The phenomenon known as immunothrombosis has garnered increased attention over the last few years. Much work has been done to characterize the cross talk between hemostasis and the innate immune system. This thesis outlines the role of the vascular endothelial cells during immunothrombotic events as regulators of coagulation, platelet-, and leukocyte recruitment. A newly developed method for investigating the interaction between endothelial cells and the blood compartment illustrated the procoagulant and proinflammatory effects elicited by tumor necrosis factor α activated endothelial cells upon exposure to whole blood. The method was utilized in evaluating treatment of endothelial dysfunction and disruption with a heparin conjugate. Damaged or hypoxic endothelial cells, in addition to basement membrane collagen, that were pretreated with the heparin conjugate prior to contact with blood were found to have reduced activation of coagulation, platelet-, and leukocyte recruitment; in contrast to unfractionated heparin, which had no effect on the aforementioned parameters. The treatment was then investigated in the setting of ischemia reperfusion injury during kidney transplantation and the heparin conjugate was found to bind cultured endothelial cells with high avidity under cold storage conditions. Furthermore, it was found to bind to the renal vasculature during static cold storage and was subsequently found to be beneficial with regard to early graft function in an experimental mouse model of syngeneic kidney transplantation. Recipients of kidneys treated with the heparin conjugate had reduced serum creatinine compared to controls 24 hours after transplantation. Lastly, the anticoagulant properties of the heparin conjugate were investigated in comparison to unfractionated heparin. While the conjugate exerted reduced capacity with regard to thrombin inhibition, it rapidly inhibited the binding of platelets to exposed collagen. The conjugate was furthermore found to preferentially locate to sites of endothelial cell activation at early stage during endotoxic shock in mice. In conclusion, this thesis demonstrates that disrupted functioning of the vascular endothelial cells actively contributes to immunothrombosis, and that it is possible to model endothelial cell function using whole blood assays. Furthermore, this thesis presents a treatment that enhances the hemocompatibility of damaged endothelial cells and subsequently improves the early renal function after kidney transplantation.

No description available.

Increases in air pollution, especially the particulate component, are associated with increased cardiovascular mortality, possibly through increases in thrombogenic mechanisms. The research presented in this thesis addresses the hypothesis that diesel exhaust particulates (DEP) increase thrombogenicity by impairing the release of tissue plasminogen activator (t-PA) from vascular endothelial cells, thus inhibiting the endogenous fibrinolytic mechanisms that promote thrombus breakdown. The initial aims of this work were to develop an in vivo model of thrombosis, to determine whether exposure to DEP did alter clotting. Initial attempts to develop the Folts’ model (which stimulates thrombus formation via arterial stenosis and mechanical injury), first in male C57/Bl6 mice and later in male Wistar rats, were unsuccessful. An alternative approach, using ferric chloride (FeCl3) to induce chemical injury to the rat carotid artery was found to produce reliable and reproducible thrombotic occlusion: this model was used for all subsequent experiments. The effects of DEP on thrombus formation were assessed in vivo by applying the FeCl3 model. DEP were administered via intratracheal instillation or tail vein injection 2, 6 or 24 hours prior to induction of thrombosis. The effects of DEP were compared with vehicle and suitable controls: carbon black (a clean carbon nanoparticle); quartz (a large non-carbon particle that causes well-characterised pulmonary inflammation). The time to thrombotic occlusion was significantly reduced 6h after intra-pulmonary instillation of DEP (0.5ml of a 1mg/ml suspension). In contrast, instillation of carbon black or quartz had no significant effect on thrombosis, despite causing greater pulmonary (increased neutrophils and levels of interleukin-6, tumour necrosis factor-α and C-reactive protein in bronchoalveolar lavage fluid) and systemic (C-reactive protein in plasma) inflammation than DEP. Direct administration of DEP (0.5mg/kg) to the blood stream resulted in an acute (2 hours after injection) increase in time to thrombotic occlusion in the absence of pulmonary inflammation. A similar (but less pronounced) effect was observed following administration of carbon black (0.5mg/kg). These data suggest that the DEP-mediated increase in thrombosis is independent of pulmonary and systemic inflammation. The mechanisms involved were addressed by measuring platelet-monocyte interactions (flow cytometry) and markers of the endogenous fibrinolytic system (ELISA). Exposure (either instillation of injection) to DEP significantly increased platelet-monocyte aggregation. Carbon black and quartz produced no such effect (but did increase platelet-platelet aggregation). t-PA antigen and activity were reduced, whilst PAI-1 and fibrinogen were increased, following either instillation or injection of DEP. The final aim was to develop a suitable dispersant for use in cell culture to determine whether DEP alter the expression (real-time polymerase chain reaction; rtPCR) and generation (enzyme-linked immunosorbent assay; ELISA) of t-PA and plasminogen activator inhibitor (PAI-1). Cell culture medium containing bovine serum albumin (0.5mg/ml; BSA) provided the best combination for DEP dispersal and maintenance of small particle size (<200nM), without detrimental effects on human umbilical endothelial cells (HUVECs). Exposure (6 and 24 hours) of HUVECs to DEP resulted in reduced basal and thrombin stimulated t-PA and PAI-1 expression. This was mirrored by reduced detection of t-PA and PAI-1 in culture medium. In conclusion, these investigations confirm that exposure to DEP is capable of increasing the rate of thrombus formation and that this is, in part, mediated by an alteration in the endogenous fibrinolytic system. These changes did not appear to be secondary to pulmonary or systemic inflammation. Whilst cell culture experiments suggested DEP could directly alter endogenous fibrinolytic activity in endothelial cells, there was no evidence from these experiments of DEP translocation into the systemic circulation. Thus, this work suggests that DEP is capable of increasing thrombus formation in vivo via several mechanisms. Similar changes may account for the increased thrombus formation in humans exposed to diesel exhaust in air pollution.

