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At sites of vessel injury, thrombin mediates coagulation by catalyzing fibrin clot formation and platelet activation. Conversely, plasmin facilitates fibrinolysis by catalyzing the degradation of fibrin clots. Thrombin generation is most frequently measured in plasma samples using small molecule substrates, these substrates have low free thrombin efficiency and specificity, cannot measure thrombin’s exosite interactions and have limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells, require anticoagulation and the addition of supraphysiological concentrations of calcium. To overcome these limitations, we have designed and characterized a fluorescence resonance energy quenching (FREQ)-based thrombin sensor (FTS) protein. Compared to small molecule substrates, the FTS demonstrated high specificity for thrombin because it is not cleaved by thrombin inhibited by α2-macroglobulin and interacts with thrombin’s anion binding exosite I. The FTS can effectively measure thrombin generation in plasma and in whole blood. The FTS does not inhibit standard thrombin generation assays. Lastly, FTS-based thrombin generation in non-anticoagulated finger-prick blood is delayed but enhanced compared to citrated plasma. Similarly, plasmin generation is also restricted to plasma samples and measured using the fluorogenic Boc-Glu-Lys-Lys-AMC, which have low free plasmin efficiency and specificity, cannot measure plasmin’s kringle domain interactions and are insensitive to the effects of plasminogen activator inhibitor-1 (PAI-1). Here, we describe the design and characterization of a (FREQ)-based plasmin sensor (FPS) protein that demonstrated high efficiency for plasmin that is not inhibited by α2-macroglobulin when compared to Boc-Glu-Lys-Lys-AMC and interacts with plasmin’s kringle domain 5. The FPS measures plasmin generation in plasma, where it demonstrated greater sensitivity to tranexamic acid compared to Boc-Glu-Lys-Lys-AMC as well as sensitivity to PAI-1 and the effects of fibrin. Therefore, the FTS and FPS will broaden our understanding of thrombin and plasmin generation in ways that are not attainable with current methods. / Thesis / Doctor of Philosophy (PhD) / Increased blood clot formation leads to strokes and heart attacks while the inability to form blood clots when needed leads to bleeding disorders. In the body, thrombin makes blood clots while plasmin breaks them down. Therefore, clotting disorders occur in the absence of proper thrombin and plasmin function and appropriate diagnostics of these processes help to determine appropriate treatment. Currently available tests of thrombin and plasmin do not just measure their active forms and are restricted to plasma assays that do not reflect physiological settings and are not useful for quick diagnosis. Here, we describe the production of novel sensors for thrombin and plasmin that are specific for thrombin and plasmin’s active forms. These sensors have potential to be developed into portable diagnostic tests of thrombin and plasmin activity and to be valuable research tools for the development of better treatment options of blood clotting diseases.

Factor XI (FXI) is the zymogen of the coagulation protease factor XIa (FXIa) that contributes to thrombin generation through the intrinsic pathway. FXI is activated by the contact pathway protease, factor XIIa (FXIIa), in a high molecular weight kininogen-dependent manner. It is also known to be activated by thrombin in a positive feedback reaction, however, the mechanism of this activation is not yet completely understood. Therefore, our objectives were to identify the role of polyanions in the thrombin mediated FXI activation, and the role of the thrombin exosites in the activation. To study this activation, we assessed the activation of FXI by thrombin in the presence and absence of the polyanions, dextran sulfate and polyphosphate (polyP). We utilized surface plasmon resonance to determine whether FXI and thrombin bind to the polyanions, and how the exosites effect thrombin’s ability to bind using thrombin exosite 1 and 2 variants. To investigate the role of the exosites in FXI activation, we analyzed the activation of FXI by the thrombin variants. In addition, we explored how inhibiting the thrombin exosites using DNA aptamers affects thrombin’s ability to bind to polyanions and activate FXI. We found that polyanions are required as a cofactor for the activation of FXI by thrombin, and stimulate the activation in a concentration dependent fashion, suggesting a template mechanism. Our findings also show that exosite 1 and 2 are required for thrombin to bind to polyanions, however, exosite 2 plays the predominant role in FXI activation. Our aptamer data showed that either exosite can be targeted to inhibit FXI activation. These findings enrich our understanding of the mechanism of FXI activation by thrombin and provides further insight on how to attenuate the activation for potential antithrombotic therapies. / Thesis / Master of Science (MSc)

