Diss. (sammanfattning) Göteborg : Univ. , 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 137-141.

Abstract Alcohol drinking may exercise both beneficial and untoward effects on the haemostatic and fibrinolytic systems. It may also predispose individuals to arterial thrombosis and trigger embolism in the brain. The aim here is to examine these problems. Methods used for evaluating platelet function were platelet aggregation and associated thromboxane B2 release, shear-induced platelet aggregation, and measurement of urinary prostaglandins. Changes in fibrinolytic system were evaluated by measuring plasminogen activator inhibitor type 1. The combined effects of alcohol drinking, physical exercise, eating a meal and circadian rhythms in healthy volunteers were examined in three experimental studies. Case-control studies were used for assessing the mechanism and etiology of ischaemic brain infarction triggered during alcohol intoxication. Alcohol drinking did not potentiate the effects of physical exercise on platelet function. Sleeping while under acute intoxication resulted in a significant activation of platelets, as shown by increased urinary excretion of a thromboxane metabolite. On the other hand, ingestion of a moderate dose of red wine seemed to attenuate platelet aggregation measured ex vivo, irrespective of whether the wine was consumed with a meal or alone. However, both red wine and a larger acute dose of alcohol in fruit juice inhibited fibrinolytic activity. In a case-control study, platelet count and function were evaluated in 426 consecutive patients hospitalized on account of acute brain infarction. Compared with the hospital-based controls, a higher than normal platelet count was observed immediately after admission. Heavy drinkers showed both higher and lower than normal platelet counts more often than the other patients with brain infarction. The changes in platelet function among the heavy drinkers reflected their recent drinking habits. Another case-control study indicated that recent heavy drinking of alcohol was an independent risk factor for cardiogenic embolism to the brain. Recent heavy drinking also seemed to predispose subjects to some other types of ischaemic brain infarction such as artery to artery embolism due to large-artery atherosclerosis and cryptogenic stroke, but these observations need to be confirmed in larger studies. In conclusion, the results show some untoward effects of acute heavy drinking of alcohol, which could contribute to the onset of brain infarction either as triggering or as predisposing factors. On the other hand, drinking of a moderate dose of red wine did not have any clear untoward effect on healthy human volunteers.

The blood of man is rich in plasminogen, the inactive precursor of plasmin, a protease (Astrup, 1956a); the most characteristic action of plasmin is the digestion of fibrin, i.e. fibrinolysis. Many tissues, including the prostate (Rasmussen and Albrechtsen, 1960a), contain substances which can activate plasminogen, and thus initiate fibrinolysis, and it has been assumed that both the excessive fibrinolysis seen in the blood of some patients with prostatic disease (Tagnon, Whitmore, Schulman and Kravitz, 1953a), and in prostatic surgery (Lombardo, 1957), is due to the release of this activator into the blood stream (Fearnley, 1965). Human semen contains a substance which can activate the blood fibrinolytic system (von Kaulla and Shettles, 1953). Indeed, when human seminal fluid is ejaculated, it undergoes a process resembling the clotting and fibrinolysis of the blood, by coagulating then liquefying spontaneously. The coagulum is formed when a fibrinogenlike protein secreted by the seminal vesicles is acted upon by a clotting enzyme from the prostate (Mann, 1964). Coagulation is followed within about 20 minutes by liquefactionliquefaction of the clots by an enzyme assumed to come from the prostate (Huggins and Neal, 1942). This enzyme resembles plasmin in that it is a protease acting on a fibrin-like substrate, and that it is derived from an inactive precursor.

Physical trauma is responsible for over six million deaths annually, and of these roughly 40 percent result from acute traumatic coagulopathy (ATC) occurring in the first few hours of incidence. Patients who have developed ATC have significantly improved survivability when treated with tranexamic acid (TXA), a chemical inhibitor of the clot lysing enzyme plasmin. Current methods of detecting ATC are inadequate, lacking in either efficient speed, sensitivity, or cost. Hyperfibrinolysis (HF) is a key component of ATC and can be a result of excess plasmin activity. The following study observes effects of plasmin on hemostasis, and explores the use of silicon-based polydimethylsiloxane (PDMS) microfluidics measuring changes in electrical resistance as a method to detect HF. Coagulation was characterized by measuring turbidity of solutions containing fibrinogen and thrombin, and plasmin was incorporated to observe fibrinolysis and other plasmin-induced effects. It was found that high concentrations of plasmin caused a delay in the turbidity increase during coagulation. This lag phase may be a contributing factor to HF and ultimately ATC. Finally, the use of PDMS microfluidics to measure changes in electrical resistance to detect coagulation and fibrinolysis activity was supported. Resistance change adhered closely to traditional substrate-enzyme kinetics and plasmin-induced effects mimicked those which were observed in turbidity measurements. Further investment and development of this method of measurement could provide a faster, more accurate, and more inexpensive alternative to current techniques for measuring fibrinolysis.

