Studying single cell protein expression is becoming increasingly important; however it is limited by the availability of highly sensitive technology. In adult cardiac progenitor cells (CPCs), expression of all the key cardiac transcription factors can be found, and both in vivo and in vitro studies have shown they can form cells of the cardiac lineage. However, effective mobilisation of these cells in situ for heart regeneration remains elusive. Close examination of CPCs for the important interactions and modifications may reveal the control mechanisms holding them in their arrested state, and therefore could lead to more effective cell therapies for heart failure. Total internal reflection microscopy is a super-resolution technique, allowing the visualisation of individual protein molecules through fluorescent labelling. It uses and immunoassay to pull down the protein of interest, detecting the protein with a secondary fluorescently labelled antibody. Whilst the assay sensitivity relies heavily on the antibody kinetics, it avoids the need to genetically modify proteins with fluorescence. Thus the assay can be adapted to look at any protein, in any cell, and can be multiplexed to examine many proteins simultaneously. In this thesis, an assay for the detection of the cardiac transcription factor, Gata-4, is developed. Gata-4 is pivotal for cardiac development; it is a part of an extensive gene regulatory network, interacting with many other key transcription factors to confer precise heart morphogenesis. It is expressed in cardiac progenitor cells, and the aim here was to quantify protein expression from single CPCs. Another facet examined here was the potential use of alternative protein capture agents. DNA oligonucleotides, encoding the consensus binding sequence for Gata-4 was investigated for its suitability in an assay for single cell protein expression. Whilst this yielded promising results it was constrained by the lack of a suitable negative control.

Plasma clot structure/function is a major determinant in cardiovascular disease risk and severity. Plasma proteins are incorporated into plasma clots via binding and factor XIII-dependent cross-linking, with complement C3 and factor H previously identified as plasma clot components using proteomics. The aim of this current project was to validate the role of C3 and factor H in fibrin structure and function and to establish a proteomics method for the identification of novel factor XIII substrates. C3 did not affect fibrin structure; however C3 induced a concentration-dependent prolongation of fibrinolysis. C3 was cross-linked to fibrin within purified and plasma clots and bound to plasma clot components. C3 was a substrate for plasmin, with cleavage occurring in the presence and absence of fibrin. C3 also influenced angiostatin production and t-PA and plasminogen interactions within fibrin clots to prevent plasminogen cleavage and plasmin generation. All of these interactions were found to influence fibrinolysis. Whereas factor H was confirmed to be a plasma clot component, was associated with inflammation and fibrin structure and function but was not associated with complement activation in individuals at risk of cardiovascular disease. Further in vitro analyses found that factor H did not affect fibrin structure or fibrinolysis. Factor H was not cross-linked to fibrin in purified and plasma clots, but did form homodimers in the presence and absence of fibrin and factor H was a substrate for thrombin and plasmin, with cleavage occurring within fibrin clots. The proteomic techniques were established for the identification of factor XIII substrates however no novel proteins were identified using these methods, suggesting the sensitivity of the technique may be insufficient to detect novel proteins. This study has added to the growing body of evidence which suggests complement and coagulation pathways interact for the purposes of preventing blood loss and pathogen invasion.

