Thesis (doctoral)--Karolinska Institutet, Stockholm, 1991. / T.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1991. / T.p. with thesis statement inserted. Includes bibliographical references.

abstract: Thrombus (blood clot) formation is at the roots of hemostasis and pathological thrombosis. Although many studies have successfully elucidated the cellular and molecular mechanisms underlying thrombus formation, there is still a void in understanding the processes limiting thrombus growth beyond that needed for stabilization. As a hemostatic thrombus grows, its surface consisting primarily of platelets changes to that composed of fibrin, which mechanically stabilizes the thrombus. Formation of fibrin ceases after some time; however, it is unclear why this fibrin is non-thrombogenic. This is puzzling since fibrin is known to support strong integrin-mediated adhesion of both platelets and leukocytes in vitro. Therefore, it would be expected that the fibrin surface of hemostatic thrombi in the circulation also support accumulation of these cells and thus continuous thrombus growth or degradation. Nevertheless, many in vivo studies did not detect any accumulation of blood cells including platelets at the fibrin surfaces of thrombi. This finding suggests the existence of natural processes that modulate the adhesive properties of fibrin to ensure proper regulation of thrombus growth, stability and degradation. In this dissertation, I document and discuss the findings supporting the existence of anti-adhesive mechanisms and their physiological relevance in surface-mediated control of thrombus growth and stability. The studies discussed in my dissertation have the potential to establish a novel aspect of hemostasis. Furthermore, it may provide new insights into the intricate and dynamic interplay between the mechanisms underlying hemostatic balance, which is essential to understanding the dysfunction of this process during pathological conditions. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2016

Orientador: Maria Elvira Pizzigatti Correa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-14T11:31:27Z (GMT). No. of bitstreams: 1 Basso_FernandaGoncalves_M.pdf: 2327570 bytes, checksum: 79b8d36d7cb580fa421c4ca5a21a76a1 (MD5) Previous issue date: 2009 / Resumo: Fibrinólise é o processo responsável pelo restabelecimento do fluxo sanguíneo no interior dos vasos, através da dissolução do coágulo formado após uma injúria vascular. Esse processo pode ser influenciado por diferentes fatores, como trauma tecidual e presença de processos inflamatórios ou infecciosos, que podem causar um aumento da atividade fibrinolítica local. Esse aumento, por sua vez, poderia causar a dissolução precoce do coágulo, aumentando o risco de eventos hemorrágicos pós-procedimentos invasivos, como extrações dentárias, principalmente em pacientes cujo processo hemostático encontra-se alterado, como aqueles sob anticoagulação oral. Portanto, o objetivo deste estudo foi o de avaliar a atividade fibrinolítica da cavidade oral de pacientes sob terapia de anticoagulação cumarínica, avaliando também fatores locais que pudessem influenciar esta atividade. Para tanto, foram selecionados 12 pacientes sob terapia de anticoagulação cumarínica com indicação para extrações dentárias, que foram submetidos a 20 procedimentos. Esses pacientes foram também submetidos à avaliação clínica e radiográfica, além de avaliação dos índices de saúde oral (Índice Gengival, índice de Placa e CPOD). Para avaliar a atividade fibrinolítica, foram coletadas amostras de saliva não-estimulada, pré e pós-procedimento de extração dentária, de sangue alveolar e de sangue periférico. Essas amostras de saliva e de sangue foram submetidas à avaliação da atividade fibrinolítica através do teste de Área de Lise em Placa de Fibrina. Para análise do nível de anticoagulação, foram realizados os testes de Tempo de Protrombina e análise da atividade dos fatores da coagulação dependentes de vintamina K (FII, FVII, FIX e FX). Nenhum evento hemorrágico foi observado no período pós-extração dentária. Os resultados do estudo da atividade fibrinolítica no sangue mostraram que esta foi maior na amostra de sangue alveolar, quando comparada ao sangue periférico (p=0,006). Essa atividade, por sua vez, apresentou correlação estatisticamente significativa com os índices de saúde oral (p=0,003/p=0,002). Os resultados do estudo da atividade fibrinolítica salivar mostraram um aumento significativo desta atividade após o procedimento de extração dentária (p=0,002/ p=0,003). Esse resultado, no