Fibrinogen aggregation is the last step in blood coagulation. Inhibition of fibrinogen aggregation could lead to anticoagulation effects. However, there is no good method for the ready evaluation of fibrinogen coagulation. A commonly used path method is slow and requires an expensive instrument. In this project, we have developed a microplate reader and In evaluating inhibitors of fibrinogen coagulations there is no good method. As an important process in hemostasis, fibrinogen coagulation is often detected by micro-plate reader. In our test of fibrinogen coagulation, we improved the observing and analyzing method by using photograph to see concentration-depended effect of thrombin inhibitors on the coagulation. Three known thrombin inhibitors, AEBSF, APMSF and PMSF, were applied to develop the method for detecting the fibrinogen coagulation. The results showed our method is of accuracy in determination of the amount of fibrin when compared with other types of methods.

Diss. (sammanfattning)--Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the comunity health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109(DE3). / Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109(DE3).

Direct factor Xa inhibitors (FXaI) account for most oral anticoagulant use. FXaI-associated bleeding events are common and are associated with substantial morbidity and mortality. Nonspecific hemostatic therapies such as prothrombin complex concentrates (PCC) are often administered for FXaI-associated bleeding. The mechanism by which these agents improve hemostasis in the setting of direct oral anticoagulation is unclear. Thrombin generation assays may effectively measure the effect of anticoagulation reversal among FXaI-treated patients when bleeding cessation would otherwise be challenging to measure. To build a research program on the utility of thrombin generation assays to measure both the impact of direct oral anticoagulation and anticoagulation reversal, we completed a review of the literature with narrative synthesis and carried out a pilot study to determine the feasibility of a full scale prospective observational study of TGA responses among patients receiving PCC for FXaI-associated major bleeding or needing urgent surgery.

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the comunity health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109(DE3). / Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109(DE3).

At sites of vascular injury, the critical blood clotting enzyme thrombin is generated from prothrombin via Prothrombinase, a macromolecular, Ca2+-dependent enzymatic complex consisting of the serine protease factor Xa and the non-enzymatic cofactor factor Va, assembled on the membranes of activated platelets. Platelets regulate thrombin formation by providing specific binding sites for the components of Prothrombinase and by releasing a platelet-derived factor V/Va molecule that is more procoagulant than its plasma counterpart and partially resistant to proteolytic inactivation. This dissertation identifies and characterizes the subpopulation of platelet-derived factor V/Va that is responsible for the observed protease resistance, and the mechanism by which Prothrombinase bound to platelets differs from a model system using vesicles composed of 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS), PCPS vesicles. Previous studies have demonstrated that activated platelets release a dissociable pool of factor V/Va and a non-dissociable, membrane-bound pool, which is covalently attached to the platelet membrane through a glycosylphosphatidyl inositol (GPI) anchor. Data described herein demonstrate unequivocally that the pool of platelet-derived factor V/Va that is resistant to proteolytic inactivation by activated protein C is provided exclusively by the non-dissociable GPI-anchored pool. Further, although this factor Va pool is susceptible to proteolysis by plasmin, the fragments formed are associated with sustained and increased cofactor activity. These observations indicate that tethering of factor Va to the membrane surface via a GPI anchor imparts resistance to proteolytic inactivation and sustained thrombin generation at sites of vascular injury. For several years it has been known that Prothrombinase assembled on PCPS vesicles does not mimic that bound to platelets. While both enzymes cleave prothrombin at Arg271 and Arg320 to form thrombin, prothrombin activation proceeds via the prethrombin-2 pathway (initial cleavage at Arg271) on the platelet surface, in contrast to the meizothrombin pathway (initial cleavage at Arg320) on PCPS vesicles. Using thrombin active site inhibitors, we demonstrate that the preference for either pathway is dictated by the conformation in which prothrombin is bound by the membrane-bound enzyme. The prethrombin-2 pathway of prothrombin activation catalyzed by platelet-bound Prothrombinase is a direct consequence of configuring prothrombin in a proteinase-like state resulting in the exposure of a pseudo-active site that can be stabilized by active site thrombin inhibitors. Conversely, prothrombin is preferentially configured in the zymogen-like state on PCPS vesicles where the meizothrombin pathway is preferred. Additional support for the differential assembly of Prothrombinase on the platelet surface is provided by observations made using prethrombin-1, an intermediate formed by cleavage of prothrombin at Arg155 by the formed thrombin. Prethrombin-1 is converted into fragment-2 and thrombin by platelet-bound Prothrombinase at a substantially higher rate than vesicle-bound Prothrombinase. The decreased rate of prethrombin-1 activation in the model system is due, in part, to inhibition of the vesicle-bound enzyme by the fragment-2 generated in the reaction. Taken together, these data not only provide important molecular insights into the mechanisms by which Prothrombinase bound to activated platelets at sites of vascular injury regulates the procoagulant response to effectively support robust thrombin generation, but also provides potential mechanistic sites that could be targeted therapeutically.

