10% on the inlet side, and 16% on the outlet side showed no difference in impeller turning speed nor flow out of the pump. / Extrakorporeal Membranoxygenering (ECMO) är en livräddande behandling för att syresätta patienters blod utanför kroppen vid svår andnings eller cirkulationssvikt. I ECMO-systemet utsätts blodet för en artificiell miljö som medför högre risk för koagulationsaktivering och blodproppsbildning. Detta arbete undersöker möjligheten att icke-invasivt mäta flödesresistanser (som proppar) utifrån att mäta förbrukningseffekten hos den elektriska blodpumpen i ECMO systemet. För att undersöka detta skrivs en kod för att ge en uppskattning av vid varierande flödesrestriktioner uppmätta värden. Dessa värden tas från en befintlig modell på KTH:s Strömningsfysiklaboratorium. Låga flödesrestriktioner påverkar varken flöde, motorrotationshastighet eller motorns effektförbrukning. Detta arbete fann att 10% av slangen till motorn och 16% av slangen från motorn kan vara täckt utan påverkan. Effektiviteten av pumpen varierar beroende på olika variabler. Detta arbete fann att effektiviteten ökar med ökat flöde 3 till 5 liter per minut (LPM). Det verkar även finnas indikationer för att effektiviteten beror på rotationer per minut (RPM), för 3 LPM fanns den högsta effektiviteten kring 2800 RPM, 4 LPM har sin högsta effektivitet vid samma område och 5 LPM har sin högsta effektivitet vid start och avtar därefter. Detta arbete fann att det är möjligt att beräkna flödesrestriktioner utifrån att kontinuerligt notera värdet på en elmätare kopplad till ECMO enheten.

Motivation In the black population of South Africa, cardiovascular disease (CVD) is rapidly increasing due to urbanisation. Stroke is usually accompanied by a prothrombotic haemostatic profile. Changing lifestyle factors that accompany the urbanisation process could have a negative impact on the haemostatic profile of black South Africans. Elevated levels of pro-coagulant factors, von Willebrand factor (vWF), fibrinogen and fibrin D-dimer have been reported in the black population, which could increase the black population’s susceptibility to CVD. However, low levels of plasminogen activator inhibitor-1 (PAI-1) previously reported in the black population could contribute towards a pro-fibrinolytic state, which may counteract the hypercoagulant state. This may have a beneficial effect on the haemostatic profile of the black population. More investigation into the haemostatic profile of black South Africans is therefore needed to determine if an altered haemostatic profile exists in this group, and if so, to what extent these alterations may relate to cardiovascular dysfunction. This study included markers of both the coagulation (vWF, fibrinogen, fibrin D-dimer) and fibrinolytic (PAI-1, fibrin D-dimer and fibrinolytic potential) systems in an attempt to investigate the haemostatic profile of the black population of South Africa, and for comparison purposes that of the white population as well. The relationship of these markers’ with selected markers of cardiovascular function was also examined to determine if they could possibly contribute to an increase in cardiovascular risk, especially in the black population. Aims The aims of this study were to first compare coagulation and fibrinolysis markers in the black and white populations of South Africa. Furthermore, to determine if associations exist between the selected components of the haemostatic system and markers of cardiovascular function, especially in the black population of South Africa, who tends to be at a higher cardiovascular risk due to altered metabolic and haemostatic profiles. Methodology The Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study was a prospective cohort study that consisted of 409 participants at baseline (2008-2009) that were equally distributed according to both ethnicity (200 black; 209 white) and gender (black, 101 men, 99 women; white, 101 men, 108 women). At follow-up (2011/2012) the cohort totalled 359 participants (170 black, 88 men and 82 women; 189 white, 93 men and 96 women). Data from baseline measurements were used for the first two manuscripts (chapters 2 and 3), while followup data was used for the third manuscript (chapter 4). vWF, fibrinogen, PAI-1, fibrin D-dimer, CLT, serum peroxides, glutathione, glutathione peroxidase and reductase activity were determined, and ambulatory blood pressure and the retinal vessel calibres were measured. The groups were stratified by ethnicity as specified by statistical interaction terms. T-tests and chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between