京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第24292号 / 人健博第107号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

<p>Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with many clinical limitations, such as the inability to inhibit fibrin-bound coagulation factors, increasing the potential for sustained procoagulant activity. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties to those of UFH. Some advantages of ATH include enhanced inhibition of surface-bound enzymes and its ability to reduce the overall size and mass of clots <em>in vivo</em>. However, the potential interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Therefore, our study utilized discontinuous second order rate constant (<em>k₂</em>) assays to determine rates of inhibition of plasmin (Pn) in the presence or absence of fibrin by AT+UFH <em>vs.</em> ATH. In addition, we monitored the rates of Pn generation in a system comprised of preformed fibrin clots with the aim of evaluating the inhibitory effect of AT+UFH or ATH in this more native system. The <em>k₂</em>values for the inhibition of Pn without fibrin were 5.74x10⁶±0.278x10⁶ and 6.39x10⁶±0.588x10⁶ for AT+UFH and ATH, respectively (p=0.36). In the presence of fibrin, the <em>k₂</em>values decreased to 1.45x10⁶±0.0971x10⁶ for AT+UFH and 3.07x10⁶±0.192x10⁶for ATH (<em>p</em></p> / Master of Science (MS)

Quebec platelet disorder (QPD) is a bleeding disorder characterized by a gain of function defect in fibrinolysis. The hallmark feature of QPD is the marked overexpression of urokinase plasminogen activator (uPA) in megakaryocytes (MK) and platelets. The genetic cause of QPD is a tandem duplication of a ~78 kb region that encompasses the uPA gene, PLAU. As the mechanism of PLAU overexpression is unknown, gene regulatory mechanisms specifically epigenetics were evaluated at the PLAU locus in QPD MK and granulocytes, a QPD unaffected lineage. The aims of the thesis were to assess if QPD is associated with 1) genome wide methylation changes of promoter CpG islands, particularly at PLAU and 2) genome wide changes of active histone modifications H3K27Ac, H3K36me3 and H3K4me2, particularly at the region of PLAU duplication. Methylation and active histone enrichment analysis revealed that in QPD and control subjects, PLAU promoter CpG island was characterized by unaltered hypo-methylation and changes in active histone peak enrichments that were within the realm of having one extra copy of PLAU in both MK and granulocytes. The findings imply that the PLAU CNV mutation does not induce altered promoter methylation status and/or significantly alter active histone markers as the reason for the marked PLAU overexpression in QPD MK. Instead, the rearrangement of an active enhancer element, particularly an H3K27Ac enhancer expressed in MK but not granulocytes, that is upstream of the second copy of PLAU might underlie the marked PLAU expression by differentiated QPD MK. The thesis provides novel insights into the epigenetic regulation of PLAU that will be crucial to identifying the mechanism underlying the aberrant PLAU expression in QPD. / Thesis / Master of Science (MSc)

Cardiovascular diseases are considered the first cause of death in westernized societies. They are directly linked to atherosclerosis, a clinical condition characterized by a thickening of the arterial wall. Atherosclerosis is in his turn linked to various genetic and environmental factors; among those factors are high oxidized LDL levels and endothelial dysfunction. In the present study, we have analyzed in vitro the effect of myeloperoxidase oxidized LDL on endothelial cells at the level of fibrinolysis and cell motility.<p>In the first part of the work, we measured fibrinolysis in real time at the surface of endothelial cells. Our results suggest that myeloperoxidase oxidized LDL interferes with the regulation of fibrinolysis by endothelial cells by decreasing their pro-fibrinolytic activity. This effect was not related to a modification in expression of major regulators of fibrinolysis such as PAI-1 and t-PA. Our data link the current favorite hypothesis that oxidized LDL has a causal role in atheroma plaque formation with an old suggestion that fibrin may also play a causal role. A model that best explains our results would be as follows: oxidized LDL increases fibrin deposition on endothelial cells which will increase their permeability resulting in more oxidized LDL infiltration into the subendothelial space of the arterial wall initiating atherogenesis. <p>In the second part of the work, we investigated the effect of myeloperoxidase oxidized LDL at the level of endothelial cell motility. We have shown that oxidized LDL is able to decrease cell migration, wound healing and tubulogenesis in endothelial cells. Those effects were not associated with any alteration at the level of neither cell viability nor proliferation. Subsequent gene expression analyses enabled us to link the oxidized LDL induced phenotypical changes in the cells to a change in expression of both microRNA-22 and Heme Oxygenase 1 genes. Our observations suggest a novel role of oxidized LDL not only as an important factor in the initiation of atheromatous lesions, but also as a potential player in the progression of the atherosclerosis disease by impeding blood vessel repair and wound healing at the sites of lesions.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

