PI3K-C2α regulates platelet biogenesis. (A) Megakaryocyte relative Pik3c2a mRNA expression normalized to β-actin cDNA (mean ± standard error of the mean [SEM]; n = 4). Western blot analysis for PI3K-C2α, p85, PI3K-C2β, and vps34 in platelet lysates. (B) In vitro platelet PI3K-C2α lipid kinase activity (mean ± SEM; n = 4; *P < .05 vs WT according to 1 sample t test). (C) Whole-blood platelet count (mean ± SEM; n = 30 mice), measured using Micros60 (Horiba ABX Diagnostics). Platelet diameter measured using line-scan analysis after α-tubulin staining on fixed resting platelets (mean ± SEM; n = 150 platelets). Surface glycoprotein expression on resting platelets (MFI, mean fluorescence intensity; mean ± SEM; n = 4). (D) Transmission and scanning electron microscopy on resting platelets. Images are representative of 5 mice of each genotype. Scale bar: 1 µm. Enlarged pictures of OCS from WT and PI3K-C2α^WT/D1268A platelets are shown. Scale bar: 200 nm. I, invaginated platelet; B, barbell shaped-proplatelets; G, α-granule; OCS, open canalicular system. Quantification on transmission electron microscopy images of normal platelets, aberrant platelets (A), and barbell shaped-proplatelets (B) (mean ± SEM; n > 400 platelets; *P < .05 and **P < .01 vs WT, according to 2-tailed Student t test. (E) α-granule number and mean area quantified on transmission electron microscopy images using ImageJ software (mean ± SEM; n = 100 platelets; ***P < .001 vs WT, according to 2-tailed Student t test). Quantification of vWF by enzyme-linked immunosorbent assay test and fibrinogen by western blotting on resting platelets (mean ± SEM; n = 3). (F) Transmission electron microscopy of mice bone marrow section (n = 3). Scale bar: 2 µm. G; α-granule. α-granule number and mean area quantified as (E) (mean ± SEM, n = 20 megakaryocytes, ***P < .001 vs WT, according to 2-tailed Student t test).