Impaired megakaryocyte development in Agk^f/fPF4-Cre mice. (A) Immunohistochemical images of CD42c in femurs and spleens of Agk^f/f mice and Agk^−/− mice. The scale bars represent 30 μm. Statistics for the megakaryocytes in femur (FM) and spleen (SP) sections of Agk^f/f mice and Agk^−/− mice are shown (n = 13; ***P < .001). (B) CD41⁺CD42b⁺ megakaryocytes in BM and spleen samples were detected by flow cytometry. Statistics for the CD41⁺CD42b⁺ megakaryocytes in the BM (n = 6; ***P < .001) and spleen (n = 5; **P < .01) samples of Agk^f/f mice and Agk^−/− mice are shown. (C) Spleens of Agk^f/f mice and Agk^−/− mice. (D) Splenectomies were performed on Agk^f/f mice and Agk^−/− mice, and circulating platelet were counted at indicated time points (n = 5; *P < .05, **P < .01, ***P < .001). (E) Megakaryocyte colony-forming unit (MK-CFU) assay using BM cells harvested from Agk^f/f mice and Agk^−/− mice (n = 3; *P < .05). (F) Megakaryocyte colony-forming unit assay using sorted MKP cells from fetal liver of Agk^f/f mice and Agk^−/− mice (n = 3; **P < .01). (G) The level of TPO in the plasma from Agk^f/f mice and Agk^−/− mice (n = 6). (H) Immunofluorescence images of α-tubulin in PPF-megakaryocytes derived from the fetal livers of Agk^f/f mice and Agk^−/− mice. Scale bars, 50 μm. Statistics for the PPF-megakaryocytes/total megakaryocytes derived from the fetal livers of Agk^f/f mice and Agk^−/− mice are shown (n = 10; ***P < .001). (I) Percentage of megakaryocytes polyploidy derived from fetal liver of Agk^f/f and Agk^−/− mice (n = 3; *P < .05, **P < .01, ***P < .001). DAPI, 4′,6-diamidino-2-phenylindole; MK, megakaryocyte; MKP, megakaryocyte progenitor.