INTRODUCTION - Plasminogen activator inhibitor type-1 (PAI-1) has a known relationship with obesity and more specifically with central obesity. Traditionally the physiological contribution of PAI-1 is seen as an indicator of fibrinolysis with increased PAI-1 levels contributing to decreased fibrinolysis. In more recent years, assays have been developed that not only uses proxy markers, such as PA-1, which is considered to be representative of fibrinolysis , but global assays that report on the global fibrinolytic potential of an individual, often reported as clot lysis time (CLT). Investigations into the relationship of CLT with obesity are scarce. Preliminary evidence shows that the relationship of CLT with obesity may differ from that of PAI-1 with obesity although in depth investigations in this regard are lacking. Therefore, the aim of this study was to investigate the association between fibrinolysis markers (PAI-1act and CLT) and various markers of body composition in the South African Prospective Urban and Rural Epidemiology (PURE) data collected during 2010. METHODS - Data collected in the PURE study in 2010 were cross-sectionally analysed. The participants (n = 1288) were apparently healthy black South-African men and women 35 years and older, residing in urban and rural settlements in the North-West Province. Experimental methods included anthropometric measurements such as height, weight, hip circumference, waist circumference, skinfolds (triceps, chest, abdominal, thigh and supra iliac skinfolds) and body composition measurements by means of air-displacement plethysmography and biolelectrical impedance analysis. Laboratory analysis of fibrinolysis markers, PAI-1act and CLT were also performed. MAIN FINDINGS - In men, similarities were seen regarding the relationship between PAI-1act and body composition markers and the relationships observed between CLT and body composition markers. In contrast, in the women more and stronger associations were observed between CLT and body composition markers compared to that observed between PAI-1act and body composition markers. CLT showed a linear relationship with body composition markers where PAI-1act levels plateaued at higher body composition categories. Possible reasons for the observed differences may be related to differences in adipose tissue distribution and sequence of accumulation between men and women. PAI-1 is associated with visceral adipose tissue (VAT) where high amounts of stromal cells are found. In men preferential accumulation of VAT may explain similarities in the relationship of PAI-1act with body composition and that of CLT with body composition. Proportionally less VAT, but more subcutaneous adipose tissue in women may explain the observed increase in CLT compared to PAI-1act levels that plateaued over body composition tertiles and categories. CONCLUSION - PAI-1act has a stronger association with central obesity while CLT has a stronger association with total body fat. In women PAI-1act and CLT showed different associations with body composition markers, whereas associations of PAI-1act and CLT with body composition were similar in men. PAI-1act is strongly influenced by type of body fat accumulation whereas CLT is associated with obesity independent of type and sequence of body fact accumulation. Significant associations observed between CLT and body composition variables are, therefore, at least in part, independent of PAI-1act. Additional factors such as, thrombin activatable fibrinolysis inhibitor (TAFI), α-2-antiplasmin, plasminogen, prothrombin and fibrin clot structure that influence CLT and are also related to obesity may additionally contribute to the link between CLT and obesity. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2014

