Disrupted GPIbα-flnA interaction does not impair megakaryocyte differentiation or proplatelet production in vitro or in the lung in vivo. (A) MK ploidy was determined for WT and FW MKs using PI staining of day-5 cultured MKs and flow cytometry. Results represent the mean ± SEM of hGPIbα^WT and hGPIbα^FW mice, (n = 5 and n = 6, respectively); P = .8347 for strain comparison, 2-way ANOVA. (B) MK differentiation was assessed by surface expression of GPIIb-IIIa and GPIb-IX using anti–CD41-PE and anti–CD42a-FITC antibodies, respectively, on day-5 cultured cells using flow cytometry, with results depicting percentage of positively stained vs total cells (mean ± SEM; n = 4 mice). (C) The number of proplatelet bearing MKs at day 4 culture was quantified for hGPIbα^WT and hGPIbα^FW mice. Results represent the mean ± SEM (n = 6-8; P = .87, unpaired t test). (D) The size of individual proplatelet terminal swellings produced by each genotype was quantified using CD42c staining and Zeiss Zen Software and/or Image J. The size represents the maximal transverse length of each proplatelet swelling, with data derived from ∼102 and 65 swellings, 16 and 13 megakaryocytes, from 2 independent hGPIbα^WT and hGPIbα^FW mice, respectively. (E) The lungs of hGPIbα^WT and hGPIbα^FW mice were sectioned and subjected to CD41 and laminin immunostaining. Representative 3D confocal images demonstrating pulmonary proplatelets in hGPIbα^WT and hGPIbα^FW mice (scale bars, 10 μm). Proplatelets were differentiated from other CD41 stained platelets by their large size, branched morphology and within alveolar septa. (F) The number of proplatelets in 4 × 1170 μm² lung areas was quantitated and the results represent the mean ± SEM of 3 mice per strain (P = .37, unpaired t test). SEM, standard error of the mean.