Thesis (M.S.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Apr. 16, 2010). Includes bibliographical references.

No description available.

A protease has been found in the bile of 11 mammalian species investigated. The protease, given the tentative name of cholelysin, has been studied intensively in Abattoir ox bile. It makes up less than 10% of the ox bile protein, and is a potent fibrinolysin, as well as being active against the substrates α-casein, and the synthetic esters of tyrosin (ATEE) and arginine (BAEE). It is inactive against trypsinogen, chymotrypsinogen and plasminogen. Isolation and purification of this protease from ox bile proved complex, and was finally achieved by an 8 step procedure which yielded a dry white powder, stable for 45 months (to date) at 4°C. Quality control of this procedure was effected by means o f a fibrin plate assay, using chymotrypsin as a reference standard: the dose response curves of cholelysin and chymotrypsin were closely similar on the fibrin plate, enabling cholelysin (units/l) to be substituted for chymotrypsin (mg/l), in equivalent diameters of fibrinolysis. Gradient elution by tris/NaCl from Whatman DE 32 produced four areas, or Peaks, of fibrinolytic activity of cholelysin, each with some differing characteristics against the various substrates. These complexities were not studied in detail, and a simplification of the procedure was discovered, using batch-wise elution with tris buffer. Thereafter, only the first peak was studied (Peak I). Studies on the inhibition of cholelysin were done using many known inhibitors, including serum of man, and 4 laboratory animals; serum of two patients with homozygous deficiency of α1-antitrypsin; α2-macroglobulin, soy bean trypsin inhibitor, and aprotonin. The serum studies were done with heated and unheated material; platelet-rich and platelet-poor plasma were also studied. Serum was fractionated by paper electrophoresis in an attempt to discover the globulin fraction containing the inhibitor. No inhibition was found in the α-globulin fractions, and inhibition was maximal in the inter-α- globulin and α-globulin fractions. ATEE-esterase activity of cholelysin was inhibited by serum as strongly as fibrinolytic activity. A limited series of studies of coagulation was done; cholelysin was only found to influence fibrinogen, being quite strongly fibrinogenolytic in vitro. A slight effect on ADP-induced aggregation of platelets was found at a low ADP concentration. Using a rat model originally devised for the study of the anticoagulant effect of ancrod, cholelysin was found to be weakly anticoagulant. The dose was low by comparison with ancrod, and the result approached, but did not reach, statistical significance. The split products of plasmin and cholelysin digestion of stabilised fibrin were studied by polyacrylamide gel electrophoresis (PAGE), and these were found to be entirely different. The kinetics of the reaction between cholelysin and fibrin were studied by means of a new technique (the composite cuvette method) and by this method it was shown that cholelysin made a two-phase attack on the fibrin molecule. The attack was studied at 2, 10, and 30 minutes following commencement of fibrinolysis, and the biphasic nature of the attack was proved by a sharp increase in the number of reaction products present between the 2 and 10-minute samples. The molecular weight of fibrin split products by plasmin were shown to agree with those found in published work. Finally, the molecular weight of cholelysin was estimated by PAGE and by column chromatography with and without SDS. It seems probable that the basic molecular weight is ~7 000, with a dimer of ~13 000 and a tetramer of ~ 28 000.

Zinc (Zn2+) is an abundant metal ion that circulates in the body. Within hemostasis, Zn2+ participates in platelet aggregation, coagulation, and fibrinolysis. At the site of injury, Zn2+ released from activated platelets accelerates coagulation and attenuates fibrinolysis. How Zn2+ regulates these processes on a molecular level has not been extensively examined. We hypothesized that Zn2+ released from platelets binds serine proteases involved in coagulation or fibrinolysis and modulates their proteolytic activity, thus controlling the rate of clot formation and lysis. We show that Zn2+ concentrations released from activated platelets are sufficient to modulate clot formation and fibrinolysis. We show in vitro that Zn2+ binds to fibrinogen with high affinity, accelerates fibrin monomer polymerization, and modifies clot structure. Zn2+ promotes clot stability by increasing fiber diameter, reducing fibrin fiber elasticity, and increases clot porosity. Although it might be predicted that these modifications would enhance clot degradation by enabling greater distribution of lytic enzymes through the more porous fibrin network, we showed the opposite. Thus, we demonstrated that Zn2+ binds to plasminogen activators and plasmin with high affinity and down-regulates their protease activity, which delays lysis. This adds to previous studies that showed that both coagulation and fibrinolysis are regulated by Zn2+ ions. These data support the functional role of Zn2+ in hemostasis. / Thesis / Doctor of Philosophy (PhD)