the components of the haemostatic system and cardiovascular function markers. This was followed by multiple linear regression analyses to investigate whether independent associations exist between the variables in both ethnic groups. P-values ≤0.050 were deemed significant. Results and conclusion of each manuscript The first manuscript (chapter 2) compares the haemostatic profiles of the black and white population to determine whether ambulatory blood pressure is related to components of the haemostatic system. The black participants displayed a prothrombotic profile with significantly higher vWF, fibrinogen, PAI-1, fibrin D-dimer and a longer CLT than their white counterparts. Furthermore, partial and multiple linear regression analyses showed a positive association of systolic and diastolic blood pressure with fibrin D-dimer in the black population, while a negative association existed between ambulatory blood pressure and CLT in the white population. These associations suggest that fibrin D-dimer may contribute, at least in part, to the high prevalence of hypertension in the black population. The second manuscript (chapter 3) determined associations between markers of the haemostatic and oxidant-antioxidant systems in the black and white populations. In addition to the prothrombotic profile that exists in the black population, this group also had significantly higher serum peroxides (oxidative stress) and lower glutathione peroxidase activity (antioxidant) levels. Multiple linear regression analyses indicated positive associations between fibrinogen and serum peroxides in both populations. In the white population, an additional positive association was found between serum peroxide and CLT. In the black population, vWF and CLT were negatively associated with GPx activity. The results suggest that there are ethnic-specific relationships between the haemostatic and oxidant-antioxidant systems. The third manuscript (chapter 4) investigated the relationships between the retinal vessel calibres and components of the haemostatic system in the black and white population. The investigation focussed specifically on arteriolar diameters in the lower median, since a narrow arteriolar diameter is known to be associated with elevated blood pressure. In both ethnic groups, a narrower arteriolar calibre was accompanied by narrower venular calibres. Independent positive associations were found between the central retinal vein equivalent (CRVE) and fibrinogen in the black population, as well as vWF and CLT in the white population. In addition, independent negative associations were found between the central retinal artery equivalent and CLT in the black population and with vWF in the white population. The results suggest that haemostatic alterations are linked to early vascular changes that may differ between ethnicities. General conclusion Ethnic-specific relationships between the components of the haemostatic system and measures of cardiovascular function are evident. The prothrombotic profile that is observed in the black population, together with the adverse associations of the haemostatic components with blood pressure, a compromised oxidant-antioxidant profile, and retinal vessel calibres may contribute, at least in part, to the high cardiovascular and cerebrovascular risk evident in this population group. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Motivation In the black population of South Africa, cardiovascular disease (CVD) is rapidly increasing due to urbanisation. Stroke is usually accompanied by a prothrombotic haemostatic profile. Changing lifestyle factors that accompany the urbanisation process could have a negative impact on the haemostatic profile of black South Africans. Elevated levels of pro-coagulant factors, von Willebrand factor (vWF), fibrinogen and fibrin D-dimer have been reported in the black population, which could increase the black population’s susceptibility to CVD. However, low levels of plasminogen activator inhibitor-1 (PAI-1) previously reported in the black population could contribute towards a pro-fibrinolytic state, which may counteract the hypercoagulant state. This may have a beneficial effect on the haemostatic profile of the black population. More investigation into the haemostatic profile of black South Africans is therefore needed to determine if an altered haemostatic profile exists in this group, and if so, to what extent these alterations may relate to cardiovascular dysfunction. This study included markers of both the coagulation (vWF, fibrinogen, fibrin D-dimer) and fibrinolytic (PAI-1, fibrin D-dimer and fibrinolytic potential) systems in an attempt to investigate the haemostatic profile of the black population of South Africa, and for comparison purposes that of the white population as well. The relationship of these markers’ with selected markers of cardiovascular function was also examined to determine if they could possibly contribute to an increase in cardiovascular risk, especially in the black population. Aims The aims of this study were to first compare coagulation and fibrinolysis markers in the black and white populations of South Africa. Furthermore, to determine if associations exist between the selected components of the haemostatic system and markers of cardiovascular function, especially in the black population of South Africa, who tends to be at a higher cardiovascular risk due to altered metabolic and haemostatic profiles. Methodology The Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study was a prospective cohort study that consisted of 409 participants at baseline (2008-2009) that were equally distributed according to both ethnicity (200 black; 209 white) and gender (black, 101 men, 99 women; white, 101 men, 108 women). At follow-up (2011/2012) the cohort totalled 359 participants (170 black, 88 men and 82 women; 189 white, 93 men and 96 women). Data from baseline measurements were used for the first two manuscripts (chapters 2 and 3), while followup data was used for the third manuscript (chapter 4). vWF, fibrinogen, PAI-1, fibrin D-dimer, CLT, serum peroxides, glutathione, glutathione peroxidase and reductase activity were determined, and ambulatory blood pressure and the retinal vessel calibres were measured. The groups were stratified by ethnicity as specified by statistical interaction terms. T-tests and chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between the components of the haemostatic system and cardiovascular function markers. This was followed by multiple linear regression analyses to investigate whether independent associations exist between the variables in both ethnic groups. P-values ≤0.050 were deemed significant. Results and conclusion of each manuscript The first manuscript (chapter 2) compares the haemostatic profiles of the black and white population to determine whether ambulatory blood pressure is related to components of the haemostatic system. The black participants displayed a prothrombotic profile with significantly higher vWF, fibrinogen, PAI-1, fibrin D-dimer and a longer CLT than their white counterparts. Furthermore, partial and multiple linear regression analyses showed a positive association of systolic and diastolic blood pressure with fibrin D-dimer in the black population, while a negative association existed between ambulatory blood pressure and CLT in the white population. These associations suggest that fibrin D-dimer may contribute, at least in part, to the high prevalence of hypertension in the black population. The second manuscript (chapter 3) determined associations between markers of the haemostatic and oxidant-antioxidant systems in the black and white populations. In addition to the prothrombotic profile that exists in the black population, this group also had significantly higher serum peroxides (oxidative stress) and lower glutathione peroxidase activity (antioxidant) levels. Multiple linear regression analyses indicated positive associations between fibrinogen and serum peroxides in both populations. In the white population, an additional positive association was found between serum peroxide and CLT. In the black population, vWF and CLT were negatively associated with GPx activity. The results suggest that there are ethnic-specific relationships between the haemostatic and oxidant-antioxidant systems. The third manuscript (chapter 4) investigated the relationships between the retinal vessel calibres and components of the haemostatic system in the black and white population. The investigation focussed specifically on arteriolar diameters in the lower median, since a narrow arteriolar diameter is known to be associated with elevated blood pressure. In both ethnic groups, a narrower arteriolar calibre was accompanied by narrower venular calibres. Independent positive associations were found between the central retinal vein equivalent (CRVE) and fibrinogen in the black population, as well as vWF and CLT in the white population. In addition, independent negative associations were found between the central retinal artery equivalent and CLT in the black population and with vWF in the white population. The results suggest that haemostatic alterations are linked to early vascular changes that may differ between ethnicities. General conclusion Ethnic-specific relationships between the components of the haemostatic system and measures of cardiovascular function are evident. The prothrombotic profile that is observed in the black population, together with the adverse associations of the haemostatic components with blood pressure, a compromised oxidant-antioxidant profile, and retinal vessel calibres may contribute, at least in part, to the high cardiovascular and cerebrovascular risk evident in this population group. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

