Os mecanismos responsáveis pelas manifestações hemorrágicas durante a leptospirose severa ainda são pouco compreendidos. Este trabalho avaliou os efeitos diretos e indiretos das leptospiras sobre moléculas do sistema de coagulação. Foi verificado que leptospiras virulentas são eficientes em bloquear a atividade da enzima trombina por meio do sequestro de seu sítio de ligação ao substrato, gerando uma menor formação do coágulo de fibrina, acarretando em sangramento e consequente disseminação do patógeno para outros sítios de infecção. Foi mostrado também que a inflamação causada pela resposta imune contra as bactérias causa uma ativação da coagulação, muito provavelmente via expressão de Fator Tissular, o que ocasiona um consumo e consequente esgotamento dos fatores e inibidores de coagulação, culminando em hemorragia e formação de trombos, os quais podem levar à falência de órgãos. Estes resultados melhoram o entendimento da patogênese da leptospirose e podem favorecer o desenvolvimento de terapias para as manifestações hemorrágicas. / The mechanisms responsible for the hemorrhagic manifestations during severe leptospirosis are still poorly understood. This work evaluated the direct and indirect effects of leptospires upon molecules of the coagulation system. It has been shown that virulent leptospires are effective in blocking the activity of the enzyme thrombin by sequestration of its substrate binding site, generating less fibrin clot formation, leading to bleeding and consequent dissemination of the pathogen to other sites of infection. It has also been shown that the inflammation caused by the immune response against the bacteria causes an activation of the coagulation, most probably via expression of Tissue Factor, which causes a consumption and consequent depletion of coagulation factors and inhibitors, culminating in hemorrhage and thrombus formation, which can lead to organ failure. These results improve the understanding of the pathogenesis of leptospirosis and may favor the development of therapies for hemorrhagic manifestations.

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INTRODUCTION: The menstrual cycle and pregnancy are processes unique to women. Both these processes involve various hormones as well as the coagulation system. Throughout normal pregnancy, platelet activation and increase in blood coagulation factors contributes to the hypercoagulable state observed on a physiological level. METHODS: Fibrin networks and platelets were analysed by electron microscopy and flow cytometry to determine any differences found in different phases of pregnancy compared to healthy control individuals. The fibrin networks from different phases of the menstrual cycle as well as different phases of pregnancy were investigated. RESULTS: It was found that ultrastructural changes in fibrin fiber morphology result from estrogen changes during the menstrual cycle. During pregnancy the minor thin fibers were prominent and thick matted layers of coagulant formation were evident. A large quantity of protein globular clusters similar to those seen in the menstrual cycle was present. Changes observed in platelet ultrastructure during pregnancy showed pregnancy-specific modifications. Platelets were activated and internal organelles showed variation from control participants. Flow cytometric analysis of platelets verified pregnancy-specific modifications. Close interactions between platelets and erythrocytes were evident. CONCLUSION: The female body is equipped to handle alterations in the coagulation system as can be extrapolated from the pregnancy-specific modifications. This study is the first to show alterations in fibrin network and platelet ultrastructure during and after pregnancy when compared to non-pregnant controls. The physiological changes during normal pregnancy can be used as a standard for comparison to abnormal or ailing pregnancy. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Physiology / Unrestricted

Fluid resuscitation with crystalloid and colloid solutions is a common treatment in perioperative medicine. However, a variety of unbalanced or balanced solutions is used in clinical practice and there is still a vivid debate going on regarding the selection of optimal fluid with minimal negative effect on coagulation. The goal of the dissertation was to investigate the adverse effect of balanced crystalloids and colloids on whole blood coagulation measured by method of rotational thromboelastometry. In the first phase of the work we had assessed the adverse effect of balanced crystalloid, hydroxyethyl starch and gelatin after dilution of blood with the solution in vitro. Parametrs of EXTEM and FIBTEM tests were evaluated by using rotational thromboelastometry. In the second phase of the work we evaluated the negative effect of infusion solution after dilution in vivo. We had obtained blood samples from 30 patients during knee arthroscopy before and after administration of 500 ml of crystalloid or hydroxyethyl starch or gelatin. Parametrs of EXTEM and FIBTEM tests were evaluated by using rotational thromboelastometry.In compliance with the results of the dissertation, hydroxyethyl starch has the most obvious negative effect on clot formation followed by gelatin and finally by crystalloids. Based on...

