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… Click to show full 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 is 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 a 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. NMDARs regulate PPF through the mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.
               
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