The objective of this study was to evaluate the root surfaces modifications after the application of different chemicals agents, and their influence on the attachment of a fibrin network and fibroblasts. From 96 anterior mandibular human incisor teeth, extracted due to severe periodontal disease, were obtained 192 dentin blocks (3x3x1 mm) of buccal and lingual surface and randomly divided into 6 groups: Control- control group, which received no treatment; Root surface scaling and root planing (SRP) - root surface was scaling and root planning with Gracey curetes; Citric acid - SRP + 30% citric acid 5 min; EDTA (ethylenediaminetetraacetic acid) - SRP + 24% EDTA gel for 1 min; Tetracycline capsule - SRP + for 3 min with a solution obtained by dissolving one 500 mg capsule of tetracycline in 2 mL of saline solution; Tetracycline gel - SRP + 50 g/mL tetracycline gel for 1 min. After dentin treatment the specimens were analyzed using 4 methodologies: 1- the demineralization level and residues of the product (n = 9); 2- the adhesion of blood components after 20 min of surface treatment (n = 9); 3- the fibroblast attachment after 24h (n = 9), were analyzed by scanning electron microscopy (SEM); and 4- the cell metabolism after 4h (n = 5) the methyltetrazolium (MTT) assay was used. Citric acid, EDTA and Tetracycline gel removed completely smear layer and smear plug on root surface, resulted in adequate demineralization .Tetracycline capsule produced great tetracycline residues with several demineralization areas on root dentin surface. Tetracycline gel and EDTA groups had more fibroblast fixation than other experimental groups. The highest mean blood clot adhesion score was observed in roots treated with a tetracycline gel, whereas, the least score was observed in roots treated with tetracycline capsule. These results demonstrated that EDTA and Tetracycline gel surface demineralization removed the smear layer over dentin surface and promoted adhesion of a fibrin network and fibroblast cells attachment. The Tetracycline capsule demonstrated less effective performance in all parameters tested. / O objetivo deste estudo foi avaliar as modificações nas superfícies radiculares após a aplicação de diferentes agentes químicos, e sua influência na inserção de rede de fibrina e fibroblastos. De 96 dentes incisivos anteriores humanos, extraídos devido doença periodontal severa, foram obtidos 192 blocos de dentina (3x3x1 mm) da superfície vestibular e lingual e aleatoriamente divididos em seis grupos: controle, que não receberam tratamento, raspagem e alisamento radicular (RAR) - superfície radicular foi raspada e alisada com curetas Gracey, Ácido cítrico - RAR + ácido cítrico 30% por 5 min; EDTA (ácido etilenodiaminotetracético) - RAR + gel de EDTA 24% por 1 min; Tetraciclina cápsula RAR + solução obtida por dissolução de uma cápsula de 500 mg de tetraciclina em 2 ml de solução salina por 3 minutos; Tetraciclina gel - RAR + 50 mg / mL de tetraciclina gel por 1 min. Após o tratamento, as amostras de dentina foram analisadas utilizando 4 metodologias diferentes: 1- o nível de desmineralização e de resíduos do produto (n = 9); 2- a adesão das componentes do sangue no tratamento de superfície após 20 min (n = 9); 3- a inserção de fibroblasto após 24 horas (n = 9), que foram analisadas por microscopia eletrônica de varredura (MEV), e 4- o metabolismo celular após 4h (n = 5), o ensaio com metiltetrazólio (MTT) foi utilizado. Ácido cítrico, EDTA e tetraciclina gel removeram completamente, smear layer e smear plug na superfície radicular, resultando em adequada desmineralização. Tetraciclina cápsula produziu grande resíduos de tetraciclina com áreas severas de desmineralização na superfície de dentina radicular. Grupos Tetraciclina gel e EDTA tiveram maior inserção de fibroblastos que os outros grupos experimentais. A maior média de adesão do coágulo sanguíneo pontuada foi observada em raízes tratadas com a tetraciclina gel, enquanto que, a menor pontuação foi observada em raízes tratadas com tetraciclina cápsula. Estes resultados demonstram que a desmineralização da superfície pelo EDTA e pela tetraciclina gel removeram a smear layer que cobria a superfície da dentina e promoveram adesão da rede de fibrina e inserção de células dos fibroblastos. A Tetraciclina cápsula demonstrou desempenho menos efetivo em todos os parâmetros testados. / Mestre em Odontologia