Ceramic membranes (CM) are robust membranes attributed with high production, long life span and stability against critical conditions. While capital costs are high, these are partially offset by lower operation and maintenance costs compared to polymeric membranes. Like any other low-pressure membrane (LPM), CM faces problems of fouling, low removal of organic matter and poor removal of trace organic compounds (TOrCs). Current pretreatment approaches that are mainly based on coagulation and adsorption can remove some organic matter but with a low removal of the biopolymers component which is responsible for fouling. Powdered activated carbon (PAC) accompanied with a LPM maintains good removal of TOrCs. However, enhanced removal of TOrCs to higher level is required. Submicron powdered activated carbon (SPAC), obtained after crushing commercial activated carbon into very fine particle, and novel activated carbon (KCU 6) which is characterized with larger pores and high surface area were employed. A pre-coating approach, which provides intimated contact between PAC and contaminants, was adopted for wastewater and (high DOC) surface water treatment. For seawater, in-line coagulation with iron III chloride was adopted. Both SPAC and KCU 6 showed good removal of biopolymers at a dose of 30 mg/L with > 85 % and 90 %, respectively. A dose of 40 mg/L of SPAC and 30 mg/L KCU 6 pre-coats were successful used in controlling membrane fouling. SPAC is suggested to remove biopolymers by physical means and adsorption while KCU 6 removed biopolymers through adsorption. Both KCU 6 and SPAC attained high removal of TOrCs whereas KCU 6 showed outstanding performance. Out of 29 TOrCs investigated, KCU 6 showed > 87 % TOrCs rejection for 28 compounds. In seawater pretreatment, transparent exopolymer particles (TEP) were found to be an important foulant. TEP promoted both reversible and irreversible fouling. TEP are highly electronegative while alumina CM is positively charged which support strong TEP–alumina binding. The influence of TEP fouling was minimized with a low dose of 0.5 – 1 mg/l Fe coagulant. Bacteria were almost completely removed; Silt Density Index (SDI) value was maintained to 2 % per minute and a constant permeate turbidity of 0.05 NTU was achieved.

The goal of this study was to evaluate the feasibility of using advanced coagulation with Fe(VI) in coagulation-flocculation-sedimentation/ flotation systems for the pretreatment of SWRO desalination plants during algal bloom events. Algal organic matter (AOM) material extracted from marine diatom species (Chaetoceros affinis) was added to Red Sea water to mimic algal bloom conditions. Low dosage of Fe(VI) (<1 mg Fe/L) was very effective at improving feed water quality containing AOM (algal bloom conditions). Based on results from both a bench-scale DAF unit and Jar testing unit, 0.75 mg Fe/L of Fe (VI) proved to be effective at improving the raw water quality which is comparable to the performance of 1 and 3 mg Fe/L of Fe (III). The removal efficiency for both testing units with the use of Fe(VI) was up to 100% for algae , 99.99% for ATP, 99% for biopolymers and 70 % for DOC. The improvement in Fe(VI) performance is related to the simultaneous action of Fe(VI) as oxidant, disinfectant and coagulant. The performance of Fe(VI) coagulant was also evaluated with the use of coagulant aids (clays). The overall turbidity, DOC, biopolymers and algal cells removal was improved via using Fe(VI) and clays at very low dose. Generally, it was found that for the same pretreatment performance achieved, a much lower Fe(VI) dose was required compared to Fe (III), which make it important to study of cost effectiveness for using Fe(VI) instead of Fe(III) and estimate cost savings during algal bloom conditions. A detailed cost comparative study was conducted for Fe(III) vs. Fe(VI) coagulation process based on the removal efficiency. The use of Fe(VI) reduced the total pretreatment cost by 77% and sludge disposal cost by > 88% compared to the use of Fe(III) in the pretreatment process. The use of Fe(VI) reduces the operational and maintenance cost in SWRO desalination plant by 7% and the production cost by 4%. This study proved that the use of Fe(VI) during high turgidity and algal bloom conditions helped providing high raw water quality to the RO process with lower chemicals and operations cost as well as low chlorine and iron residuals.

On vessel wall injury the complex process of blood coagulation is set off. It is composed of vasoconstriction, primary hemostasis, secondary hemostasis and fibrinolysis. This work enriches current model of primary hemostasis of Storti. The previous model used ALE formalism for tracing of development of platelet plug. The phase field method is used for tracing of the development of interface blood-thrombus. Storti's primary hemostasis was extended to capture the fact, that the platelets can be activated in the blood flow in the area of reactive surface not only by influence of chemical agents like thromboxane, ADP and thrombin but also by their exposure to elevated values of shear stress. In our first approach we deal the emerging thrombus as a fluid with very high viscosity. In the second approach it was assumed, that platelet plug develops as a viscoelastic material according to constitutive equations of clot introduced by Kempen. In this manner platelet clot matures into blood clot. In both approaches the blood is represented as a non-Newtonian fluid. The framework of the phase field method was applied also to the model of high shear rate thrombosis of Weller. Weller's original model of Weller took advantage of the cylindrical symmetry of computational domains for its computations, hence the...

