Mutant PRKACG leads to defective PPT formation, which is rescued by wild-type PRKACG overexpression. (A-E) In vitro MK differentiation was induced from control or patient peripheral blood CD34⁺ progenitors in the presence of TPO and SCF. (B-E) CD34⁺ cells of patients II-1 and II-2 were transduced with a lentiviral vector harboring wild-type (wt) or mutant PRKACG cDNA (used as a control of experiment) at days 1 and 2 of culture. (A-B) The percentage of PPT-forming MKs was estimated by counting MKs exhibiting ≥1 cytoplasmic processes with areas of constriction at day 13 of culture. A total of 200 cells per well were counted. The histograms show 1 of 2 independent experiments with similar results. Each experiment was performed in triplicate. Data represent mean ± SD of triplicate. *P < .05, 2-tailed Mann-Whitney test. (C-D) Immunoconfocal analysis of platelet-like structures formed by PPTs generated from patients (C) II-1 and (D) II-2. MKs overexpressing wt or mutant PRKACG PPT-forming MKs were allowed to adhere on fibrinogen for 2 hours at day 13 of culture and stained with anti-tubulin (red) and rabbit anti-VWF (green) antibodies. Confocal imaging was performed on a Leica TCS SP8 inverted laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany), equipped with a 405-nm UV laser diode and visible optically pumped semiconductor lasers (488 and 552 nm). All images were acquired using an oil immersion 63× objective (1.4 numeric aperture). (E) At least 5 MKs for each condition were analyzed, and the size of platelet-like structures was measured by LAS AF version 2.4.1 software. Data are presented ± SEM. **P < .005 and ***P < .0001, unpaired Student t test with Welch’s correction.