Single-nucleotide mutation within Mpig6b results in severe macrothrombocytopenia and impaired MK maturation. Platelet count (A) and volume (B) in 10-week-old female and male WT and Mpig6b^mut mice were assessed with an automated blood cell analyzer. Values are the mean ± SD. Unpaired, 2-tailed Student t test. ***P < .001. (C) Identification of a mutation in a splice acceptor site of Mpig6b in Mpig6b^mut mice by whole-exome sequencing. The G>A single-nucleotide exchange in Mpig6b was present in all reads in mutant but not in WT mice. (D-E) Absence of G6b-B was validated in Mpig6b^mut platelets by flow cytometry (D) and immunoblot analysis (C-terminal antibody) using enhanced chemoluminescence (E). (F) WT and Mpig6b^mut MKs were differentiated in vitro in the presence of TPO and analyzed by brightfield microscopy. Mean MK diameter was determined manually with ImageJ software. At least 30 MKs per culture were analyzed. Values are mean ± SD (n = 3). Unpaired, 2-tailed Student t test. *P < .05. (G) The αIIbβ3⁺ cell population in whole BM of WT or Mpig6b^mut mice was analyzed by flow cytometry. Values are mean ± SD (n = 8). Unpaired, 2-tailed Student t test. ***P < .001. (H) Mean size of native MKs was analyzed ex vivo by flow cytometry. Values are mean ± SD (n = 4). Unpaired, 2-tailed Student t test. ***P < .001. (I) Demarcation membrane system maturation in WT and Mpig6b^mut BM MKs was visualized by transmission electron microscopy, constrasted by osmium tetroxide and stained with uranly acetate/lead citrate. Bars represent 3 µm; insets: 1.5 µm. Nuclear (J) and DMS (K) fraction in relation to cell size were quantified manually using ImageJ software. At least 7 MKs per mouse were analyzed. Values are mean ± SD (n = 3). Unpaired, 2-tailed Student t test. *P < .05; ***P < .001.