MK culture from probands carrying GALE p.Lys78ValfsX32, p.Thr150Met, p.Val128Met, and p.Leu223Pro variants showed normal polyploidization and maturation but impaired proplatelet formation. MKs were differentiated from peripheral blood progenitor cells from probands A.II.4 and B.II.2 through a 14-days culture, in parallel with healthy controls. (A) MK differentiation assessed as CD61⁺ cells were measured by MFI by flow cytometry. (B) MFI of GALE, evaluated by flow cytometry, in permeabilized MKs from controls, and probands A.II.4 and B.II.2. Data was relativized with 1 control value. (C) Immunoblotting of MK lysates. Control, A.II.4, and B.II.2 samples were probed with anti-GALE and anti-β3 integrin (internal control). (D) Assessment of MK viability and apoptosis rates at the end of the culture using Annexin V–propidium iodide(PI) labeling. The percentage of CD61⁺ MKs that are negative for both markers (viable cells), Annexin V⁺ PI⁻ (apoptotic cells), and double-positive (dead cells) is represented. (E) Percentage of polyploid MKs after PI labeling. (F) Representative images of mature and polynuclear MKs labeled with an anti–β3 integrin antibody (green fluorescence). Hoechst (blue) was used for counterstaining nuclei. Scale bars, 20 μm. (G) Diameter measurement of MK from A.II.A, B.II.2, and healthy controls, in fibrinogen-coated coverslips (n = 100). (H) Characterization of impaired proplatelet formation in fibrinogen-coated coverslips in probands A.II4 and B.II.2 vs control, (1) Rate of proplatelet formation measured as the proportion of MKs displaying ≥1 proplatelet with respect to the total number of MKs; (2) Number of bifurcations of proplatelet shafts per MK; (3) Number of proplatelet-free ends (tips); (4) Diameter measurement of proplatelet-free ends (tips) from A.II.4, B.II.2, and healthy control (n = 100). Box and whiskers graphs are shown. The central line represents the median value, whereas percentiles 25 to75 is included in the box. Whiskers represent the minimum and maximum values. (I) Representative image of proplatelet formation in optical and immunofluorescence microscope. MKs was plated on fibrinogen-coated coverslips and incubated for 16 hours at 37°C and 5% CO₂. Cells were stained with an anti-CD61 antibody (green). Hoechst (blue) was used for counterstaining nuclei. Scale bars, 20 μm. ∗∗∗P < .001; 2-tailed Student t test.