Impact of Grin1 deletion on megakaryopoiesis and thrombopoiesis in vivo and ex vivo. (A) Line graph showing peripheral platelet counts from Pf4-Grin1^−/− and WT mice after injection with antiplatelet serum (APS). Three independent experiments were performed, with a total of 12 to 15 biologic replicates per group. (B) Line graph showing platelet half-life of Pf4-Grin1^−/− and WT mice. Platelets were labeled in vivo after IV injection of DyLight488-conjugated anti-CD42 (×488) antibodies, and the proportion of positively labeled platelets was measured by flow cytometry. Four independent experiments were performed, with a total of 10 biologic replicates per group per time point. (C-E) Representative images of BM sections from WT and Pf4-Grin1^−/− mice under bright-field microscopy stained with hematoxylin and eosin (C), immunofluorescence staining of GPIX (megakaryocytic marker [green]) and CD105 (endothelial cell marker [red], with framed areas enlarged in the right panels; nuclei stained with 4′,6-diamidino-2-phenylindole [blue]) (D), and CD61 immunostaining (Ei). Scale bars are shown. (Eii) Bar graph showing MK counts (CD61⁺ cells per 20× field counted from sections shown in panel Ei) from WT and Pf4-Grin1^−/− mice. (F) Bar graph showing level of nuclear ploidy (%) in WT and Pf4-Grin1^−/− mice examined by flow cytometry; 2N, 4N, 8N, 16N, 32N, and 64N indicate ploidy classes. Five independent experiments were performed with 5 biologic replicates per group. (G) Bar graphs showing number of MK colonies observed per matrix, examined using the MegaCult assay. MKs were stained with acetylthiocholiniodide solution; colonies were counted and scored as small (3-20 cells), medium (20-50 cells), or large (>50 cells). Four independent experiments were performed. (H) Examination of PPF by MKs migrating out of BM explants. (Hi) Representative images of MKs scored as round, protrusion forming, or proplatelet forming (indicated by arrows). Scale bar is shown. (Hii) Bar graphs showing percentage of MK forms per explant. Three independent experiments were performed. Triplicate measures were recorded per biologic replicate with a total of 3 biologic replicates per group. Line and bar graphs show mean ± SEM in panels A-B and E-H. Statistical significance is shown (2-way analysis of variance for panels A and F; analysis of covariance for panel B; 2-tailed Student t test for panels Eii, G, and Hii).