3D organization of the granules in stimulated platelets. To study exocytosis, platelets were stimulated with ADP (5 µM) or thrombin (0.1 or 1 U/mL), fixed at time points corresponding to (A) shape change or (B) half-maximal aggregation, stained with tannic acid, and prepared for FIB-SEM. Tannic acid coats the cell surface, fills the OCS, and enters the granules undergoing secretion. Two images from FIB-SEM slices of stimulated platelets (right, detail of the granules) and a 3D reconstruction of stained granules (in red) and unstained granules (in yellow) are shown for each condition. Scale bars in the first column, 500 nm; scale bars in the second column, 200 nm. Images are representative of at least 3 different experiments. (A) Maximal platelet shape change. (i-iii) Platelets stimulated with ADP (5 µM; 7 seconds). Note that single granules and OCS remained in separate compartments. (iv-vi) Platelets stimulated with 0.1 U/mL thrombin for 15 seconds. Some single granules are tannic acid–positive. (vii-ix) Platelets stimulated with 1 U/mL thrombin for 10 seconds. (*) Indicates stained aggregates of granules. Note that the connections with the platelet surface pass through narrow OCS channels (arrows), resulting in small pores at the cell surface. (B) Half-maximal aggregation. (i-iii) In platelets stimulated with ADP (5 µM; 30 seconds), the granules remained unstained. (iv-vi) In platelets stimulated with 0.1 U/mL thrombin for 70 seconds, 2 pools of granules are visible. (vii-ix) In platelets stimulated with 1 U/mL thrombin for 70 seconds, the fused granules form large intracellular compartments (*) which are all in contact with the external space, creating large fusion pores at the platelet surface (arrows). Bar graphs represent the quantification of the exocytosis events (ie, no granular fusion [white bar], single granular fusion [gray bar], or granule-to-granule fusion [black bar], in response to different degrees of platelet stimulation and different time courses of activation). Three experiments were performed for each condition with >150 platelets in each case. Vertical bars indicate mean ± standard error of the mean. (C) Representative SEM images of (i) unstimulated platelets, (ii) platelets stimulated with 0.1 U/mL thrombin and fixed at time points corresponding to shape change (15 seconds), and (iii) half-maximal aggregation (70 seconds). The fusion pores are indicated with black arrows, and OCS openings are indicated with a white arrow. Scale bars, 1 µm. (D) Time course of α granule cargo release from platelets stimulated with 0.1 and 1 U/mL thrombin. At the indicated times (in seconds), thrombin stimulation was stopped with hirudin (100 U/mL), and platelets were immediately centrifuged at 16 000g for 5 minutes. The releasates were assayed for PF4 content (square), fibronectin content (triangle), and VWF activity (circle) using enzyme-linked immunosorbent assay kits. Each time point corresponded to 3 separate experiments.