Atherosclerosis is a chronic inflammatory disease characterized by plaque or clot build-up in the arterial vessel wall, which leads to blood vessel occlusion and consequently heart attack and stroke. Diabetes is a metabolic disorder characterized by elevated sugar (hyperglycemia), which is a known risk factor for the development and exacerbation of atherosclerosis. Recent studies identified fibrinolytic factors (e.g. plasminogen activator inhibitor 1 (PAI-1), thrombin-activatable fibrinolysis inhibitor (TAFI)) that are linked with worsening of atherosclerosis and diabetes. In addition, since activated TAFI (TAFIa), possesses both antifibrinolytic and anti-inflammatory properties, it is a molecule of interest within the context of atherosclerosis progression. Therefore, we hypothesize that fibrinolysis is influenced by atherosclerosis and diabetes. To test this, we used mice that are prone to developing atherosclerosis (ApoE-/-) and hyperglycemia (Ins2+/Akita) from which the heart and plasma samples were collected at 15- or 25-weeks of age. Overall, no differences in plasma clot lysis times were observed between male (hyperglycemic) and female (normal glycemic) ApoE-/-:Ins2+/Akita mice. Quantitation of plaque volume showed a significant increase at 25-weeks compared with 15-weeks, consistent with previous reports. Closer examination revealed that in 15-week-old mice, plaque volume and PAI-1 levels displayed a trend with lysis time, but not in 25-week-old mice. The lysis times showed no difference with hyperglycemia or age. When TAFIa was inhibited, 15-week-old mice had longer lysis times compared with 25-week-old mice independent of hyperglycemia. In addition, elevation of cholesterol and triglyceride levels from hyperglycemia were only observed at 15-weeks. Total PAI-1 levels appeared to decrease with age. TAFI zymogen levels did not change with hyperglycemia or age. Fragment 1.2 levels, which indicate coagulation activation/thrombin generation increase with age but were not correlated with hyperglycemia. Overall, hyperglycemia does not appear to impact fibrinolysis directly, but rather indirectly through atherosclerosis in ApoE-/-:Ins2+/Akita mice. / Thesis / Master of Science (MSc) / Atherosclerosis is a disease of the large arteries that leads to unwanted blood clot formation that restrict blood flow to the heart and the brain causing heart attack and stroke, respectively. Diabetes mellitus is a metabolic disorder characterized by elevated blood sugar levels (hyperglycemia). Hyperglycemia directly alters protein and cellular structure, and indirectly influences cardiovascular complications (e.g. exacerbation of atherosclerosis progression). Although fibrinolysis, the process of removing clots, has also been linked with hyperglycemia, the mechanism remains unclear. To study the effect of hyperglycemia- mediated atherosclerosis exacerbation on fibrinolysis, we used mice that are prone to atherosclerosis with and without hyperglycemia and quantified: (a) atherosclerosis (lesion size, lipids), (b) blood sugar, and (c) fibrinolysis (lysis times, coagulation/fibrinolytic factors). We found strong correlation between lesion size and lysis time but not with other fibrinolytic markers. Hyperglycemia also did not affect fibrinolysis directly. Therefore, hyperglycemia may indirectly influence fibrinolysis through atherosclerosis.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Observational studies have consistently demonstrated associations between exposure to air pollution and increased cardiovascular morbidity and mortality. These associations are strongest for fine particulate matter (PM), of which particulates from the combustion of fossil fuels are an important component. In Europe, the contribution to urban PM from diesel emissions is increasing with the popularity of diesel engines for road transport. Despite the strength of the epidemiological evidence and the emergence of promising hypotheses, the important constituents and biological mechanisms responsible for the cardiovascular effects of air pollution are largely unknown. It is possible that nanoparticulates or soluble components of PM may translocate into the bloodstream, resulting in direct effects on the vascular endothelium and thrombotic pathways. I investigated the potential for inhaled radiolabelled nanoparticulates to translocate into the circulation in man. Using two unique human exposure facilities I assessed the effects of exposure to combustion-derived particulates in dilute diesel exhaust and concentrated ambient fine and ultrafine particles on vascular endothelial, endogenous fibrinolytic and myocardial function in healthy volunteers and patients with stable coronary artery disease. In total, forty-two healthy men and thirty-two patients with stable coronary artery disease were exposed to particulates or filtered air for 1-2 hours in a series of double blind randomised crossover studies. At levels encountered in an urban environment, inhalation of dilute diesel exhaust impaired two important and complementary aspects of vascular function in man: the regulation of vascular tone and endogenous fibrinolysis. Vascular dysfunction persisted for up to 24-hours following exposure and was associated with an increase in systemic inflammatory cytokines. In patients with coronary heart disease exposure to diesel exhaust did not aggravate pre-existing vasomotor dysfunction, but did exacerbate exercise-induced myocardial ischemia and reduce acute endothelial tissue plasminogen activator release. In contrast, exposure to concentrated ambient particulates, low in combustion component, did not affect vascular function in either healthy volunteers or patients. I found little evidence that inhaled radiolabelled nanoparticles translocate into the circulation and suggest the adverse vascular effects of combustion derived air pollution are mediated primarily by their soluble components rather than by a direct interaction between nanoparticles and the vasculature. My findings have identified ischemic, vascular and thrombotic mechanisms that may explain in part the observations that exposure to combustion-derived air pollution is associated with adverse cardiovascular events including acute myocardial infarction. Ongoing research in this area will provide further insight into the adverse effects of PM, with the possibility of targeted interventions, such as the use of retrofit particle traps on diesel powered vehicles, to reduce the impact of environmental air pollution on cardiovascular disease a realistic goal.