entanto, não pôde ser correlacionado à variação do fluxo salivar e tampouco aos índices de saúde oral (IG e IP). Quando correlacionados a atividade fibrinolítica do sangue periférico e o nível de anticoagulação, estes não apresentaram correlação positiva (p=0,28). A correlação entre a atividade fibrinolítica do sangue alveolar e o nível de anticoagulação se mostrou limítrofe (p=0,053). A atividade fibrinolítica da cavidade oral parece estar fortemente associada aos fatores locais, como trauma tecidual e eventos inflamatórios, não apresentando a mesma associação com a anticoagulação. / Abstract: Fibrinolysis is a part of the haemostatic process that is responsible for reestablish the blood flow, by the dissolution of the fibrin clot formed after a vascular injury. This process can be altered by several factors, such as tissue trauma and presence of inflammatory or infectious process, which can increase the local fibrinolytic activity and, by that, cause precocious clot dissolution. This could increase the hemorrhagic risk after invasive procedures, like teeth extractions, especially in patients under oral anticoagulation. The aim of this study was to evaluate the oral fibrinolytic activity of patients under oral anticoagulation with cumarin agents and also to evaluate the local factors that could be involved on this activity. Twelve patients under oral anticoagulation who needed teeth extractions were enrolled on this study and submitted to twenty teeth extractions. These patients were submitted to clinical and radiographic evaluation and oral health analysis, by the measurement of oral health indexes (Gingival Index, Plaque Index and Decayed, Missing and Filled Teeth). Samples of non-stimulated saliva were collected before and after each procedure and samples of alveolar and peripheral blood were also collected. These samples were submitted to fibrinolytic activity analysis, by the Fibrin Plate Method. For the anticoagulation analysis, prothrombin time assay and analysis of activity of vitamin-K-dependent coagulation factors (II, VII, IX and X) were performed. As result, no hemorrhagic event was observed after the procedures. The results of the blood fibrinolytic activity analysis showed that the alveolar blood presented a higher fibriolytic activity than the peripheral blood (p=0,006). This activity also showed a positive correlation with the oral health indexes (p=0,003 - GI/ p=0,002 - PI). The salivary fibrinolytic activity showed a significant increase after the tooth extraction (p=0,002 - supernatant fraction/p=0,003 - precipitated fraction). This activity, however, could not be associated with the oral health indexes. The level of anticoagulation showed no correlation with the fibrinolytic activity of peripheral blood (p=0,28) and showed a bordering correlation with the fibrinolytic activity of the alveolar blood (p=0,053). The fibrinolytic activity of the oral cavity seems to be strongly associated to local factors, such as local trauma and local inflammatory conditions, not showing the same association to the oral anticoagulation itself. / Mestrado / Patologia / Mestre em Estomatopatologia

Orientador: Marcos Jun Watanabe / Coorientador: Carlos Alberto Husnni / Banca: Juliana de Moura Alonso / Banca: Paula Alessandra Di Filippo / Resumo: Nos equinos, os distúrbios gastrointestinais (GI) são as causas mais comuns de problemas de coagulação, podendo gerar complicações fatais. Atualmente, uma das ferramentas mais sensíveis para avaliação da hipercoagulabilidade e hiperfibrinólise em cavalos é a determinação das concentrações de dímeros-D. Dessa forma, objetivou-se relacionar as concentrações plasmáticas e peritoneais de dímeros-D com as variáveis clínicas (frequência cardíaca, frequência respiratória, cor das membranas mucosas, tempo de preenchimento capilar, temperatura retal, grau de dor e tempo de evolução do quadro) e laboratoriais (hematócrito, proteína plasmática total, plaquetas, fibrinogênio e leucócitos sanguíneos, além de proteína, fibrinogênio, células nucleadas, bactérias e glicose no líquido peritoneal) de equinos com síndrome cólica e com o diagnóstico e prognóstico desses casos. Foram utilizados 86 equinos com idade mediana de 6,5 anos e com peso mediano de 400 Kg. Os animais foram submetidos ao exame clínico e coleta de amostras de sangue e líquido peritoneal (LP) na admissão. As concentrações plasmáticas e peritoneais de dímeros-D foram avaliadas através de um ensaio semiquantitativo de aglutinação em látex. Apesar das concentrações plasmáticas e peritoneais de dímeros-D terem demonstrado um sentido biológico relacionado à gravidade dos casos de cólica na análise descritiva, isso não foi comprovado estatisticamente através da análise multivariada. Portanto, concluímos que a determinação das conc... