Polyphosphate is retained on the platelet surface after degranulation. (A) Time series of platelet adhesion and spreading on immobilized VWF under flow. Polyphosphate was tracked with DAPI (blue) and SYTOX (orange). Degranulation was detected by an anti-CD63 antibody (green). Microscopic images were acquired at a magnification of ×1000. A scale bar is shown in the upper left panel (10 μm). All images are shown at the same magnification. Times (above) represent the time course, starting from the moment of first stable platelet adhesion in the image field (indicated by arrow in upper left panel). (B-C) Individual spreading platelets were visually inspected (n = 25, derived from videos of >3 separate experiments). Approximately 4 minutes after initial adhesion, both CD63 and polyphosphate appeared on the platelet surface (“Presentation”'). The fluorescence of both these markers persisted for the remainder of the experiments (“Retention”). (D) Localization of polyphosphate and CD63 on spreading platelets after >20 minutes. T, time.