Le rôle physiologique du caillot est d’éviter un épanchement excessif de sang en présence d’une brèche vasculaire. Une fois cette fonction remplie, il doit pouvoir être facilement détruit, afin qu’il ne passe pas dans le système veineux et ne gêne la circulation sanguine. La formation d’un caillot de fibrine et sa lyse, processus clés de l’hémostase, impliquent à la fois la polymérisation des monomères de fibrinogène en un réseau de fibres de fibrine, et la résorption du réseau de fibres de fibrine constitué. Bien que ce réseau contrôle l’ensemble des propriétés physiques et mécaniques du caillot, sa structure aux échelles inférieures au micron est très mal caractérisée. Le principal verrou à la caractérisation physique du caillot en environnement clinique est l’absence de méthode de mesure quantitative, fiable, sensible et reproductible. Il est donc nécessaire de produire une méthode de mesure adéquate, couplée à un système de mesure sensible. Nous avons démontré dans ce travail, grâce à notre méthode utilisant plusieurs longueurs d’onde, que l’analyse du spectre de lumière visible transmis à travers un caillot permet de déterminer simultanément, quantitativement et en conditions quasi-physiologiques, plusieurs paramètres essentiels de structure du caillot de fibrine, à savoir le nombre de protofibrilles par fibre de fibrine, le rayon et la densité de ces fibres, ainsi que les temps de formation et de lyse du caillot. Cette technique a été validée via les résultats avec des CV inférieurs dans l’ensemble à 6% sous plusieurs conditions de tests et différents profils plasmatiques : normaux, hypo/hyper coagulants et hypo/hyper fibrinolytiques, attestant de la robustesse et de la fiabilité de la technique de mesure aussi bien pour le suivi de la coagulation que de la lyse. Cette méthode de spectrophotométrie a pu être implantée sur un automate modifié à des fins de diagnostic et à vocation hospitalière pour des plasmas de patients présentant des troubles de l’hémostase. Les informations cliniques et intérêts attendus de ce nouveau test, concernent à la fois la qualité du réseau de fibrine, sa lyse accélérée ou sa résistance à la fibrinolyse ainsi que la résultante de la balance coagulo-lytique. / The physiological role of the clot is to avoid excessive bleeding in the presence of a vascular breach. Once this function is filled, the clot must be able to be easily destroyed, so that it is not transported in the venous system and does not hamper blood circulation. The formation of a fibrin clot and its lysis are key processes of hemostasis, implying simultaneously the polymerization of the fibrinogen monomers in a fibrin fibers network, and the destruction of this constituted network.Although this network controls the physical and mechanical properties of the clot, its structure at scales smaller than the micron is poorly characterized. The main problem in the physical characterization of clot in clinical settings is the current absence of a quantitative, sensitive and reproducible measurement method.We demonstrated in this work, thanks to our method using several wavelengths, that the analysis of the visible spectra of light transmitted through a clot allows to determine simultaneously, quantitatively and in quasi-physiological conditions, several essential parameters of structure of the fibrin clot, namely the number of protofibrils per fibrin fibers, the radius and the density of fibers, and various times of clotting and lysis of the clot. This method was validated by the results with CV inferior to 6 % under all test conditions and various plasmatic profiles: normal, hypo / hyper coagulant and hypo / hyper fibrinolytic. This demonstrates the robustness and reliability of the measurement method when measuring both clotting and clot lysis.This spectrophotometric method was implemented on a modified automaton dedicated to diagnosis of patients presenting hemostatic disorders. The clinical information and the interests expected from this new test concern at the same time the quality of the fibrin network, its accelerated lysis or its resistance to fibrinolysis, and the resultant of the coagulo-lytic balance.