Introduction and Background: Central venous catheters (CVCs) are extensively used worldwide. Mechanical, infectious and thrombotic complications are well described with their use and may be associated with prolonged hospitalisation, increased medical costs and mortality. CVCs account for an estimated 90% of all catheter-related bloodstream infections (CRBSI) and a host of risk factors for CVC-related infections have been documented. These include, most importantly, the duration of catheterisation. The duration of use of CVCs remains controversial and the length of time such devices can safely be left in place has not been fully and objectively addressed in the critically ill patient. Over the past few years, antimicrobial impregnated catheters have been introduced in an attempt to limit catheter-related infection (CRI) and increase the time that CVCs can safely be left in situ. Recent meta-analyses concluded that antimicrobial-impregnated CVCs appear to be effective in reducing CRI. Materials and Methods: This was a prospective randomised double-blind study performed in the adult multidisciplinary Intensive Care Unit (ICU) at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) over a four year period. The study entailed a comparison of standard triple-lumen versus antimicrobial impregnated CVCs on the rate of CRI. The aim was to determine whether the duration of catheter insertion time could safely be increased from the standard practice of seven days at the CMJAH adult multidisciplinary ICU to 14 days, to assess the influence of the antimicrobial impregnated catheter on the incidence of CRI, and to elucidate the epidemiology and risks of CRI. Results: One hundred and eighteen critically ill patients were included in the study which spanned 34 951.5 catheter hours (3.99 catheter years). Sixty-two patients received a standard triple-lumen catheter and 56, a chlorhexidine-silver sulfadiazine (CSS) impregnated triple-lumen catheter. The mean duration of placement for the full sample of 118 CVCs was 12.3 days (range, 1-14). No statistically significant difference in CRI rates between the two types of catheters could be demonstrated. The most common source of primary CRBSI was skin, followed by hub and infusate. The site of CVC insertion (internal jugular versus subclavian vein) and the use of parenteral nutrition were not noted to be risk factors for catheter infection. There was no clinical evidence of catheter-related thrombosis in either of the study groups. Conclusion: This study was unable to demonstrate that antimicrobial catheters provided any significant benefit over standard catheters, which it is felt, can safely be left in place for up to 14 days with appropriate infection control measures. The most common source of CRI was the skin. The administration of parenteral nutrition and the site of catheter insertion (internal jugular vein versus subclavian vein) were not noted to be risk factors for CRI. There was no clinical evidence of thrombotic complications in either of the study groups. This study offers direction for the use of CVCs in critically ill patients and addresses many of the controversies that exist.

