Abstract/Summary

The C-type lectin-like receptor-2 (CLEC-2) is a platelet receptor for the endogenous ligand podoplanin. This interaction contributes to several pathophysiological roles, such as lymphangiogenesis, preservation of blood and lymphatic vessel integrity, organ development and tumour metastasis, however the mechanism of this interaction is poorly understood. The aims of this thesis were to develop a reconstituted artificial lipid membrane platform as an experimental approach to investigate what determinates the biochemical properties of platelet signalling; and to identify a small-molecule inhibitor of the CLEC-2-podoplanin interaction and to characterise their effect on human platelet activation. To achieve the aims, supported lipid bilayers were used to mimic the platelet membrane. Fluorescent recovery after photobleaching and total internal reflection fluorescence microscopy were used to investigate bilayer mobility and platelet�mediated clustering of human podoplanin. The AlphaScreen-based high-throughput screen with 18,476 compounds from Pivot Park Screening Centre library was used to identify a small-molecule inhibitor of the CLEC-2-podopolanin interaction. Molecular docking and bio-layer interferometry were used to predict the binding sites and affinity between small molecule and CLEC-2. Light transmission aggregometry, flow cytometry, platelet spreading, and phosphorylation assays were used to evaluate the effect of the small molecule on CLEC-2-mediated platelet activation. Human dermal lymphatic endothelial cells were used to investigate the small molecule effect on platelet adhesion to cells endogenously expressing podoplanin. A semi-high-throughput artificial lipid membrane platform has been successfully developed as an experimental approach to investigate CLEC-2-mediated clustering of its endogenous ligand, podoplanin. An identified small molecule inhibitor of the CLEC-2-podoplanin interaction, MAS9, showed an inhibitory effect on the human platelet function and selectivity for the CLEC-2 receptor. This study provides the opportunity to take full advantage of the supported lipid bilayer model to understand CLEC-2-mediated signalling and clustering. Identified in a high-throughput screen, MAS9, may be a good candidate for a novel anti-platelet drug having therapeutic potential.