Das Endothel bildet eine einschichtige Zellbarriere zwischen Blut und interstitiellem Gewebe, deren Durchlässigkeit entscheidend durch die sekundären Botenstoffe Ca2+ und cAMP reguliert wird. Während Ca2+ durch eine verstärkte Kontraktion der Endothelzellen die Permeabilität erhöht, fördert cAMP die Adhäsion der Zellen und unterstützt somit die Barrierefunktion. Es ist bekannt, dass Thrombin durch einen Anstieg der intrazellulären Ca2+-Konzentration und vermutlich auch durch eine Hemmung der cAMP-Konzentration zu einer Permeabilitätserhöhung führt. Ziel dieser Arbeit war es, Thrombin-induzierte Änderungen der cAMP-Konzentration in Echtzeit in lebenden Endothelzellen mittels Fluorescence-Resonance-Energy-Transfer (FRET) zu untersuchen. Hierfür wurden Human-Umbilical-Vein-Endothelial-Cells (HUVECs) mit dem FRET-basierten cAMP-Sensor Epac1-camps transfiziert. Die Bindung von cAMP an Epac1-camps führt zu einer Konformationsänderung des Sensors und damit zu einer Abschwächung des FRET. Mit Hilfe dieses Sensors kann die cAMP-Konzentration mit hoher zeitlicher Auflösung in einzelnen lebenden Zellen gemessen werden. Untersucht wurde der Effekt von Thrombin auf die cAMP-Konzentration in Endothelzellen, deren cAMP-Konzentration durch Stimulierung endogener β-Rezeptoren erhöht war. Thrombin erniedrigte Ca2+-abhängig die cAMP-Konzentration um ca. 30 %. Dieser Abfall der cAMP-Konzentration folgte zeitlich verzögert dem Thrombin-induzierten Ca2+-Signal. Die cAMP-Konzentration erreichte ca. 30 s nach der Thrombinzugabe ein Minimum und stieg danach wieder an. Durch die Herunterregulierung der durch Ca2+ direkt inhibierten Adenylatzyklase 6 (AC6) mittels siRNA wurde die Thrombin-induzierte Abnahme der cAMP-Konzentration vollständig aufgehoben. Dies bestätigte, dass Thrombin durch die Ca2+-vermittelte Inhibierung der AC6 eine Abnahme der cAMP-Konzentration verursacht. Ohne β-adrenerge Stimulation führte die Applikation von Thrombin zu einem langsamen Anstieg der cAMP-Konzentration, der mehrere Minuten anhielt. Dieser cAMP-Konzentrationsanstieg beruhte auf der Ca2+-abhängigen Aktivierung der Phospholipase A2 (PLA2). Diese setzt Arachidonsäure aus Membranphospholipiden frei, die als Substrat für die Synthese verschiedener Prostaglandine dient. Durch die pharmakologische Beeinflussung von Zyklooxygenasen und Prostazyklinrezeptoren konnte gezeigt werden, dass die Synthese von Prostazyklin und die anschließende Stimulation Gs-gekoppelter Prostazyklinrezeptoren zum Thrombin-induzierten Anstieg der cAMP-Konzentration führte. Da die Physiologie der Endothelzellen im Gefäß stark von Faktoren aus der unmittelbaren Umgebung beeinflusst wird, ist die Messung der Änderungen der cAMP-Konzentration in Endothelzellen, die sich innerhalb eines Gewebes befinden, von sehr großer Bedeutung. Deshalb war die Generierung transgener Mäuse mit einer gewebespezifischen Expression des FRET-Sensors Epac1-camps in Endothelzellen ein weiteres Ziel dieser Arbeit. Durch Anwendung eines Cre-Rekombinase/loxP-Ansatzes konnten transgene Mäuse generiert werden, die Epac1-camps spezifisch in Endothelzellen exprimierten. An isolierten pulmonären Endothelzellen konnte die Funktionalität des transgen exprimierten Sensors Epac1-camps nachgewiesen werden. Die Echtzeitmessung der Thrombin-induzierten Änderungen der cAMP-Konzentration verdeutlichte ein zeitlich sehr komplexes Wechselspiel zwischen Ca2+- und cAMP-Signalen, das die Barrierefunktion des Endothels maßgeblich beeinflussen wird. Die