<p> The fibrinolytic system has an important role in maintaining vascular patency by restricting fibrin clot formation to prevent occlusion of the blood vessel. Plasminogen activation is the central event in fibrinolysis and is tightly regulated by activators and inhibitors. Histidine-rich glycoprotein (HRG) is an abundant plasma protein that has been proposed to have a regulatory role in many biological processes, including fibrinolysis. Approximately 50% ofplasminogen in the blood circulates in complex with HRG. Conflicting reports dispute the role of HRG in fibrinolysis, specifically whether it promotes or inhibits plasminogen activation. To elucidate the role of HRG in fibrinolysis, we isolated HRG from human plasma and analyzed its effect on plasminogen activation by tissue-type plasminogen activator in a kinetic assay. HRG had no significant effect on plasminogen activation by tissue-type plasminogen activator once contaminating plasminogen was eliminated from our HRG preparations. Based on these results, the focus of our research was redirected to analyzing the effect of HRG on additional plasminogen activators, namely urinary-type plasminogen activator and factor (F) Xlla. HRG inhibited plasminogen activation by both activators. HRG had the greatest inhibitory effect on FXIIa activity. This novel finding led us to explore the relationship between HRG and FX.IIa by measuring the affinity of HRG for FXIIa by surface plasmon resonance, and by analyzing the effect of HRG on FXIIa activity in various contact pathway reactions. ZnCh was also included in these reactions because it plays an important role in enhancing both HRG-and FXII-mediated interactions and is released by activated platelets. In the presence of 12.5 μM ZnCl2, FXIIa bound to the histidine-rich region of HRG with very high affinity (Kd = 56 ± 8.9 pM). Interestingly, HRG does not bind to FXII. Functional analysis of HRG revealed that it significantly inhibits a number of contact pathway reactions, including FXII autoactivation, kallikreinmediated FXII activation, and FXIIa-mediated FXI activation. Conversely, HRG enhanced FXIIa-mediated prekallikrein activation. Based on these findings, we hypothesize that HRG binds to an exosite on FXIIa, which is not expressed by the zymogen FXII, and alters FXIIa activity. The mechanism of HRG-mediated FXIIa inhibition is not fully understood and needs to be further analyzed by both binding and functional assays. These observations raise the possibility that the main function of HRG is to modulate FXIIa activity, rather than plasminogen activation. Because of its abundance, HRG may function as a modulator of haemostasis through its effect on coagulation and fibrinolysis. </p> / Thesis / Master of Science (MSc)

The demonstration of antithrombotic efficacy in man is challenging. Most techniques evaluate specific plasma or cellular components under static conditions in vitro. In contrast, in vivo thrombus initiation and growth occur in whole blood, under conditions of continuous flow and in the presence of vascular injury. An in vivo model for use in clinical studies presents significant safety issues and does not currently exist. The Badimon chamber is an ex vivo model of thrombosis that is suitable for use in clinical studies and has previously been used to assess novel antithrombotic regimens. Although well-established, previous characterisation studies were performed in a porcine system and using methodology that has since been superceded. In addition, it has a number of disadvantages that limit its broader applicability and has not previously been used to assess fibrinolysis. Having established The Badimon Chamber within my own institution, I developed the methodology and performed careful validation and characterisation studies with a particular emphasis on reproducibility. These developments allowed more efficient data analysis and the accurate addition of compounds to the extracorporeal circuit, both of which broaden the applicability of the technique. In subsequent studies, using a series of double-blind randomised controlled crossover studies in healthy volunteer cohorts, I utilised the updated methodology to address questions in separate but overlapping areas of cardiovascular medicine. The dynamic regulation of intravascular thrombus formation by the endogenous fibrinolytic system is central to the pathogenesis of acute atherosclerotic events, particularly within the coronary circulation. Previous work within our institution has provided novel insights into the role of endogenous fibrinolysis. Despite a growing body of evidence, a key limitation of studies to date is that the effects of acute endogenous t-PA release on in situ thrombus formation have not been demonstrated. Is endogenous endothelial t-PA released under agonist stimulation functionally active and able to enhance fibrinolysis of in situ thrombus? Firstly, I demonstrated that the addition of exogenous t-PA into the extracorporeal circuit of The Badimon Chamber results in a dose dependent increase in plasma D-dimer associated with a dose dependent reduction in thrombus formation, consistent with enhanced fibrinolysis. Having validated the model, I proceeded to investigate whether freshly released endogenous t-PA would have similar effects to exogenous t-PA. By combining intraarterial infusion of bradykinin into the human forearm in order to stimulate acute release of endogenous t-PA with an assessment of thrombus formation in the Badimon Chamber, I demonstrated that endogenous t-PA released acutely from the human vascular endothelium enhances fibrinolysis and limits in situ thrombus formation. These data validate the forearm model as a relevant model with which to assess acute fibrinolytic capacity, confirm the functional significance of t-PA released during agonist stimulation and suggest that further studies to explore its therapeutic manipulation are warranted. I went on to evaluate a promising small molecule PAI-1 inhibitor, PAI-749, using assessments of ex vivo thrombosis complimented by extensive in vitro studies. Interestingly, in contrast to the promising results seen with this compound in preclinical models, we were unable to demonstrate efficacy in any of the clinical models used, highlighting the potential pitfalls of relying solely on in vitro and preclinical models during early compound development. In the final phase of this work, I used the chamber to explore the prothrombotic effects of exposure to air pollution. A plethora of observational data exist to suggest that acute exposure to particulate air pollution can trigger vascular events including myocardial infarction although the underlying mechanisms are only partly understood. Using a unique human exposure facility, we demonstrated that inhalation of diesel exhaust causes platelet activation and enhances thrombus formation. These data provide a plausible mechanism linking exposure to particulate air pollution with acute cardiovascular events including myocardial infarction. Furthermore, in a separate study we were able to demonstrate that reducing the particulate component of the exposure using a commercially available particle trap prevents the detrimental effects on ex vivo thrombosis and endothelial function. These data support calls for the application of particle traps to diesel-powered vehicles in order to limit a range of adverse cardiovascular effects that result from exposure to traffic-derived air pollution.

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