N-methyl-D-aspartate expression in megakaryocytes and platelets

Abstract

Megakaryocytes are specialized hematopoietic precursor cells that are responsible for the production of platelets. Preliminary studies have highlighted the significance of glutamate signalling in the normal function of megakaryocytes and platelets. Glutamate has long been recognised as one of the most important neurotransmitters for inter-neuronal communication in the central nervous system. Neuronal-N-methyl-D-aspartate (NMDA) receptors, a glutamate receptor type, have recently been discovered in megakaryocytes and platelets. The knowledge of the detailed expression characteristics of NMDA receptor in these blood lineages is very limited. The main objective of this study was to characterize NMDA receptor expression in human platelets and megakaryocytic cells. First, megakaryocytic cell lines MEG-01, SET-2 and K562 were used. NMDA receptor expression in human megakaryocytes within bone marrow samples, normal and neoplastic, was also tested. The types of human malignancies tested were chronic myeloproliferative neoplasms where megakaryocytes are abnormal: Essential Thrombocythaemia, Primary Myelofibrosis and Chronic Myelod Leukaemia. Material from patients with Acute Megakaryoblastic Leukaemia was also examined. The techniques used were Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. The expression of the subunits NR1, NR2A and NR2D was detected in platelets using all three techniques. The subunits NR1, NR2A, NR2C and NR2D were detected in all three cell lines by RT-PCR. MEG-01 also showed some transcription of NR2B. The protein detection by Western blotting was challenging for cell lines but the expression of NR1, NR2A and NR2D proteins was observed at a cellular level by immunohistochemistry. The expression of subunits NR1, NR2A and NR2D in megakaryocytes from normal bone marrow samples was also detected. The megakaryocytes in neoplastic samples were only tested for the subunit NR1 that was detected by immunohistochemistry in all these types of disorders. Some variations between individuals were present but reasons would need to be elucidated further. My data confirmed the expression of NMDA receptor in human platelets, megakaryocytic cell lines and primary human megakaryocytes, both normal and neoplastic. Further studies are required to establish functionality and specific roles of NMDA receptor in the megakaryocytic and platelet lineages. My results will provide a foundation for such targeted functional studies.