VAMP8 mediates compound exocytosis in stimulated platelets. (A) VAMP8 immunogold staining. (i) Unstimulated platelets or (ii) platelets stimulated with 0.1 U/mL thrombin for 15 seconds were fixed for IEM and stained with an anti-VAMP8 antibody (detected with 10-nm gold particles). VAMP8 labeling was mostly associated with α granules in unstimulated platelets and with large multigranular compartments in thrombin-stimulated cells. The insert in (i) illustrates double immunogold labeling (VAMP8¹⁰  and P-selectin [Psel]¹⁵ ). P-selectin is present in the δ granule membrane,³⁷  and labeling allows the identification of δ granules. This image shows that VAMP8 was also present on δ granules. Scale bars, 200 nm. (B-D) FIB-SEM analysis of VAMP8^−/− platelets stimulated by 0.1 U/mL thrombin. Platelets were fixed at half-maximal aggregation (70 seconds). (B) Representative 2D images from FIB-SEM slices and 3D reconstructions of WT and VAMP8^−/− platelets showing that granule-to-granule fusion (*) occurred in WT but not in VAMP8^−/− platelets. Scale bars in the first column, 2 nm; scale bars in the second column, 500 nm. (C) Incubation with tannic acid revealed the presence of single-stained granules in stimulated VAMP8^−/− platelets and multigranular compartments in WT platelets, visible on a 3D reconstruction (red) and 2D images from FIB-SEM slices (arrow in insert). α granules (yellow), δ granules (black), mitochondria (blue). (D) Left panel: 3D quantification of the exocytosis events (ie, no granular fusion [first bar], single granular fusion [second bar], and granule-to-granule fusion [third bar]) in WT (gray) and VAMP8^−/− (blue) platelets stimulated with 0.1 U/mL thrombin (>61 and <96 platelets). Right panel: 3D quantification of the number of organelles per whole platelet in WT (gray) and VAMP8^−/− (blue) stimulated platelets (n = 12). Note the difference in the y-axis mean ± standard error of the mean (*, P < .05). Vertical bar of the right figure: organelles/platelet. (E-F) FIB-SEM analysis of VAMP8^−/− platelets stimulated by 1 U/mL thrombin. Platelets were fixed at half-maximal aggregation (70 seconds). (E) Representative 2D images from FIB-SEM slices and 3D reconstructions of WT and VAMP8^−/− platelets. The difference in compound exocytosis was less visible when platelets were stimulated with 1 U/mL thrombin. Scale bars in the first column, 2 nm; scale bars in the second column, 500 nm. (F) 3D quantification of (left panel) the exocytosis events and (right panel) the number of organelles per whole platelets in WT (gray) and VAMP8^−/− (blue) -stimulated platelets (n = 12). Vertical bar of the right figure: organelles/platelet. Multigr., multigranular.