Defective platelet production and granule distribution in Vps34-deficient platelets. (A) Whole blood platelet count was measured by using a HORIBA ABX Micros 60 analyzer (mean ± SEM; n = 38 mice for WT and 49 for Pf4-Cre-Pik3c3^lox/lox mice [Vps34]); ***P < .001 vs WT according to 2-tailed Student t test) (left). Quantification of the percentage of mice with a mean platelet volume ranging from 4 to 7 µm³ and from 7 to 10 µm³ (mean ± SEM; n = 38 mice for WT and 49 for Pf4-Cre-Pik3c3^lox/lox mice [Vps34]) (middle). TEM of resting platelets (right). Images are representative of 5 mice of each genotype. Scale bar represents 2 µm. (B) Mice were intravenously injected with a dylight⁴⁸⁸–anti-GPIbβ immunoglobulin derivative antibody. The percentage of labeled platelets in blood samples was measured at various time points after injection. (C) Thrombocytopenia in mice was induced by intraperitoneal injection of anti-GPIbα antibody (left). The platelet count was measured in blood samples collected 6 hours after injection (time = 0) and at various time points. The mouse serum TPO level was quantified by immunoassay (middle). Platelets were incubated with 50 ng/mL of TPO at the indicated times and after fixation with a rat antibody against the extracellular domain of Mpl and an anti-rat Alexa Fluor⁴⁸⁸ antibody (right). The graph is expressed as the percentage of the mean fluorescence intensity (MFI) resting (0) values after flow cytometry analysis (mean ± SEM; n = 4-6 mice of each genotype, *P < .05 vs WT according to 2-way ANOVA). (D) TEM of resting platelets. Images are representative of 5 mice of each genotype. Scale bar represents 1.5 µm. Arrows indicate α-granules. Platelet α- and dense (δ)-granule numbers and mean areas were measured on TEM images by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-tailed Student t test).