The platelet is an anucleate cell fragment released into the blood from its progenitor cell the megakaryocyte (MK). It was the aim of this thesis to explore the relationship between platelet functional responses and variation in the platelet transcriptome. Rigorous quality assessment of platelet RNA was completed to determine its suitability for DNA microarray investigation. The transcriptomes of individuals homozygous for the major and minor haplotypes of the collagen receptor GPVI, which show differences in signalling responses, were compared using cDNA microarrays. Fifty-two differentially expressed transcripts were identified and ten were selected for further validation. Two transcripts were confirmed as differentially expressed between the haplotypes and the relationship between protein and transcript abundance was investigated. In a second, larger study, platelet RNA from 37 individuals from the ‘extreme ends’ and the mid-range of the distribution of platelet response was isolated. Using whole genome coverage DNA microarrays, 69 transcripts were identified that showed variation in abundance that correlated with platelet response. The proteins encoded by the 69 transcripts were investigated in detail and functionally related proteins were identified, including groups of proteins involved in transcription, cell signalling, metabolism and membrane interactions. Finally, the catalogue of transcripts identified as present in the platelet transcriptome was studied. This data was compared to published platelet and MK transcriptomes and examined for known and novel platelet transcripts, focusing on transcripts encoding transmembrane proteins. In conclusion, this thesis describes in detail the platelet transcriptome in healthy individuals. In addition, genes have been identified whose transcript abundance correlates with platelet functional response, thus identifying genes that play key regulatory roles in platelet function. A number of these genes have since been shown to be risk factors for coronary artery disease thus highlighting the role of platelets in this common disease.

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No description available.

Alpha<SUB>1</SUB>-antitrypsin is the most abundant proteinase inhibitor in plasma and the archetypal member of a large family of serine proteinase inhibitors known as the serpins. All serpin members have a common molecular architecture which consists of three β-sheets surrounded by eight α-helixes with an exposed mobile reactive centre loop. In this thesis I show that it is possible to induce and purify an inactive latent α<SUB>1</SUB>-antitrypsin conformation which can be reactivated after denaturation and refolding. An inactive latent component was also shown to exist in commercial α<SUB>1</SUB>-antitrypsin pasteurised for use as replacement therapy in patients with α<SUB>1</SUB>-antitrypsin deficiency. Another consequence of the mobile reactive centre loop is the formation of inactive loop-sheet polymers which form when the reactive centre loop of one molecule is inserted into the β-sheet of another. This interaction accounts for the retention of Z (Glu<SUP>342</SUP>→Lys) α<SUB>1</SUB>-antitrypsin in the endoplasmic reticulum of hepatocytes with accompanying plasma deficiency. I show here that the plasma deficiency associated with the common S α<SUB>1</SUB>-antitrypsin allele (Glu <SUP>264</SUP>→Val), which is present in 19% of individuals of Spanish descent, can also be attributed to loop-sheet polymerisation. The structural basis of this loop-sheet linkage was clarified by the solving here of the crystal structures of intact wildtype and a thermostable mutant of α<SUB>1</SUB>-antitrypsin. Both structures show the reactive centre loop in a canonical conformation that docks perfectly with its cognate proteinase. The rest of the reactive centre loop is held in a β-strand conformation via a stabilising salt bridge with the body of the molecule. In this conformation the reactive loop can be readily modelled into the A β-sheet of a second molecule, providing strong support for A β-sheet linkage as the mechanism for loop-sheet polymerisation. Finally, loop-sheet polymers of α<SUB>1</SUB>-antitrypsin have also been shown to occur within the small airways and alveoli of the lungs of Z α<SUB>1</SUB>-antitrypsin homozygotes. This findings provides another mechanism for the inactivation of α<SUB>1</SUB>-antitrypsin, thereby exacerbating the proteinase-antiproteinase imbalance in the lung disease emphysema.

