Langan, Stacey Anne (2011)
M.Res. thesis, University of Birmingham.
The platelet receptor CLEC-2 is a ligand for the transmembrane protein podoplanin, which is present on human lymphatic endothelial cells (HLEC). Mice lacking CLEC-2 or podoplanin display a blood-lymphatic mixing phenotype, possibly due to incomplete separation of the developing vessels. Previous work demonstrated that podoplanin mediates polarised migration of HLEC in response to VEGF-C and that platelets inhibit HLEC migration. Therefore, the aim of this work was to assess whether the interaction between podoplanin and CLEC-2 altered the migration of HLEC. Transfilter migration assays were used to assess the effect platelets and an anti-human podoplanin antibody on HLEC migration.
We found that platelets inhibit VEGF-C mediated HLEC migration in a count-dependent manner and that crosslinking podoplanin with a specific antibody and an appropriate secondary also gave a reduction in HLEC migration. Crosslinking podoplanin in the presence of the Rho kinase inhibitor Y27632 gave no further inhibition of migration. Using Y27632 in conjunction with platelets gave a partial recovery in the effect of platelets, suggesting platelets require activation of the RhoA signalling pathway to exert their anti-migratory effects.
In summary, crosslinking podoplanin with platelets or antibodies inhibits VEGF-C mediated HLEC migration and this may be dependent on the RhoA signalling pathway.
Tetraspanins are transmembrane proteins that are thought to have a number of roles in the organisation of cell surface proteins. The platelet collagen receptor GPVI has been identified as a component of tetraspanin microdomains. Similarly, its sheddase, ADAM10, has been shown to be tetraspanin-associated and previous work has found that ADAM10 is regulated by a specific subgroup of tetraspanins, known as the 8-Cys tetraspanins. Therefore, the aim of this work was to determine whether ADAM10 activation altered its localisation within the tetraspanin microdomain. We also wanted to confirm whether ADAM17 was associated with CD9 and assess whether the megakaryocyte-like HEL cell line would be a suitable model in which to study tetraspanin regulation of GPVI cleavage by ADAM10.
We found that ADAM10 was associated with platelet tetraspanins regardless of its activation state. From our data, ADAM17 does not appear to be associated with CD9 in platelets, but this may be due to low ADAM17 expression on these cells. HEL cells are not a suitable for studying GPVI cleavage and ADAM10 regulation by tetraspanins. Attempts to knockdown ADAM10 with siRNA were not successful and GPVI staining was lower than in platelets.
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