La thrombose est la formation d’un caillot sanguin (thrombus) dans le système cardiovasculaire. Il s’agit d’un des principaux problèmes des dispositifs biomédicaux en contact avec du sang. Toutefois, les biomatériaux utilisés jusqu’à présent dans ces dispositifs déclenchent la coagulation à travers le système de contact. Ce système n’a pas été pris en compte dans les modèles de thrombose dédiés aux dispositifs, alors que son importance pour la thrombose a été récemment remarquée. L’objectif de cette thèse est d’introduire les réactions de coagulation initiées par le système de contact dans la modélisation de la thrombose liées aux dispositifs. Un nouveau modèle réduit pour la génération de thrombine est proposé en incluant l’activation par contact. Le modèle arrive à calculer la formation de thrombine dans du plasma physiologique et du plasma déficient en prothrombine. Une fois réalisée, l’approche réduite est incluse dans un modèle de croissance du thrombus basé sur l’activité des plaquettes. Cette nouvelle stratégie est utilisée pour calculer la formation du thrombus dans une configuration académique. / Thrombosis, which is the formation of a blood clot (thrombus) in the vascular system, is one of the major issues of blood-coated medical devices. To reduce thrombosis risk in this type of devices, computational fluid dynamics has been used. Up to date, bio-material surfaces used in blood coated devices initiate blood coagulation through the contact activation system. This system has not been considered in models dedicated to devices, even though its importance in thrombosis has been recently highlighted. The objective of this thesis is to introduce device-triggered coagulation reactions in the modeling of device-related thrombosis.A novel reduced kinetic model including the contact activation system is proposed. The model correctly captures thrombin formation in physiological and prothrombin deficient platelet-poor-plasma. The reduced set of reactions is then included in a platelet-based model for thrombus formation considering platelet activation by thrombin. This approach is used to study thrombus formation in an academic configuration.

The increased risk of atherothrombosis in chronic kidney disease (CKD) has been under extensive examination for decades now. However, a treatment tailored for CKD patients is yet to be found. Current management plans can only tackle comorbidities and mostly fail. This thesis study examines the current literature related to CKD and thrombosis. The aim is to find a target suitable for therapeutic exploration. Normalizing the risk of thrombosis in CKD patients could curb a huge margin of their morbidity and mortality. In recent years, molecular biology studies attributed the extreme thrombogenicity in CKD to the retained uremic toxins. Indolic compounds are uremic toxins with a well described point of thrombotic activation. Of them, indoxyl sulfate is to be highlighted since it was shown to that it is one of the strongest pro-thrombotic uremic toxin. It is possible to therapeutically target this CKD specific cause of hyperthrombogenicity. Further research is very much needed in this area.

Orientador: Carlos Alberto Hussni / Banca: Pierre Barnabé Escodro / Banca: Regina Moura / Resumo: As tromboses podem ocorrer frequentemente em equinos como decorrência de coagulopatia intravascular disseminada, hipóxia, endotoxemia, cólica e laminite ou ainda por processos iatrogênicos. Porém, há poucos estudos em relação à cirurgia vascular e alterações morfológicas deste processo em equinos. Este trabalho teve por objetivo estudar a morfologia e os aspectos histopatológicos da veia cefálica em dois modelos experimentais. Foram utilizados 10 equinos hígidos, divididos em dois estudos: Estudo Trombose (ET), avaliou-se os aspectos histopatológicos da veia cefálica na tromboflebite aguda induzida. Estudo Fogarty (EF) avaliou-se os aspectos histopatológicos após a aplicação imediata do cateter de Fogarty. Para obtenção de base morfológica para avaliação das túnicas, coletou-se uma amostra controle (AC) de veia cefálica hígida. Nos animais do ET, foi realizada a indução química da trombose na veia cefálica e após 24 horas procedeu-se a coleta de segmento venoso para avaliações histopatológicas. Nos animais do EF, foi realizada a passagem do cateter de Fogarty na veia cefálica hígida e coleta imediata do segmento venoso. Os segmentos da veia cefálica foram divididos em segmento proximal e distal e analisados quanto à morfologia e histometria. Os segmentos do ET apresentaram espessamento da parede vascular, com desorganização das células musculares lisas, maior quantidade de matriz extracelular e alteração morfológica das células endoteliais em relação à AC. Os segmentos do EF apresentaram alterações morfológicas das células endoteliais e menor quantidade de células musculares lisas em relação à AC. A partir destes resultados, podemos concluir que a trombose induziu resposta inflamatória na parede vascular e espessamento das túnicas associada à presença do trombo. O EF apresentou alteração morfológica das células endoteliais em resposta imediata à passagem... / Abstract: Thrombosis can occur frequently in horses as a consequence of disseminated intravascular coagulopathy, hypoxia, endotoxemic, colics and laminitis or a iatrogenic process. However, there are few studies regarding vascular surgery and morphology changes of this process in horses. This research aimed to study the morphology and histopathological aspects of the cephalic vein in two experimental models. Ten healthy horses, was divided into two studies: Thrombosis study (ET), were used to evaluate the histopathological aspects of the cephalic vein in induced acute thrombophlebitis. Fogarty study (EF) was evaluated histopathological aspects after the immediate application of the Fogarty catheter. To achieve morphological based measures of the coats, were collected a sample control (AC) from a healthy cephalic vein. The animals of ET, chemical induction of thrombosis was performed in the cephalic vein and 24 hours proceeded the segment collection of the vessel for histological evaluation. The animals of EF, were submitted to the passage of a Fogarty catheter in the patent and healthy cephalic vein, followed by immediate collection of a segment venous. The cephalic vein segments were divided into proximal and distal and analyzed for morphology and histometry. The ET segments showed thickening of the vascular wall, with disorganization of the smooth muscle cells, increased amount of extracellular matrix and morphological alteration of endotelial compared to AC. The segments EF showed morphological changes in endothelial cells and fewer smooth muscle cells in relation to AC. From these results, we can conclude that thrombosis induced inflammatory response in vascular wall thickening and coats related to the presence of thrombus. The EF presented morphological alteration of endothelial cells in immediate response to the passage of the catheter, but so did the segment where there was the passage of the catheter / Mestre

