Analysis of α-granules and their content in proplatelet-forming MKs by electron and confocal microscopy. (A) Low magnification of a representative control proplatelet-forming MK by scanning electron microscopy showing proplatelet-like territories emerging from the main core of the cell. (B-C). Higher magnification of the circled areas in (A) seen by TEM. The developing proplatelets contain MVBs, precursors of α-granules. (D-E) Immunolabeling of CD41 and VWF in proplatelet-forming MKs by confocal microscopy in control (D) and Nbeal2^−/− (E) cells. VWF is restricted to the MVBs bodies observed in (B-C). The inset at the top-right corner in (E) highlights the presence of a thin MK filament bridging several emerging platelet-like particles. (F-G) Immunolabeling of CD41 in proplatelet-forming MKs previously cultured in the presence of fluorescein isothiocyanate (FITC)-fibrinogen. Fibrinogen is endocytosed and packaged into α-granules in control (F) and Nbeal2^−/− (G) MKs, and transported through the proplatelet shafts. (H-I) Proplatelet-forming MKs cultured with FITC fibrinogen, as stated in (F), and P-selectin-immunolabeled in control (H) and Nbeal2^−/− (I) samples. (J-K) Proplatelet-forming MKs cultured in the presence of FITC-fibrinogen, as stated in (F), and VWF-immunolabeled in control (J) and Nbeal2^−/− (K) samples. (L) Quantitation of proplatelets bud in MK culture. (M) Percentage of proplatelets buds containing the α-granule protein VWF. (D-K) Insets represent a higher magnification of platelet-like particles showing a similar granular distribution of P-selectin, VWF, and fibronogen in control and Nbeal2^−/− cells. (D-K) The nucleus was stained with 4,6 diamidino-2-phenylindole (blue). Representative images were selected from 5 different mice per group.