The SCL gene encodes a basic helix-loop-helix protein that has a critical role in the normal development of all haematopoietic lineages, as well as an oncogenic role in T cell leukaemogenesis. SCL is suspected to function as a tissue specific transcription factor, but the mechanisms by which SCL mediates its functions are unclear. Identification and characterisation of protein-protein interactions SCL should begin to unravel the roles of this protein. As SCL is expressed in committed erythroid, mast and megakaryocytic cells as well as early haematopoietic progenitor cells, yeast-two-hybrid screens of haematopoietic cell line cDNA libraries were conducted to identify potential SCL interaction partner proteins. cDNA libraries were generated from an early haematopoietic progenitor cell line and a megakaryocytic cell line. These screens using both these libraries and a mouse E10.5 embryo cDNA library identified a range of potential interaction partners for the SCL protein. PolyA<SUP>+</SUP> RNA was isolated from a wide range of haematopoietic cell lines, and embryonic tissues and Northern blot analysis was then used to investigate the expression patterns of these potential interaction partners. The combination of this expression data and data gathered from sequence analysis using bioinformatics programs was used to assess which of the proteins identified was the most promising candidate SCL interaction partner. The association between SCL and the IFNα inducible protein IFI60 was investigated.

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Expression arrays and chromatin immunoprecipitation in a <i>Drosophila</i> blood cell line had previously identified high confidence direct targets of Notch signalling. Therefore one aim of the research was to validate a selection of these putative Notch targets. These were shown to contain Notch responsive enhancers that direct expression in the lymph gland, implicating these novel targets in blood cell regulation. Further analysis of two of these novel Notch targets, <i>hindsight (hnt) </i>and <i>klumpfuss (klu) </i>indicate that their expression overlaps with a known crystal cell-lineage marker, <i>lozenge (lz). </i>This suggests that these, and most likely the other targets, are involved downstream of Notch in precursor cells during crystal cell specification, as part of the gene cascade that results in terminal differentiation. My work has found that <i>hnt</i> and <i>klu</i> are both necessary for crystal differentiation, acting downstream of Notch and Lz. Furthermore, <i>klu</i> is sufficient to induce crystal cell fates in cells of another lineage. My work further suggests that Notch and Lz act in a co-ordinate manner. The model that emerges is that expression of <i>lz</i> confers competence to receive the Notch signal, so that the receiving cells activate genes required to promote crystal cell differentiation, including <i>hnt</i> and <i>klu</i>. The requirement of Notch and Lz in <i>Drosophila </i>hematopoiesis echo that of NOTCH1 and the homologue of Lz, Runx1, in the mammalian system, suggesting that novel targets identified here may be relevant to the regulatory networks involved in mammals.

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This thesis examines the mechanisms underlying the activation of the store-operated calcium entry (SOCE) pathway in human platelets and how they relate to the Ca²⁺ signals elicited in platelets stimulated with physiological agonists and phorbol esters. A novel role for an increase in the activity of the cysteine protease calpain in the activation of SOCE is demonstrated. Data is also presented dissociating the role of the actin cytoskeleton in the store-operated Ca²⁺ entry from the store-operated Mn²⁺ and Na⁺ entries and demonstrate that the role of the cytoskeleton is not in the activation of the store-operated channel but in regulating Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX). These data therefore provide strong evidence against the <i>de novo </i>conformational coupling hypothesis in human platelets. During further examination of the role of the NCX in SOCE in human platelets, it is demonstrated that SOCE elicits dense granule secretion in human platelets and inhibiting the platelets response to secreted autocrine messengers greatly reduces TG-evoked Ca²⁺ entry, suggesting an important role of autocrine modulation of SOCEI. These results therefore question the specificity of using thapsigargin as a specific activator of this Ca²⁺ entry pathway. The results also suggest the presence of two pharmacologically-distinct NCX subpopulations in human platelets. Results from experiments investigating the autocrine modulation of the Ca²⁺ responses of platelets stimulated with physiological agonists such as ADP and thrombin are also presented. These experiments lead to the suggestion that a rise in [Na²⁺]_i may play an important role in potentiating the activation of human platelets. Lastly data is presented suggesting the presence of the TRPV1 ion channel in human platelets and provides preliminary evidence towards a role for this ion channel in modulating the secretion of serotonin from these cells.