James, Nicola Jane; (2002) Cellular signalling by tissue factor and lipoproteins in the pathogenesis of atherosclerosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Atherosclerosis is a chronic disease advanced by repeated episodes of endothelial injury. Upon endothelial injury, tissue factor: factor VIIa complexes (TF: FVIIa) are formed that promote production of fibrin and induce intracellular signalling. A role for the cytoplasmic domain of TF in the induction of cytosolic Ca2+ transients was investigated. Wild type TF was cloned and a cytoplasmic deletion mutant was generated. Wild type and mutant TF were successfully expressed in Chinese Hamster Ovary K1 cells (CHO K1), however, unstable basal cytosolic Ca2+ levels precluded determination of the role of the cytoplasmic domain of TF in signalling. The procoagulant activity of TF is inhibited by the apolipoprotein B100 moiety of native low density lipoprotein (LDL). The inhibitory motif is located in a 6-mer region of apo B100 (KRAD6). KRAD6 exhibited concentration-dependent inhibition of TF: FVIIa-induced cellular signalling. Furthermore, a 14-mer peptide, containing the KRAD6 sequence, decreased the formation of cellular networks in an in vitro model of angiogenesis. The KRAD6 peptide inhibits both the procoagulant and signalling functions of TF: FVIIa. The KRAD6 may thus be of therapeutic use in diseases whose pathologies are linked with angiogenesis, such as atherosclerosis and metastasis. TF activity and oxLDL are important risk factors for atherosclerosis. Activation of TF: FVIIa by oxLDL may contribute to the proatherogenic activity of oxLDL. The involvement of TF in the induction of cytosolic Ca2+ transients by LDL was investigated. Ca2+ responses to nLDL were independent of TF and a role for TF in Ca2+ signalling by oxLDL was equivocal. In addition, the data show that Ca2+ elevations to nLDL were transient, whereas oxLDL elicited sustained Ca2+ elevations. These distinct patterns of cytosolic Ca2+ flux enable cells to respond appropriately, in accordance with the oxidation status of LDL.

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