Christodoulou, M.; (2007) Endothelial cell responses to thrombin and shear stress. Doctoral thesis , University of London.

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Abstract

Endothelial cells alter their morphology in response to a combination of chemical and mechanical signals. Both thrombin and shear stress have been reported to activate integrin receptors, calcium channels and downstream Rho GTPases. The aim of the investigation was to contribute in the understanding of the mechanisms involved in the transduction of these signals. Biochemistry and microscopy-based techniques were employed to study the contribution of focal adhesion proteins (FAK, Paxillin, Src). Both thrombin and shear stress altered the phosphorylation status of FAK. paxillin and Src and the response was more rapid under shear stress conditions. The patterns of phosphorylation followed by specific tyrosine residues were similar for the two responses, but shear stress responses were cell type and magnitude-dependent. Despite similarities in focal adhesion protein phosphorylation patterns, changes in cell morphology differed between thrombin- and shear stress-treated endothelial cells. It was found that, thrombin-induced morphological changes preceded its effects on the phosphorylation of FAK, Src and paxillin while the shear-stress-induced morphological adaptations followed changes in the phosphorylation status of these proteins. Thrombin induced increased activity of RhoA. not Racl, but, only in the shear stress response, was stress fibre formation associated with increased phosphorylation of myosin light chain protein. These results suggest that the two stimuli signal through distinct pathways leading to different morphological changes. The role of the calcium-dependent proteases calpain-1 and calpain-2 was investigated using calpain inhibitors and isoform specific siRNAs. It was found that calpains and specifically calpain-2 plays an important role in the regulation of focal adhesion turnover. Calpain-2 could thereby play a central role in both the thrombin and shear stress responses in endothelial cells.

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