INTRODUCTION - Plasminogen activator inhibitor type-1 (PAI-1) has a known relationship with obesity and more specifically with central obesity. Traditionally the physiological contribution of PAI-1 is seen as an indicator of fibrinolysis with increased PAI-1 levels contributing to decreased fibrinolysis. In more recent years, assays have been developed that not only uses proxy markers, such as PA-1, which is considered to be representative of fibrinolysis , but global assays that report on the global fibrinolytic potential of an individual, often reported as clot lysis time (CLT). Investigations into the relationship of CLT with obesity are scarce. Preliminary evidence shows that the relationship of CLT with obesity may differ from that of PAI-1 with obesity although in depth investigations in this regard are lacking. Therefore, the aim of this study was to investigate the association between fibrinolysis markers (PAI-1act and CLT) and various markers of body composition in the South African Prospective Urban and Rural Epidemiology (PURE) data collected during 2010. METHODS - Data collected in the PURE study in 2010 were cross-sectionally analysed. The participants (n = 1288) were apparently healthy black South-African men and women 35 years and older, residing in urban and rural settlements in the North-West Province. Experimental methods included anthropometric measurements such as height, weight, hip circumference, waist circumference, skinfolds (triceps, chest, abdominal, thigh and supra iliac skinfolds) and body composition measurements by means of air-displacement plethysmography and biolelectrical impedance analysis. Laboratory analysis of fibrinolysis markers, PAI-1act and CLT were also performed. MAIN FINDINGS - In men, similarities were seen regarding the relationship between PAI-1act and body composition markers and the relationships observed between CLT and body composition markers. In contrast, in the women more and stronger associations were observed between CLT and body composition markers compared to that observed between PAI-1act and body composition markers. CLT showed a linear relationship with body composition markers where PAI-1act levels plateaued at higher body composition categories. Possible reasons for the observed differences may be related to differences in adipose tissue distribution and sequence of accumulation between men and women. PAI-1 is associated with visceral adipose tissue (VAT) where high amounts of stromal cells are found. In men preferential accumulation of VAT may explain similarities in the relationship of PAI-1act with body composition and that of CLT with body composition. Proportionally less VAT, but more subcutaneous adipose tissue in women may explain the observed increase in CLT compared to PAI-1act levels that plateaued over body composition tertiles and categories. CONCLUSION - PAI-1act has a stronger association with central obesity while CLT has a stronger association with total body fat. In women PAI-1act and CLT showed different associations with body composition markers, whereas associations of PAI-1act and CLT with body composition were similar in men. PAI-1act is strongly influenced by type of body fat accumulation whereas CLT is associated with obesity independent of type and sequence of body fact accumulation. Significant associations observed between CLT and body composition variables are, therefore, at least in part, independent of PAI-1act. Additional factors such as, thrombin activatable fibrinolysis inhibitor (TAFI), α-2-antiplasmin, plasminogen, prothrombin and fibrin clot structure that influence CLT and are also related to obesity may additionally contribute to the link between CLT and obesity. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2014

The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.

The birth of complexity research derives from the logical progression of advancement in the scientific field afforded by reductionist theory. We present in silico models of two complex physiological processes, wound healing and coagulation/fibrinolysis based on two common tools in the study of complex physiology: ordinary differential equations (ODE) and Agent Based Modeling (ABM). The strengths of these two approaches are well-suited in the analysis of clinical paradigms such as wound healing and coagulation. The complex interactions that characterize acute wound healing have stymied the development of effective therapeutic modalities. The use of computational models holds the promise to improve our basic approach to understanding the process. We have modified an existing ordinary differential equation model by 1) evolving from a systemic model to a local model, 2) the incorporation of fibroblast activity, and3) including the effects of tissue oxygenation. Possible therapeutic targets, such as fibroblast death rate and rate of fibroblast recruitment have been identified by computational analysis. This model is a step toward constructing an integrative systems biology model of human wound healing. The coagulation and fibrinolytic systems are complex, inter-connected biological systems with major physiological roles. We present an Agent Based Modeling and Simulation (ABMS) approach to these complex interactions. This ABMS method successfully reproduces the initiation, propagation, and termination of blood clot formation and its lysis in vitro due to the activation of either the intrinsic or extrinsic pathways. Furthermore, the ABMS was able to simulate the pharmacological effects of two clinically used anticoagulants, warfarin and heparin, as well as the physiological effects of enzyme deficiency/dysfunction, i.e., hemophilia and antithrombin III-heparin binding impairment, on the coagulation system. The results of the model compare favorably with in vitro experimental data under both physiologic and pathophysiologic conditions. Our computational systems biology approach integrates reductionist experimental data into a cohesive model that allows rapid evaluation of the effects of multiple variables. Our ODE and AMBS models offer the ability to generate non-linear responses based on known relationships among variables and in silico modeling of mechanistic biological rules on computer software, respectively. Simulations of normal and disease states as well as effects of therapeutic intervention demonstrate the potential uses of computer simulation. Specifically, models may be applied to hypothesis generation and biological advances, discovery of new diagnostic and therapeutic options, platforms to test novel therapies, and opportunities to predict adverse events during drug development. The ultimate aim of such models is creation of bedside simulators that allow personalized, individual medicine; however, a myriad of opportunities for scientific advancement are opened through in silico experimentation.

