Defining the Role of NMDA Receptors in the Thrombopoietic Lineage Using a Conditional Knockout Mouse Model

Abstract

Processes of platelet production and function are incompletely understood. Glutamate pathways and its receptors have arisen as potential novel regulators of these processes, but details are limited. The main objective of this thesis was to characterise the function of the glutamate receptor called N-methyl-D-aspartate (NMDA) receptor in megakaryocytes and platelets. We set out to create a conditional knockout of the NMDA receptor in the platelet lineage using the CreloxP system by crossing the Grin1-loxP and Pf4-Cre mouse strains. Grin1 encodes the obligate NMDA receptor subunit (GluN1) and Pf4 promoter is megakaryocyte-specific. Therefore, with the appropriate combination of genes, Cre would cut Grin1 out in megakaryocytic cells, preventing assembly of functional NMDA receptors. Mice with Pf4-driven Grin1 deletion (Pf4-Grin1-/- mice) were successfully created in this thesis through a two-stage breeding approach guided by PCR-based genotyping. Pf4-Grin1-/- mice were born in a Mendelian manner, implying no loss of knockout mice in utero. Pf4-Grin1-/- mice did not display obvious signs of bleeding, appeared healthy and gained weight well, excluding major health problems. Through specialised testing, we determined that Pf4-Grin1-/- mice had lower platelet counts, prolonged bleeding time, reduced platelet activation and smaller spleen size. Bone marrow and megakaryocyte morphology was grossly normal in Pf4-Grin1-/- mice, with megakaryocytes present in preserved numbers and no change in megakaryocyte ploidy. Deletion of the Grin1 gene was documented in megakaryocytic cells in Pf4-Grin1-/- mice by a conventional PCR with an associated reduction in Grin1 transcripts. At the protein level, we saw a reduction in GluN1 expression in megakaryocytes by immunofluorescence however flow cytometric analysis showed no change in expression of GluN1 in platelets. Overall, our results suggest that Pf4-driven Grin1 deletion led to the knockdown of GluN1 in megakaryocytes and platelets, although this will require further evaluation. The phenotype observed in Pf4-Grin1-/- confirmed an important role for NMDA receptors in megakaryocytes and platelets. Our results suggest that the NDA receptor may be involved in thrombopoiesis and platelet function.