| Summary: | Uncontrolled activation of the coagulation cascade following lung injury has been implicated in both lung inflammation and fibrosis. In addition to its role in coagulation, thrombin exerts pluripotent cellular effects via the activation of its high-affinity receptor, proteinase activated receptor-1 (PAR_1). PAR_1 is a seven transmembrane domain G protein-coupled receptor that exhibits the ability to couple to multiple G protein family subunits, including G\alpha_{i/o}, G\alpha_q and G\alpha_{12/13} within the same cell type. Activation of PAR_1 on fibroblasts, a key effector cell in lung fibrosis, results in the induction of several mediators, including the potent monocyte and fibrocyte chemoattractant CCL2. In this thesis, the G-protein and downstream signalling pathways involved in PAR_1-mediated CCL2 production and release were examined. Using a novel PAR_1 antagonist which blocks the interaction between PAR_1 and G\alpha_q, this thesis shows for the first time that PAR_1 coupling to G\alpha_q is essential for thrombin (10nM)-induced CCL2 gene expression and protein release in murine lung fibroblasts (MLFs). The work presented here further demonstrates that these effects are mediated via the cooperation between ERK1/2 and Rho kinase signalling pathways: a calcium-independent PKC, c-Raf and ERK1/2 pathway was found to mediate PAR_1-induced CCL2 gene transcription; whereas PLC, calcium, calcium-dependent PKC and Rho kinase pathway influences CCL2 protein release. This thesis represents the first demonstration of the cooperation between two pathways in mediating the stimulatory effects of thrombin, or indeed any other extra cellular stimulus, on the induction and release of the potent chemoattractant, CCL2. This thesis also examined the signalling receptor and downstream effectors involved in thrombin-induced CCL2 production in primary human lung fibroblasts (pHALFs). The results demonstrate that PAR_1 coupling to G\alpha_q is also both necessary and sufficient in mediating thrombin-induced CCL2 production at low concentration of the proteinase, whereas at high concentrations, these effects may be partially PAR-independent. This discrepancy between MLFs and pHALFs may be explained by differences of PAR receptor expression between species. Taken together, this thesis proposes that targeting the interaction between PAR_1 and specific G proteins may allow more selective blockade of PAR_1 pro-inflammatory and pro-fibrotic signalling, whilst preserving the essential role of other PAR_1-mediated cellular responses.
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