transgene Expression von Epac1-camps in Endothelzellen ermöglicht in Zukunft die Untersuchung der Thrombin-verursachten Änderungen der cAMP-Konzentration und der Permeabilität innerhalb eines intakten Gefäßes. / Endothelial cells form a semi permeable barrier between blood and interstitial tissues. The permeability of this barrier is mainly regulated by the second messengers Ca2+ and cAMP. While Ca2+ increases the permeability by inducing cell contraction, cAMP increases the adherence of the cells and, thereby, supports the barrier function. The Ca2+-elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. The aim of this thesis was to investigate thrombin-induced changes of the cAMP concentration in real time in living endothelial cells via fluorescence resonance energy transfer (FRET). Therefore, human umbilical vein endothelial cells (HUVECs) were transfected with the FRET-based cAMP sensor Epac1-camps. Binding of cAMP to the binding domain of Epac1-camps induces a conformational change of the sensor that results in a decrease of FRET. With help of this sensor, changes in cAMP concentration can be monitored with high temporal resolution. First, the influence of thrombin on cAMP levels was investigated after elevating cAMP levels by stimulation of β-adrenergic receptors. Thrombin led to a Ca2+-dependent decrease of cAMP levels by approximately 30 %. The decrease of cAMP levels was delayed compared to the thrombin-induced Ca2+ signal. This decrease was also transient and reached a minimum value 30 s after thrombin stimulation. A siRNA-mediated downregulation of the Ca2+-inhibited adenylyl cyclase 6 (AC6) completely abolished the thrombin-induced decrease of cAMP concentration. This provided the first direct evidence that the Ca2+-mediated inhibition of AC6 accounts for the thrombin-induced decrease in cAMP levels. In the absence of a β-adrenergic-mediated increase of cAMP concentration, thrombin led to a slow increase in cAMP concentration that lasted for several minutes. This increase in cAMP concentration was caused by the Ca2+-dependent activation of phospholipase A2 (PLA2). PLA2 releases arachidonic acid, which represents the substrate for prostaglandin synthesis. It was confirmed by pharmacological interference of cyclooxygenases and prostacyclin receptors that the synthesis of prostacyclin and subsequent stimulation of Gs-protein-coupled prostacyclin receptors caused the thrombin-induced increase in cAMP concentration. The real time monitoring of changes in cAMP concentration in endothelial cells within the vascular system is highly important as the physiology of endothelial cells in vivo is strongly influenced by factors contained in the surrounding blood or tissue. Therefore, a further aim of this thesis was the generation of transgenic mice expressing the FRET-based sensor Epac1-camps specifically in endothelial cells. Using a Cre-recombinase/loxP-approach transgenic mice were generated that specifically expressed Epac1-camps in endothelial cells, and the functionality of the transgenic sensor was proven in isolated pulmonary endothelial cells. Real time monitoring of thrombin-induced changes of cAMP concentration in endothelial cells revealed a temporally complex crosstalk between Ca2+ and cAMP signals that will affect endothelial barrier function. The transgenic expression of Epac1-camps opens the door for the investigation of thrombin-induced changes of cAMP levels and of endothelial permeability within intact vessels.