Megakaryocytes (MKs) of the probands exhibit defective SDF1-driven migration on fibrinogen and type I collagen and altered proplatelet formation. (A-C) SDF1-driven migration of MKs was investigated using a Transwell assay. Transwell systems having a polycarbonate membrane with an 8-µm pore size were coated with fibrinogen or type I collagen. Aliquots of 1 × 10⁴ MKs were seeded in the upper chamber of the Transwell insert, whereas the lower chamber was filled with medium containing 100 ng/mL SDF1. After incubation for 16 hours at 37°C and 5% CO₂, cells that migrated to the lower face of the membrane were labeled with an anti–β1-tubulin antibody (red) and counted using fluorescence microscopy with an Olympus BX-51 microscope. Samples of the 2 probands were processed in parallel with those of 3 HCs. (A) Representative images of microscopic fields of migrated cells. Scale bars, 30 µm. (B-C) MK migration was quantified as the number of migrated MKs per field (mean ± SD) by analyzing the entire polycarbonate membrane area. The assays were performed in triplicate wells for each condition. (D-G) Proplatelet formation was analyzed on fibrinogen-coated coverslips after incubation of MKs for 16 hours at 37°C and 5% CO₂. Cells were stained with an anti–β1-tubulin antibody (red). Hoechst (blue) was used for counterstaining nuclei. Samples of the probands were processed in parallel with those of 3 HCs. (D) Representative images of the morphology of proplatelets extended by probands II-1 and II-2. Proplatelets from HCs are shown in the top row comparison. Scale bars, 30 µm. (E) The rate of proplatelet formation (%PPF) was measured, using fluorescence microscopy, as the proportion of MKs displaying ≥1 proplatelet with respect to the total number of MKs (mean ± SD). The number of proplatelet free ends (tips) (F) and the number of bifurcations of proplatelet shafts (G) per MK were measured using image analysis that investigated ≥25 MKs for each individual (patient or control). *P < .0001, 2-tailed Student t test.