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Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation
James I Hearn1, Taryn N Green1, Colin L Hisey2
1Blood and Cancer Biology Laboratory, Department of Molecular Medicine and Pathology, School of Medical Sciences, and.
Abstract:
The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.
Insights
N-methyl-D-aspartate receptors (NMDARs) are crucial for platelet production and function. Genetic deletion of NMDARs in mice impairs megakaryocyte maturation and platelet formation, impacting blood clotting.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- Proplatelet formation (PPF) is essential for platelet production, requiring megakaryocyte (MK) interaction with the extracellular matrix (ECM) and cytoskeletal dynamics.
- Intracellular calcium ion (Ca2+) fluxes regulate MK-ECM interaction and PPF.
- Glutamate-gated N-methyl-D-aspartate receptors (NMDARs) are highly permeable to Ca2+ and have been shown to inhibit MK maturation ex vivo.
Purpose of the Study:
- To investigate the in vivo role of NMDARs in megakaryopoiesis, thrombopoiesis, and platelet function.
- To elucidate the mechanism by which NMDARs regulate proplatelet formation.
Main Methods:
- Generated a platelet lineage-specific knockout mouse model (Pf4-Grin1-/-) using the Cre-loxP system to reduce NMDAR function in MKs and platelets.
- Assessed platelet function and production in vivo and ex vivo using established assays.
- Analyzed megakaryopoiesis, thrombopoiesis, platelet counts, bleeding time, platelet activation, spreading, and cytoskeletal organization (F-actin, α-tubulin).
Main Results:
- Pf4-Grin1-/- mice exhibited defects in megakaryopoiesis, thrombopoiesis, and platelet function, including reduced platelet counts, impaired platelet production during thrombocytopenia, and prolonged bleeding time.
- Platelet activation by various agonists was impaired, associated with reduced Ca2+ responses and defective platelet spreading.
- MKs showed reduced colony and proplatelet formation, linked to impaired reorganization of F-actin and α-tubulin, and decreased expression of ECM-related transcripts.
Conclusions:
- Provides the first genetic evidence for an active role of NMDARs in platelet function and production in vivo.
- NMDAR signaling regulates proplatelet formation by influencing MK-ECM interaction and cytoskeletal reorganization.
- NMDARs are critical for guiding proplatelet formation in vivo.