Transparent exopolymer particles (TEP) have been reported as one of the main factors of membrane fouling in seawater reverse osmosis (SWRO) process. Research has been focused on algal TEP so far, overlooking bacterial TEP. This thesis investigated the effects of coagulation on removal of bacterial TEP/TEP precursors in seawater and subsequent reduction on TEP fouling in ultrafiltration (UF), as a pretreatment of SWRO. Furthermore, the performance of pretreatment (coagulation + UF) has been investigated on a bench-scale SWRO system. TEP/TEP precursors were harvested from a strain of marine bacteria, Pseudoalteromonas atlantica, isolated from the Red Sea. Isolated bacterial organic matter (BOM), containing 1.5 mg xanthan gum eq./L TEP/TEP precursors, were dosed in Red Sea water to mimic a high TEP concentration event. Bacterial TEP/TEP precursors added to seawater were coagulated with ferric chloride and aluminum sulfate at different dosages and pH. Results showed that ferric chloride had a better removal efficiency on TEP/TEP precursors. Afterwards, the non-coagulated/coagulated seawater were tested on a UF system at a constant flux of 130 L/m2h, using two types of commercially available membranes, with pore sizes of 50 kDa and 100 kDa, respectively. The fouling potential of coagulated water was determined by the Modified Fouling Index (MFI-UF). Transmembrane pressure (TMP) was also continuously monitored to investigate the fouling development on UF membranes. TEP concentrations in samples were determined by the alcian blue staining assay. Liquid chromatography-organic carbon detection (LC-OCD) was used to determine the removal of TEP precursors with particular emphasis on biopolymers. Finally, SWRO tests showed that TEP/TEP precursors had a high fouling potential as indicated by MFI-UF, corresponding to the TMP measurements. Coagulation could substantially reduce TEP/TEP precursors fouling in UF when its dosage was equal or higher than 0.2 mg Fe/L. The flux decline experiments showed that coagulation + UF pretreated water had a smaller fouling potential than MF pretreated water. This thesis also provides useful and practical information on controlling bacterial TEP/TEP precursors fouling in UF and RO systems.

Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis. / Thesis / Doctor of Philosophy (Medical Science)

To separate plasma/serum from blood cells, blood samples are centrifuged. Currently, there is no standard centrifugation protocol available. However, there are several centrifugation recommendations from the tube manufacturers. The aim of this study is to evaluate centrifugation program with a shorter centrifugation time and higher centrifugal force and how it affects measured analyte concentrations. 120 sample pairs were collected in lithium-heparin tubes and sodium citrate tubes from 120 participants. One sample of each pair was then centrifuged with reference program (2000g, 10min) for routine chemistry samples and (2500g, 15min) for coagulation samples. The other sample of each pair was centrifuged with programs (3000g, 7min). The statistical analysis was performed using the Student’s t-test and the Holm-Bonferroni method. P-value less than 0.05 was considered statistically significant.There was no significant difference between data from BD tubes and Greiner tubes, therefore the corresponding data from both groups were merged. No statistically significant difference was observed for the following parameters; Cobalamin (B12), High-density lipoprotein (HDL), Lactate dehydrogenase (LD), Phosphate, Triglycerides (TG), Thyroid stimulating hormone (TSH), Activated partial thromboplastin time (APTT), Fibrinogen (Fib) and Prothrombin complex PK (INRR). However, the statistical analysis showed a significant difference regarding potassium (K). Centrifugation program (3000g, 7min) can replace the programs (2000g, 10min) and (2500g, 15min) which are recommended by the tube manufacturers BD &amp; Greiner, with the exception of Potassium which requires more research. A trace of increasing hemolysis, still within acceptable limits, was detected in samples centrifuged with programs (3000g, 7min). The centrifugation programs with a shorter time or less acceleration is preferred. / Blodprover centrifugeras för att separera plasma/serum från blodceller. Inget standardprotokoll för centrifugering är tillgänglig dock finns flertal centrifugeringsrekommendationer från rörtillverkarna. Syftet med denna studie är att utvärdera centrifugeringsprogram med en kortare centrifugeringstid och högre g-tal, samt hur det påverkar uppmätta analytkoncentrationer. 120 provpar samlades i litium-heparin-rör respektive natrium-citrat-rör från 120 deltagare. Det ena provet av varje provpar centrifugerades med referensprogram (2000g, 10 min) för rutinkemi respektive (2500g, 15min) för koagulation medan det andra centrifugerades med program (3000g, 7min). Statistiska analysen utfördes med hjälp av Student t-test och Holm-Bonferroni metoden. P-värde mindre än 0,05 ansåg vara statistiskt signifikant. Ingen betydande skillnad fanns mellan data från BD-rör och Greiner-rör, därför har motsvarande data från både gruppen slagits ihop. Ingen statistiskt signifikant skillnad observerades för följande undersökta parametrar; Kobalamin (B12), High-density lipoprotein (HDL), Laktatdehydrogenas (LD), Fosfat, Triglycerider (TG), Tyroideastimulerande hormon (TSH), Aktiverad partiell tromboplastintid (APTT), Fibrinogin (Fib) och Protrombinkomplex PK(INR). Däremot visade den statistiska analysen en signifikant skillnad avseende kalium (K). Centrifugeringsprogram (3000g, 7min) kan ersätta programmen (2000g, 10min) och (2500g, 15min) som rekommenderas av rörtillverkarna BD &amp; Greiner, med undantag av Kalium som kräver mer undersökning. Ett spår av ökande hemolys, dock fortfarande inom acceptabla gränser, upptäcktes i prover centrifugerades med program (3000g, 7min). Därpå är centrifugeringsprogram med kortare tid eller mindre hastighet att föredra.