AbstractThis study investigated the role of NMDARs in the differentiation of MEG-01 cells and in the activation of human platelets. This investigation demonstrated that the NR1, NR2D and NR3 subunit proteins are expressed in human platelets, with the NR1 subunit also expressed in MEG-01 cells. The NR2A subunit protein was not detectable in either MEG-01 cells or human platelets. PMA-induced differentiation of MEG-01 cells did not appear to stimulate changes in expression of any of the subunit proteins tested. Using assays to measure the changes in [Ca2+]i and ATP secretion, it was determined that donors could be separated into those who responded to the agonists applied and those who did not; responses also decreased over time in both assays. Human platelets from responding donors demonstrate an increase in [Ca2+]i in response to extracellular glutamate, and that increases in ATP secretion are detected at a 10-fold lower concentration. The same is also true with extracellular glycine. Increases in [Ca2+]i were elicited on the addition of extracellular NMDA; extracellular D-serine had no effect. NMDAR inhibitors, MK-801 and D-AP5, inhibited ATP secretion evoked by either glutamate alone or in combination with glycine. D-serine inhibited responses elicited by extracellular glycine. NMDARs play a role in MK differentiation, with the adhesion of MEG-01 cells cultured on a fibrinogen-surface and differentiated with PMA reduced by both inhibitors. PMA-treated MEG-01 cells increased both in size and irregularity, with the addition of NMDAR-specific inhibitors having no effect. S-nitrosylation also inhibits activation of NMDAR, and a new molecule has been developed which can detect S-nitrosylated proteins through a single step process in live cells. Overall, this study has shown that both human platelets and MEG-01 cells express NMDAR subunits, which have been demonstrated to form functional receptors in human platelets.
|Date of Award||1 May 2010|
|Supervisor||Amanda Mackenzie (Supervisor)|
The Functional Expression of N-Methyl-D-Aspartate Glutamate-Type Receptors by Megakaryocytes and Platelets
Hobbs, C. (Author). 1 May 2010
Student thesis: Doctoral Thesis › PhD