Records of all neurosurgical inpatients admitted to Royal North Shore Hospital since 1976 have been prospectively kept in a relational database. Demographic details, diagnoses, operations and complications have been entered continuously since 1982 by the author of this study. Complications are monitored at monthly review meetings attended by medical staff. The recurrence of deep vein thrombosis (DVT) and pulmonary embolism (PE) at these meetings, despite continual improvements in patient care, prompted this study. It aims to use the database to study changes in the incidence of DVT and PE over the previous twenty years; to find what database variables predict these complications; and whether use of mechanical and pharmacological agents has had an impact on DVT and PE rate. Univariate analysis of the incidence of DVT and PE by age, sex, length of stay (LOS), admission month, diagnosis, operation and surgeon over time was run. Any significant variables were then analysed by multivariate logistic regression. The DVT rate was low by world standards, but rose from 0.6% in 1979-83 to 1.2% in 1984-88, then rose exponentially to 3.60% in 1994-98 with a significantly increasing trend over the twenty years (c2 MH =114.20, with IDF, P<0.001). PE rate doubled significantly over the twenty years from 0.6% to 1.2% (c2 MH =17.94 with 1DF, P<0.001). Age, LOS, diagnosis, operation and surgeon were significant predictors of DVT and PE. After adjustment for LOS, time period and age, vascular surgery was found to be the strongest predictor of DVT (OR=2.82, 95% CI: 2.08-3.82, c2 =43.91, P<0.01). Vascular diagnosis was the strongest diagnosis predictor. No effect of sex or month of admission was shown. After adjustment for LOS, time period and age, spinal fusion was the strongest predictor of PE (OR=4.04, 95% CI: 1.81-9.03). Anterior communicating artery aneurysm was the diagnosis most highly associated with PE. The rise in DVT rate may be due to increased complexity of surgical and nursing management, and some screening of patients with the introduction of duplex scanning. The doubling of PE rate is unexplained. The risk of brain or spinal cord haemorrhage makes prophylactic anticoagulation a difficult choice. This study reveals groupings which can be used to determine appropriate prophylaxis. Use of mechanical and pharmaceutical agents is not recorded consistently in the database, but it is known approximately when they were introduced. No impact on the rate of DVT and PE can be demonstrated by these agents. More vigilant and widespread use of mechanical prophylaxis might be just as effective in controlling DVT and PE.

No description available.

Lupus anticoagulants (LA) have been identified as antiphospholipid antibodies which can alter certain membrane-related endothelial activities, resulting in increased thrombogenesis. The effects of LA-containing patient plasmas on selected thrombosis-related endothelial functions were examined; 68% of 25 patient plasmas exhibited significant antiphospholipid antibody (APA) IgG and/or IgM directed against one of the four phospholipids tested in vitro; 44% exhibited anti-endothelial antibody. Eighty percent of plasmas with anti-endothelial IgM exhibited APA reactive with phosphatidyl inositol; antiphospholipid IgG did not correlate well with antiendothelium IgG. Patient plasmas that significantly stimulated EC PGI2 secretion uniformly exhibited anti-phosphatidyl serine IgM.Multiple mechanisms of induction of LA, and strong association of anti-PS and anti-EC antibody with thrombosis and related disorders were observed.Ball State UniversityMuncie, IN 47306