cFN modulates MK adhesion on collagen type I, but not on fibrinogen. Mature MKs were differentiated from human cord blood CD34⁺ cells as described in “Methods.” Mature MKs were then incubated on type I collagen or fibrinogen-coated coverslips for 16 hours at 37°C in an atmosphere of 5% CO₂ in the presence of TPO. (A) After 3 and 16 hours of incubation, coverslips were fixed and MK spreading and pro-platelets were evaluated as described in “Methods.” (B) Representative image of MK forming pro-platelets upon fibrinogen adhesion. Cells were stained with an anti–α-tubulin antibody (green) and Hoechst 33288 for nuclear staining (blue). Scale bar = 20 μm. (C) After 3 hours of incubation, coverslips were fixed, permeabilized, and stained with a polyclonal antibody against cFN. Subcellular localization of cFN (red) was analyzed by confocal microscopy. Scale bar = 10 μm. (D) Immunoprecipitation of released FN in supernatants harvested after 3 hours of MK adhesion to type I collagen (lane 2) or fibrinogen (lane 3). FN from human plasma was used as a positive control (lane 1), while unconditioned StemSpan medium was used as a negative control (lane 4). Actin was revealed by Western blotting in adherent cells on both matrices to ensure the same number of cells. (E) Scanning electron microscopy of MKs spread on type I collagen after 3 hours of incubation. Right panel, immunogold staining with polyclonal FN antibody. Arrows indicate FN exposure on the MK membrane. Scale bar = 1 μm. *P < .05