Es wurde ein zweistufiger, indirekter enzymatischer Assay (Fibrinolytische-Kapazität, FC) in zwei Varianten (basal, aktiviert) vorgestellt, der summarisch Störungen der Fibrinolyse erfasst, da in ihn die Gesamtaktivität der Aktivatoren und Inhibitoren des Plasmas einfließt. In der ersten Stufe wird Plasma Urokinase zugeführt, welche mit Plasminogenaktivatorinhibitoren interagiert. Die noch freie Urokinase aktiviert Plasminogen zu Plasmin. Die plasmatischen Antiplasmine, hauptsächlich alpha 2-Antiplasmin, werden oxidativ mit Taurin-Chloramin inaktiviert. Schließlich wird die resultierende Plasminmenge mit einem chromogenen Substrat quantifiziert. In einer zweiten Variante wird die kontaktphasenabhängige Fibrinolyse vorher sehr potent mit Dextransulfat stimuliert. Zur Validierung wurde der Einfluss von PAI-1, Fibrinogen und Plasminogen untersucht. Störgrößen wie Antioxidantien, parenterale Antikoagulantien, Phenprocoumon, Aprotinin, Tranexamsäure, Thrombozyten und Bilirubin wurden ebenfalls untersucht. Zusätzlich wurde der Test anhand eines Normal-, Thrombose- und Schwangerenkollektives sowie zweier kleiner Kollektive (Schwangere und Patienten unter oraler Antikoagulation) im Zeitverlauf untersucht. Beide FC-Varianten bilden dabei die prothrombotischen Faktoren unterschiedlich ab. In der Regressionsanalyse reagiert die basale FC eher auf Veränderungen der PAI-1- und Plasminogenkonzentrationen, die aktivierte FC eher auf Plasminogen und Thrombose. Thrombose wird durch die aktivierte FC besser als durch die basale FC diagnostiziert (beta-Koeffizienten für Thrombose -0,12 vs. -0,26, Zusammenhangsmaß Eta² von FC und Thrombose 5,6% vs. 9,9%, Entscheidungsgrenze (Cut-Off) für Thrombose 33,0% vs. 66,2% für basale bzw. aktivierte FC). Beide FC-Varianten besitzen ähnliche Sensitivität, Spezifität, prädiktive Werte und relative Risikos für Thrombose bei FC-Werten unterhalb der Entscheidungsgrenze. Die Thromboseerkennbarkeit ist für beide Varianten gleichwertig bei einer Übereinstimmung untereinander von 61,3% (Cohen-Kappa-Koeffizient). Bei der Abklärung einer akuten Thrombose ist dieser Fibrinolyse-Globaltest in der Lage, Ursachen innerhalb des fibrinolytischen Systems zu erkennen. / A two-step indirect enzymatic assay (fibrinolytic capacity, FC) was presented in two variations (basal, activated) detecting the total fibrinolytic disturbances by its ability to assess the entire plasmatic activity of activators and inhibitors. In the first step urokinase is added to plasma, which interacts with plasminogen-activator-inhibitors. The remaining urokinase activated plasminogen to plasmin. The plasmatic antiplasmines, mainly alpha 2-antiplasmine were oxidative inhibited with taurine-chloramine. Finally the resulting amount of plasmin was quantified using a chromogenic substrate. In a second variation the contact-phase fibrinolysis was highly stimulated with dextran-sulfate. The influence of PAI-1, fibrinogen and plasminogen were analysed including disturbing substances such as antioxidants, parenteral anticoagulants, phenprocoumon, aprotinine, tranexamic acid, platelets and bilirubine. In addition, validation was performed including healthy individuals, patients with thrombosis and pregnant women and two small cohorts (pregnant women and patients under oral anticoagulation) over time. The prothrombotic factors were differently represented by the two FC-variations. In the regression analysis the basal FC reacted predominantly to alterations in the concentration of PAI-1 and plasminogen. In contrast the activated FC was more likely affected by plasminogen and thrombosis. The activated FC was more sensitive in the detection of thrombosis than the basal FC (with a beta-coefficient for thrombosis -0,12 vs. -0,26, a coefficient of strength of association eta² from FC with thrombosis 5,6% vs. 9,9% and a cut-off for thrombosis 33,0% vs. 66,2% for basal and activated FC respectively). Below these cut-offs both FC-variations had equal sensitivity, specificity, predictive values and relative risks in the detection of thrombosis by FC-values. The ability to detect thrombosis were equally with a correspondence of 61,3% (Cohen-Kappa-coefficient). This fibrinolytic global-test is able to identify the underlying cause within the fibrinolytic system for the a clarification of acute thrombosis.