Fibrinogen is a vital component of coagulation; cleavage of fibrinogen yields fibrin monomers that polymerise to form a network of fibres, constituting the blood clot. Human fibrinogen is secreted from hepatocytes in its phosphorylated form, with 20-25 % of circulating fibrinogen phosphorylated exclusively at Aα chain Ser3 and Ser345. Phosphorylation of fibrinogen is elevated in acute phase conditions, venous thrombosis and ovarian cancer, but little is known about the regulation and effects of this modification. The aims of this PhD project were to characterise the cellular mechanism and functional role of fibrinogen phosphorylation in vivo. Human hepatoma cells were incubated in the presence and absence of IL-6 and the phosphate content of secreted fibrinogen was analysed by western blotting. Interleukin-6 caused a 3.1-fold increase in fibrinogen phosphorylation, demonstrating for the first time that the up-regulation of this modification in acute phase conditions is regulated at the cellular level. Using real-time PCR, IL-6 was found to significantly enhance (6.0-fold) the expression of Golgi casein kinase Fam20A, whose recognition sequence matches the Ser3 and Ser345 phosphorylation sites. Expression of other potential fibrinogen kinases, including CK2, Fam20B and Fam20C, were unchanged. This finding suggests that Fam20A plays an important role in the hepatocellular response to acute phase conditions and may phosphorylate fibrinogen in vivo. Chromatographic enrichment of phosphorylated human plasma fibrinogen was conducted for functional analyses. Binding and activity assays found no effect of fibrinogen phosphorylation on FXIII cross-linking of fibrin α and γ chains, plasmin(ogen) binding to fibrinogen, or α2-antiplasmin incorporation. Analysis by SDS-PAGE revealed a small decrease in the rate of fibrinogen degradation by plasmin with increasing phosphorylation, indicating a possible role in protection from fibrinolysis. Scanning electron microscopy and turbidimetric assays revealed thinner fibres and more extensive branching in clots with a higher phosphate content, which typically represents a pro-thrombotic structure. This work highlights the importance of fibrinogen phosphorylation in maintaining the balance between clot formation and lysis. Investigations have shown that increased intracellular kinase activity leads to elevated fibrinogen phosphorylation in acute phase conditions. The observed alterations to clot phenotype with elevated fibrinogen phosphorylation suggest this modification may help to stem bleeding following trauma. Furthermore, it may have important implications in the development of thrombosis, which would make it a valuable target for therapeutic intervention in associated pathologies.

1. Many studies which have examined reflexes from the left ventricle have failed adequately to localise the pressure stimulus to that area, and have caused simultaneous changes in coronary pressure. Recent studies demonstrate that localized changes in pressure, over a physiological range, to the coronary arteries results in reflex vascular responses. The coronary arteries thus function as baroreceptors. However, there has been no study to compare the characteristics of coronary baroreceptors with those of aortic arch and carotid sinus receptors. This study was therefore designed to compare and contrast these three baroreceptor regions. 2. The experiments described in this thesis were all performed in anaesthetized dogs attached to perfusion circuits. This allowed independent control of pressures perfusing the coronary arteries, aortic arch and carotid sinuses. A cardiopulmonary bypass and an oxygenator incorporated into some of the circuits allowed control of the pulsatility of the pressures applied to the coronary arteries. Reflex responses were assessed from changes in perfusion pressure to the systemic and hind limb circulations and from changes in sympathetic efferent nerve discharge recorded from renal and lumbar nerves. 3. The time courses of the vascular responses to loading and unloading coronary baroreceptors were investigated and compared with responses from aortic arch and carotid sinus baroreceptors. All three baroreceptor groups when stimulated initiated rapid reflex vasodilatation Coronary baroreceptor unloading resulted in vasoconstriction that was significantly slower than that from either aortic arch or carotid sinus baroreceptors. This effect was not altered by increasing the duration at high coronary pressure from 30 s to 8 minutes or by making the stimulus pressure non-pulsatile. The time course of the responses from aortic and carotid baroreceptors were rapid and not different from each other. 4. A further series of experiments examined the mechanism of the slow coronary induced vasoconstriction. Electrophysiological recordings of sympathetic efferent nerves showed that they responded rapidly to increases in coronary pressure but slowly to decreases, thus mirroring the vascular response. Sympathetic efferent nerve responses to changes in carotid and aortic pressure were rapid. 5. Further experiments compared the responses of each baroreceptor group to non- pulsatile and pulsatile stimuli and their operating ranges. There was no difference between the response curves to non-pulsatile and pulsatile stimulation of coronary baroreceptors. Stimulation of carotid receptors with pulsatile pressures shifted the response curve to lower carotid pressures. Pulsatile aortic pressures induced greater vasodilatation over the mid-range of the baroreceptor response curve compared to non-pulsatile pressures. The threshold for responses from coronary baroreceptors was extremely low with responses obtained at the lowest pressures tested. The operating range of the coronary baroreceptors was over significantly lower pressures than for aortic or carotid baroreceptors, which both had similar operating ranges. 6. These results demonstrate that coronary baroreceptors operate mainly in hypotensive situations initiating a slow vasoconstriction to unloading and were active at extremely low pressures. They are insensitive to changes in pulse pressure whereas both aortic and carotid baroreceptors were sensitive.