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In horses, gastrointestinal (GI) disorders are the most common cause of coagulation problems, which can lead to fatal complications. One of the most sensitive tools for assessing hypercoagulability and hyperfibrinolysis in horses is the determination of D-dimer concentrations. The aim of this study was to correlate the plasma and peritoneal D-dimer concentrations with the clinical variables (heart rate, respiratory rate, mucous membranes color, capillary filling time, rectal temperature, pain degree, and time frame evolution) (hematocrit, total plasma protein, platelets, fibrinogen and blood leukocytes, as well as protein, fibrinogen, nucleated cells, bacteria and glucose in the peritoneal fluid) of horses with colic syndrome and with the diagnosis and prognosis of these cases. A total of 86 horses with a median age of 6.5 years and with a median weight of 400 kg were used. The animals were submitted to clinical examination and collection of blood and peritoneal fluid (LP) samples at admission. Plasma and peritoneal concentrations of D-dimers were evaluated by a semi-quantitative latex agglutination assay. Although plasma and peritoneal concentrations of D-dimers demonstrated a biological significance related to the severity of colic cases in the descriptive analysis, this was not statistically demonstrated through multivariate analysis. Therefore, we concluded that the determination of plasma and peritoneal concentrations of D-dimers using semi-quantitative latex agglutinati... (Complete abstract click electronic access below) / Mestre

Optimal induction of coronary thrombolysis depends in part upon the nature of the specific plasminogen activator used. The two general classes of plasminogen activators available clinically differ in a fundamental respect delineated by the term, clot selectivity. Clot selective agents are less prone to induce plasminemia and consequent occult activation of the coagulation cascade than are non-selective agents. However, under clinical conditions, all plasminogen activators result in some activation of the cascade with consequent generation of thrombin. Accordingly, optimal therapy requires the use of conjunctive anticoagulation to preclude the deleterious effects of rebound generation of thrombin, which has been well documented biochemically. The potential value of antiplatelet agents that can attenuate the positive feedback loop between activation of platelets and markedly amplified generation of thrombin in the setting of coronary thrombolysis is under active exploration. With appropriate monitoring of the efficacy of such agents in vivo it should be possible to enhance even further the benefits that can be conferred by pharmacologically induced coronary thrombolysis.

The endothelial protein C receptor (EPCR) binds to protein C (PC) and increases the rate of activated protein C (APC) generation by the thrombin-thrombomodulin (TM) complex. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects, which are EPCR-dependent. The thrombin-TM complex is also a potent activator of thrombin activable fibrinolysis inhibitor (TAFI), leading to impaired clot lysis. Mutations and polymorphisms identified in the EPCR gene, which can affect the efficiency of PC activation, have been associated with an increased risk of thrombosis. In this thesis we investigate the impact of impaired PC binding to EPCR on coagulation, inflammation, and fibrinolysis using novel in vitro and in vivo models. Using a murine model that harbours a variant of EPCR that does not bind PC (R84A), we demonstrate that upon thrombotic challenge, there is an increase in thrombin generation and fibrin deposition in the lungs. Upon inflammatory challenge, impaired PC/EPCR interactions also result in increased thrombin generation and increased neutrophil infiltration into the lungs. Using cells that express TM and a human variant of EPCR that does not bind PC (R96C), we demonstrate that clot lysis is delayed in normal plasma independent of TAFI activation, suggesting PC and TAFI do not compete for activation by the thrombin-TM complex. In contrast, delayed clot lysis in plasma deficient of PC is a result of greater TAFI activation by the thrombin-TM complex. Taken together, impairment of the PC pathway contributes to thrombosis through pro-coagulant, pro-inflammatory