Les plaquettes sont la principale source de CD40L soluble (sCD40L), une molécule thrombo-inflammatoire dont les taux élevés chez les patients atteints des maladies coronariennes (CAD) prédisent des événements athérothrombotiques. Nous avons déjà démontré que le sCD40L active deux voies de signalisation dans les plaquettes, la p38 MAPK et le facteur nucléaire-κB (NF-κB), non affectées par l’activité antiplaquettaire de l'aspirine (ASA). Nous avons montré aussi que le sCD40L, en réponse à des doses sous-optimales d'agonistes plaquettaires comme la thrombine et le collagène, potentialise l'agrégation des plaquettes via le récepteur CD40 et son effecteur cible présent en aval le NF-κB. En effet, le NF-κB appartient à une famille de dimères cytoplasmiques inactivés par l'inhibiteur IκB et régulés par l’IκB kinase (IKK). Suite à des signaux immunologiques, l’IKK phosphoryle l’IκB, ce qui entraîne sa dégradation par le protéasome. Par conséquent, le NF-κB, une fois libre, se déplace vers les noyaux des cellules immunitaires où il agit sur le génome pour maintenir la survie et la prolifération cellulaires. Contrairement aux cellules immunitaires, la régulation et les fonctions de NF-κB dans les plaquettes, fragments cellulaires dépourvus de génome, sont moins bien comprises. Dans ce projet, nous proposons l'hypothèse que l’axe sCD40L/NF-κB/protéasome amorce les plaquettes en les prédisposant à une activation et une agrégation prononcées. Nos objectifs seront donc (i) d'étudier l'interaction entre le NF-κB et le protéasome en réponse au sCD40L et (ii) d'examiner les rôles de cet axe sur les fonctions plaquettaires. Pour cela, des plaquettes ont été fraîchement isolées à partir du sang des donneurs humains sains qui ne prennent pas des médicaments antiplaquettaires ou anti-inflammatoires. Ces plaquettes ont été par la suite stimulées par le sCD40L. En étudiant l'activation de NF-κB, l'analyse par Western blot a montré que le sCD40L induit la phosphorylation d’IκB et la dégradation de sa forme phosphorylée (p-IκB) dans un délai de 30 minutes, d'une manière similaire à l’action de la thrombine, l’agoniste plaquettaire le plus puissant. Le prétraitement des plaquettes avec deux classes d'inhibiteurs de NF-κB, le répresseur d’IKK (BAY 11-7082) et l’antagoniste du protéasome (MG132), a montré que l'activation du NF-κB dans les plaquettes est régulée par l’IKK et le protéasome, identiquement aux cellules nucléées. L'examen des événements de signalisation induits par le sCD40L a montré que l'inhibition du NF-κB plaquettaire n'affecte pas la phosphorylation de p38 MAPK ou le clivage protéasomique de la Taline-1 qui est impliquée dans le changement de la forme des plaquettes. Concernant les effets sur les fonctions plaquettaires, l'inhibition de NF-κB ou du protéasome n'a pas influencé l'agrégation des plaquettes induite par des doses élevées de collagène ou de thrombine. Cependant, elle a considérablement réduit l'agrégation plaquettaire suite à un amorçage avec le sCD40L, suivie d'une stimulation avec des doses sous-optimales de collagène ou de thrombine. Nous avons également constaté que le sCD40L exacerbe la rétraction des caillots fibrino-plaquettaire formés par de faibles doses de thrombine. Cet effet d'amorçage est régulé par l’axe NF-κB/protéasome. En bref, l’axe sCD40L/NF-κB/protéasome fonctionne de manière non génomique, en amorçant les plaquettes, induisant ainsi la potentialisation de l'agrégation plaquettaire et l'augmentation de la rétraction des caillots fibrino-plaquettaire. Par conséquent, le ciblage de cet axe dans les plaquettes pourrait avoir un rôle thérapeutique chez les patients atteints de CAD et dont les plaquettes ne répondent pas ou sont moins sensibles aux traitements antiplaquettaires conventionnels. / Platelets are the principal source of soluble CD40L (sCD40L), a thrombo-inflammatory molecule whose high levels in coronary artery disease (CAD) patients predict atherothrombotic events. We have previously demonstrated that sCD40L activates two signaling pathways in platelets, p38 MAPK and the nuclear factor-κB (NF-κB), both of which were unaffected by the anti-platelet activity of aspirin (ASA). We have also shown that sCD40L, in response to suboptimal doses of platelet agonists like thrombin and collagen, potentiates platelet aggregation through CD40 receptor and its downstream effector target NF-κB. Indeed, NF-κB belongs to a family of cytoplasmic dimers that are inactivated by the inhibitor IκB and regulated by IκB kinase (IKK). Following immunological signals, IKK phosphorylates IκB, driving the degradation of its phosphorylated form (p-IκB) by the proteasome. Consequently, NF-κB becomes free to translocate