The aim of the present thesis was to assess the occurrence of early lower extremity arterial disease (LEAD) in patients with diabetes and to assess novel potential risk markers for development or worsening of LEAD in the same patients. In parallel different measures of impaired peripheral circulation were evaluated. The measurement of ankle-to- brachial blood pressure index (ABI) to screen for asymptomatic LEAD in diabetic subjects is unreliable since a large proportion of patients have stiff ankle arteries (mediasclerosis) and thus may display a too high ABI. We studied type 1-, type 2 diabetic and non-diabetic subjects without a previous history of LEAD and a composite variable of ankle – plus toe blood pressures and indices was compared to ABI alone in detecting LEAD. Significantly more subjects with reduced peripheral circulation were detected using the composite variable compared to ABI alone. This was particularly true in diabetic subjects, about 30% of whom had signs of impaired peripheral circulation. Thus, it was found that toe blood pressure measurements, alone or in combination with ankle blood pressure measurements, increase the sensitivity for finding early asymptomatic LEAD in diabetic subjects. No significant difference in reproducibility between measurements of absolute ankle- and toe blood pressure and indices was found, but a correlation between systemic (brachial) and toe blood pressure variations over time may suggest that indices are more correct in assessing peripheral arterial circulation. Furthermore, toe blood pressure measurements can be performed using either the great toe or dig II and a strong concordance is found between these measurements. In addition, since the pole-test, another non-invasive method to measure peripheral blood pressure which is less sensitive to the presence of mediasclerosis compared to ABI, correlated significantly with toe blood pressure measurements this method may be used as an alternative screening method in subjects with previously known LEAD. Age, hypertension and glycemic control are well known risk factors and, in addition, high tissue plasminogen activator (tPA) activity turned out to be a novel early marker for asymptomatic LEAD in diabetic subjects, particularly in patients with type 2 diabetes. Age and hyperglycemia are the most important risk factors for development and progression of subclinical lower extremity arterial disease in type 2 diabetic subjects. No independent associations between markers of inflammation, such as CRP, interleukin-6 and TNF-α and early asymptomatic LEAD were seen among non-diabetic or diabetic subjects. In conclusion, impaired arterial circulation in the lower extremities is common in diabetic subjects even in the absence of symptoms. Including toe blood pressure measurement when screening for asymptomatic LEAD in diabetic subjects improves the ability to detect reduced peripheral circulation and this method avoids falsely elevated blood pressures readings due to mediasclerosis in the ankle arteries. Moreover, an altered fibrinolytic activity should be further evaluated as an early marker of atherosclerosis and LEAD.