A simple, precise "reaction rate" assay for plasma cholinesterase based on a succiny1choline substrate has been developed. It's ability to define individuals at risk of succiny1choline sensitivity and identify those who had experienced apnoea was superior to the previously best available assay. However it was not able to identify abnormal forms of cholinesterase which could hydrolyze conventional assay substrates but not succinylcholine. It was-concluded that if these forms exist their numbers are small. The failure to identify such cholinesterase types may have been because'the substrate concentration chosen for the assay was higher than that found"pharnacologically. However investigation of the kinetics of the succiny1choline-cholinesterase interaction showed that this was not the case. The assay was applied to the assessment of liver dysfunction and compared to three established methods was superior. All assays identified patients with severe liver disease but the succinylcholine-based one identified more patients with moderate/mild disease. The assay was also used to investigate the clinical observation that children require a higher dose of succiny1choline for muscle relaxation than adults. Infants were found to have higher succiny1choline activities than adults which is compatible with their relative resistance to the drug. Finally cholinesterase measurements were made, using a range of substrates including succinylcholine, in a variety of animal species. Results show that only when succiny1choline is used as the substrate for'the assay of cholinesterase does enzyme activity correlate with tolerance to it's muscle relaxant properties. The choice of procedure for the analysis of any biochemical variable depends on a number of criteria including ease of assay, precision, accuracy and cost; however the primary consideration should be the ability of the method to provide clinically useful information. Based on all these criteria, in particular the latter, succiny1choline must be considered as the substrate of choice.

No description available.

During synthesis Von Willebrand Factor (VWF) undergoes O-linked glycosylation (OLG) but the functional significance of this has not been fully explored. In this study the 10 OLG sites in VWF were mutated to alanine, individually and as clusters on either or both sides of the A1 domain: Clus-1 (N-terminal side), Clus-2 (C-terminal side) and double cluster (DC). All mutants demonstrated normal expression, multimeric structure and collagen and heparin binding functions. Furthermore, mass spectroscopy analysis showed that the OLG structures presented on recombinant VWF were similar in structure to those on plasma derived VWF including the presence of a rare disialosyl motif. The variants T1486 (C-terminal side of the A1 domain), Clus-2 and DC were less susceptible to ADAMTS13 proteolysis under static and pseudo-shear stress conditions and showed increased binding to ADAMTS13 when in solution. Using an optimised plate-based ELISA assay, it was demonstrated that mutation of the OLG sites on the N-terminal side of the VWF-A1 domain enhanced sensitivity to ristocetin-mediated binding to recombinant GPIbα. Increased GPIbα binding was also observed with desialylated VWF and this effect was attributed to sialic acid residues on the OLGs. Similar results were obtained using the conventional ristocetin cofactor assay. To further investigate these findings, an in vitro flow assay was optimised to analyse the interaction of VWF and platelets under shear stress. Mutation of the OLG sites on the N-terminal side of the A1 domain, increased platelet rolling velocity but did not significantly alter the dissociation rate of the VWF-A1:GPIbα bond. However, they did decrease the number of transient tethering events as a function of time when VWF was immobilised directly on to the flow surface. Interestingly, when compared with wtVWF the T1255A and Clus-1 variants mediated increased platelet capture to collagen under high shear stress. The data presented in this thesis demonstrates that the OLG clustered at the C-terminal side of the A1 domain alter susceptibility of VWF to ADAMTS13 cleavage. Whilst OLGs clustered at the N-terminal side of A1 modulate its interaction with GPIbα both under static and shear conditions. A unifying hypothesis would be that VWF OLG alter conformational stability around the A1 domain and modulate its interactions with adjacent domains and with other molecules. However different effects may predominate under static and flow conditions. Importantly, VWF-platelet interaction in flow assays depends on the way VWF has been immobilised.