to the nuclei of immune cells where it acts genomically to maintain survival and proliferation. That is the case in immune cells, but in platelets which are devoid of genome, the regulation and functions of NF-κB are less understood. Herein, we hypothesized that sCD40L/NF-κB/proteasome axis primes platelets, predisposing them to pronounced activation and aggregation. We thus aimed to (i) assess the interplay between NF-κB and proteasome in response to sCD40L and (ii) investigate the roles of this axis at the level of platelet functions. For this purpose, platelets were freshly isolated from the blood of healthy human donors who are not on anti-platelet or anti-inflammatory medication and then stimulated with sCD40L. In monitoring the activation of NF-κB, western blot analysis showed that sCD40L induces the phosphorylation of IκB and the degradation of p-IκB during a 30-mins time window, in a similar fashion to the most potent platelet agonist thrombin. The pre-treatment of platelets with two classes of NF-κB inhibitors, an IKK repressor (BAY 11-7082) and a proteasome antagonist (MG132), showed that the activation of platelet NF-κB is regulated by IKK and the proteasome as in nucleated cells. The examination of the functional signaling events induced by sCD40L showed that NF-κB inhibition does not affect the phosphorylation of p38 MAPK or the proteasomal cleavage of shape change-involved Talin-1. In functional studies, NF-κB or proteasomal inhibition did not influence the aggregation of platelets induced by high doses of collagen or thrombin; however, it markedly reduced platelet aggregation primed with sCD40L followed by stimulation with sub-optimal doses of collagen or thrombin. We also found that sCD40L exacerbates the retraction of fibrin-platelet clots formed by low doses of thrombin. This priming effect was regulated by NF-κB/proteasome dyad. In brief, sCD40L/NF-κB/proteasome axis primes platelets and functions non-genomically, inducing the potentiation of platelet aggregation and augmenting the retraction of fibrin-platelet clots. Hence, targeting this axis in platelets might have a therapeutic potential in CAD patients whose platelets are not or less responsive to conventional antiplatelet therapies.

<p>  </p> <p>Thrombosis is the pathological formation of a blood clot in the body that blocks blood circulation, leading to high morbidity and mortality rates. Thrombolytic drugs that offer rapid clot dissolution are promising treatments yet current drugs are often associated with limited efficacy and high bleeding risks. While numerous animal thrombosis models have been developed for drug screening, the translation of therapeutic agents into and through clinical trials remains limited. This is largely due to animal models’ poor reproducibility and distinctive physiology to that of humans. <em>In-vitro</em> flow models that utilize both human blood components and physiologically relevant flow conditions can provide for a more representative testing environment to screen thrombolytic drugs. Developing better <em>in-vitro</em> models may not eliminate the need for preclinical animal testing but can help exclude inefficient agents earlier in the drug development pipeline to expedite the drug evaluation process. Existing <em>in-vitro</em> thrombolysis flow models are not ideal as they either adopt over-simplified clot substrates or utilize small-length-scale geometries that insufficiently mimic native hemodynamics. Thus, we propose to first develop a static fluorescently labeled clot lysis assay for an initial high throughput screening of thrombolytic drugs, and ultimately engineer a highly reproducible, physiological scale, flowing clot lysis model for more human relevant drug efficacy evaluation. Developing the static clot lysis assay not only helps to understand the mechanism of how diversified clotting conditions affect clot properties but also offer a chance to well-characterize fluorescence conjugations to fibrins. The ultimate flow model combines an <em>in-vivo</em>-like fluorescence incorporated synthetic clot (FISC) and a human-relevant flow system. Guided by results from static clotting experiments diversified FISCs are fluorescently optimized and fabricated dynamically using a Chandler loop setup at various conditions. The flow system is a tubing-based structure that comprises of a peristaltic pump, and a well-controlled flow chamber to provide for physiological shear and pulsatile levels. Therefore, the proposed synthetic clot model is a versatile platform that can mimic a variety of thrombosis conditions and offer representative drug testing and dosing results across numerous thrombolytic agents.</p>