Membrane-associated polyphosphate is incorporated into platelet aggregates. Microscopic images were acquired at a magnification of ×1000 (A-D) and ×630 (E). Scale bar is shown in the left panels (10 μm). All images are shown at the same magnification; insets show indicated areas at higher magnification. Times (above) represent the time course, starting from the moment of first stable platelet adhesion in the image field. (A) Time series of spreading and degranulation of platelets in citrated plasma on immobilized fibrinogen at 25 s⁻¹ shear rate during activation by PAR4-activating peptide (PAR4-AP). Polyphosphate was visualized with SYTOX (orange); PAR4-AP enters the flow chamber after 15 minutes of perfusion (indicated below the images). (B) Time series of platelet adhesion and spreading on immobilized VWF under flow. Polyphosphate was visualized with SYTOX (orange); PPX (500 µg/mL) enters the flow chamber after 20 minutes of perfusion (indicated below the images). (C) Time series of platelet adhesion and spreading on immobilized VWF under flow. Polyphosphate was visualized with Alexa488-conjugated PPX_Δ12 (green). (D-E) Cross-section images of SYTOX-stained platelet aggregates, formed by perfusing citrated whole blood over immobilized collagen at 1600 s⁻¹ shear rate for 10 minutes. (D, bright-field/fluorescence microscopy image of unfixed aggregates; E, confocal microscopy image of fixed aggregates) Green, anti-CD42b; orange, SYTOX. Images are representative of experiments that were performed >3 times.