The plasma concentration of platelet specific releasable proteins, as measured in vitro, might reflect the degree of platelet aggregation and release within the circulation. One such protein, β-thromboglobulin, has recently been isolated. The aim of this study was to develop an assay to measure the plasma concentration of β-thromboglobulin in normal subjects and patients. Although quantitative immunoeleotrophorotic, radiolimmuno diffusion and haemagglutination inhibition assays were set up, those were insufficiently sensitive to detect β-thromboglobulin in plasma and a sensitive and specific radioimmunoasoay was therefore developed. Using the radioimmunoassay, further evidence for the platelet specificity of β-thromboglobulin was obtained. To measure the concentration of β-thromboglobulin in platelet poor plasma its release in vitro was inhibited by collecting blood into tubes containing EDTA, prostaglandin E1 and theophylline at 0-4°c, and centrifuged for one hour at 1900g. With this sample collection procedure the mean plasma β-thromboglobulin concentration from 160 healthy subjects was 30.50 ± 12.59n&/ml and the individual results were normally distributed. The plasma levels in males were similar to those in females and they were not related to the whole blood platelet count. Of interest was the observation that the plasma β-thromboglobulin concentration rose with age. When 51 chromium platelet survival studies were undertaken in 10 healthy subjects the platelet mean lifespan was positively correlated with the reciprocal of the plasma β-thromboglobulin concentration. Studies in patients with a variety of disorders revealed several differences when compared with normal individuals. In patients with myeloproliferative disorders a positive correlation was noted between the whole blood platelet count and the plasma β-thromboglobulin concentration; further, a change in the platelet count during treatment was accompanied by a corresponding alteration in the plasma β-thromboglobulin concentration. Some patients with thromboembolic disease had raised plasma concentrations of β-thromboglobulin, but these did not correlate with the chromium platelet mean lifespan estimations. Raised plasma levels were also observed in some patients with rheumatic heart disease and prosthetic cardiac valves, but the significance of these was not clear. The studies reported in this thesis suggest that knowledge of the plasma β-thromboglobulin concentration may, in future, be useful in the diagnosis and management of a variety of disorders in which platelets participate.

No description available.

Approximately half of all deaths in the developed world arise from cardiovascular disease, primarily caused by the deposition of atheroma within major arteries. It has been observed that atheroma is deposited preferentially in regions along the outer wall of bifurcations, and along the distal part of the inner wall of bends. These are regions associated with disturbances of the blood flow that display abnormal shear rate (spatial velocity gradient at the vessel wall). Thus, in order to facilitate clinical diagnoses, it is important to visualise the structure and haemodynamic properties of arteries and veins. Magnetic resonance imaging (MRI) is well suited for volume imaging and can be made sensitive to flow. Quantitative velocity measurements are possible using phase contrast (PC) MRI. The aim of this project was the provision of a method that provides information on wall shear rate vectors using MRI. To handle the large number of images acquired in PC MRI automated flow detection algorithms were developed. Three different algorithms were identified: one operating on magnitude MRI images only and two methods which additionally use the velocity information generated from <i>in-vivo</i> and <i>in-vitro</i> acquisitions. These algorithms are based on an edge detection method and were tested on phantoms. The post processing steps necessary to calculate wall shear stress involved the fit of smooth functions to the velocity data, the detection of walls and the calculation of the wall shear rate vector based on that information. Fitting a smooth function removed residual noise and allowed the calculation of spatial derivatives. The velocity data was satisfactorily described by a segmented fifth order polynomial fit. One method of vessel wall reconstruction was based on the fitted velocity field, while another one utilised the detected flow regions. Using the surface position and normals, the wall shear rate was calculated from the shear stress tensor. All post-processing steps were integrated in a purpose built program that enabled graphical user interactions. The calculated wall shear rate values were quantitatively verified with experiments on various phantoms and simulations, and qualitatively compared with computational fluid dynamics calculations. It is shown that a method to calculate reliably wall shear rate directly from time averaged PC MRI acquisitions has been established.