and anti-fibrinolytic mechanisms. Interestingly, mice with EPCR variant R84A, develop bone marrow failure and splenomegaly, revealing a novel role for EPCR in the bone marrow. Taken together, PC/EPCR interactions regulate the coagulation, inflammation, and fibrinolytic pathways, which may have a significant impact on maintaining hematopoietic homeostasis. / Thesis / Doctor of Philosophy (PhD) / Under normal conditions, blood is maintained in a fluid state. Upon injury or infection, the blood begins to form a clot to prevent bleeding. Once bleeding has stopped the clot is dissolved and blood regains its fluid state. The formation of a blood clot is a serious and potentially life threatening disease. A blood clot formed inside a blood vessel can block the flow of blood through the circulation, leading to organ damage. Approximately 50% of blood clots are caused by known genetic or environmental factors, leaving 50% of blood clots caused by unknown factors. In this thesis we investigate the unknown factors that contribute to blood clotting. In patients who have experienced blood clots with no known cause, we have identified genetic mutations in a blood vessel wall protein, known as the endothelial protein C receptor (EPCR) that renders it non-functional. We demonstrate both in vitro and in vivo that non-functional EPCR not only leads to the formation of a blood clot but also delays the removal of the blood clot. Our in vivo studies have also revealed a previously unknown role for EPCR in the bone marrow, likely through its effects on blood coagulation. Taken together, loss of EPCR function contributes to the development of clot formation and likely impacts other organ systems.

Plasmin is the principal endogenous protease in bovine milk. Distribution of overall proteolytic potential among milk fractions was determined using Coumarin Substrate as a synthetic substrate. Casein containing fractions had a higher amidolytic potential. However, preparation of casein by acid treatment produced increased dissociation of plasminogen and plasmin from casein. The variable results obtained from milks of different cows could be due in part to the influence of inhibitors and activators of the fibrinolytic system present in milk. We have shown, for the first time, the occurrence of α₂-M in bovine skimmilk (using SRID) at a level (using ELISA) of 1.54 +/- 0.91 mg/ml. This inhibitor appeared to primarily associate with the acid whey fraction. A high level (< 1 mg/ml) of α₂-M was also detected in human skimmilk. The other major fibrinolytic inhibitor, α₂-AP, as well as the complex formed between this inhibitor and plasmin were also shown to occur in human milk. We used a coupled colorimetric assay to demonstrate the occurrence of a fibrin-independent plasminogen activator (similar to u-PA) in bovine skimmilk. The occurrence of a u-PA like activator in bovine milk was further confirmed in co-polymerized gel electrophoresis. Moreover, u-PA could also be detected in a sample of human skimmilk. However, the electrophoretic gel patterns also contained additional zones of clearing which may be due to the occurrence of other activators in bovine milk. These plasminogen activators may be fragments of u-PA, or t-PA (shown to occur in sow milk) which retain catalytic activity. The occurrence of such high levels of α₂-M (~ 4% of the total protein) and plasminogen activators may be of tremendous significance to the dairy industry, as they may not only influence plasmin-mediated proteolysis of milk proteins, but may also interfere with the action of milk clotting enzymes. / Master of Science

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot. / Thesis (Master, Biochemistry) -- Queen's University, 2008-01-31 17:04:50.447

Background: Cardiovascular disease (CVD) is the leading global cause of death. CVD risk factors are considered intermediaries for the association between dietary fatty acids and CVD. Raised plasma total cholesterol, low density lipoprotein (LDL) cholesterol, raised triglycerides and decreased levels of high density lipoprotein (HDL) cholesterol, as well as reduced fibrinolytic potential (measured as increased clot lysis time) are known risk factors for CVD. Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of the fibrinolytic process and an elevated PAI-1 level is therefore considered to be a potential risk factor for CVD. The growing number of controversies around the role that fat intake (more specifically the type of dietary fat) plays in CVD risk, is making it increasingly difficult for consumers and practitioners alike to form conclusions, and make recommendations and decisions regarding fat intake. Knowledge of the intake of individual fatty acids, fatty acid status (as opposed to subgroups of fat such as polyunsaturated fatty acids) and their associations with blood lipids, PAI-1act and fibrinolytic potential is lacking in black South Africans and other populations. Therefore we aimed to investigate dietary fatty acid intake, as well as plasma phospholipid fatty acid status and their associations with blood lipids, PAI-1act and clot lysis time, as a marker for fibrinolytic potential. Methods: Cross-sectional data analysis within the Prospective Rural Urban Epidemiology (PURE) baseline study of apparently healthy black South African men and women (n=1950, 35– 70 years) from rural and urban areas in the North West Province, from whom dietary data were collected. Blood lipid analyses, as well as laboratory analyses of fibrinolysis markers such as PAI-1act and clot lysis time were also performed. Plasma phospholipid fatty acid extraction and isolation were performed on a random subsample (n = 716). Results: The intake of individual fatty acids was significantly higher in urban than rural dwellers. However, the intake of omega-3 polyunsaturated fatty acids was below recommendations in all groups (rural and urban males, and rural and urban females). Total cholesterol and LDL cholesterol were higher in females than in males, with no rural‒urban differences. Intake of alpha-linolenic acid was positively associated with total cholesterol (β=0.143) and triglycerides (β=0.256) in males. The risk of having elevated LDL cholesterol also increased with increased intake of alpha-linolenic acid (OR 1.49, 95% CI 1.04, 2.14). In females, dietary arachidonic acid and eicosapentaenoic acid (EPA) were positively associated with total cholesterol and LDL cholesterol, whereas docosahexaenoic acid (DHA) was negatively associated with total cholesterol and LDL cholesterol. Dietary alpha-linolenic acid was positively correlated with plasma EPA (males r = 0.19, p = 0.002, females r = 0.25, p < 0.001) and DHA (males r = 0.33, p < 0.001, females r = 0.30, p < 0.001). Plasma DHA was positively associated with triglycerides in males (β = 0.410, p< 0.001) and in females (β = 0.379, p< 0.001). PAI-1act was positively associated with clot lysis time, and plasma myristic acid and DHA were positively associated with PAI-1act in females. However, these fatty acids were not associated with clot lysis time. Different types of plasma fatty acids were associated with PAI-1act than with clot lysis time. Plasma alpha-linolenic acid (β = 0.123, P = 0.037), mead acid (β = 0.176, P = 0.019), arachidonic acid (β = 0.253, 0.025) and omega-3 docosapentaenoic acid (omega-3 DPA) (β = 0.224, P = 0.002) were positively associated with clot lysis time, while both myristic acid (β = - 0.130, P = 0.016) and EPA (β = -0.131, P = 0.021) were negatively associated with clot lysis time in male subjects. Plasma oleic acid (C18:1n9) (β = -0.411, P = 0.001) and omega-6 DPA (C22:5n6) (β = -0.285, P = 0.001) were negatively associated with clot lysis time, while dihomogamma- liolenic acid (DGLA) (C20:3n6) were positively associated (β = 0.178, P = 0.001) with clot lysis time in females. Conclusions: These results suggest that specific individual dietary fatty acids might be associated with blood lipids in males differently than in females, irrespective of rural or urban dwelling. It is not known however, if associations would still be present under conditions of greater intake of alpha-linolenic acid. Our results further suggest that a higher percentage of alpha-linolenic acid might be converted to DHA in this population with low intake of essential and long-chain polyunsaturated fatty acids compared to populations with a high intake of these fatty acids. These results suggest that plasma phospholipid fatty acids should not be used in isolation as biomarkers for intake of fat, without taking dietary intake data into consideration also. Associations between fatty acids and clot lysis time might be independent from PAI-1act. The association between mead acid and clot lysis time indicates that clot lysis time might increase with an essential fatty acid deficiency. This may be of particular concern in this population with a documented lower fat intake. Because the study design of this study is crosssectional, it is not able to determine cause-and-effect, and results should therefore be verified with a randomised controlled trial. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015