We observed that platelets, which mediate thrombus formation, also enhance fibrinolysis by single-chain urokinase (scuPA).  Preliminary data suggested that this enhancement was due to platelet thrombospondin (TSP), which, depending upon the specifics of the environment, changed conformation, influencing its role in the fibrinolytic system.  Results showed that the activity of scuPA was enhanced by platelets, regardless of platelet treatment or protein release, and that TSP could not explain the platelet effect.  Investigation of the underlying mechanism, using the non-cleavable mutant scuPA (K158E) and protease inhibitors, showed that the platelet-dependent enhancement of scuPA arose from the activation to uPA by a serine protease.  Factor VII activating protease (FSAP) was not the protease responsible, since inhibition of platelet FSAP with function-blocking antibodies did not inhibit the platelet effect.  Comparison of plasminogen and plasma kallikrein, using an array of inhibitors, showed that both candidates mirrored the platelet effect.  Further results, including those from assessment of protease activity on platelets against chromogenic substrates and from the evaluation of uPA formation over time, were consistent with the involvement of plasminogen.  In addition, experiments with platelets from plasminogen-deficient mice showed that platelets lacking plasminogen no longer activated scuPA.  The enhancement of scuPA was found to be platelet-dependent, even in plasma; scuPA activation was more efficient when plasminogen was associated with the platelet membrane, compared to in solution; and the presence of membranes was essential to induce rapid lysis by scuPA.  Our findings indicate that platelets stimulate fibrinolysis by scuPA via local activation to uPA by platelet-associated plasminogen, which is activated by scuPA, consistent with a system of reciprocal activation.

No description available.