Impaired platelet function in Nbeal2^−/− mice. (A-B) Ultrastructure of platelets from control mice showing abundant α-granules (AG), among other organelles. (C-D) Ultrastructure of Nbeal2^−/− platelets showing a significant reduction of α-granules, several vacuoles (V) and membrane inclusions (MI). (E-F) Immunolabeling of VWF (red) and CD41 (green) in control (E) and Nbeal2^−/− (F) platelets. Images correspond to the maximum intensity projections of an image stack across an entire platelet section. Insets: A higher magnification of the circled platelets is shown for clarification. VWF is restricted to the α-granules in control platelets, whereas its distribution is more diffuse in Nbeal2^−/− platelets. Note also the absence of VWF in some Nbeal2^−/− platelets. (G) Representative immunoblots of α-granule proteins VWF, THBS1 and PF4 in control and Nbeal2^−/− platelet lysates. CD63 (LAMP-3), a lysosomal/dense granule marker, and β-actin and glyceraldehyde-3-phosphate dehydrogenase included as loading controls. Bottom graphs show the densitometry analysis performed using ImageJ (n = 3). *P < .05. (H) Flow cytometry analysis of platelet activation induced by 1 μg/mL collagen-related peptide and 1 U/mL thrombin (n = 4) showing equivalent fibrinogen binding to Nbeal2^−/− and control platelets (top panel, only shown for collagen-related peptide) in contrast reduced P-selectin exposure in Nbeal2^−/− platelets with both agonists (middle and bottom panels). Plots with vertical lines correspond to basal platelet activation in the absence of agonist. For all graphs, bars represent mean ± standard error of the mean. Scale bars represent 100 nm (A, C) and 500 nm (B, D). *P < .05. NS, nonsignificant; MFI, mean fluorescent intensity.