Plasminogen localizes in “caps” of PS-exposing platelets. Platelets (0.5 × 10⁸/ml) were adhered to a collagen (0.6 µg) ± thrombin (3 pmol) or ± TRAP-6 (0.45 µmol) coated slide ± GPRP (5 mM) or ± tirofiban (1 µg/ml). After 40 minutes incubation, plasminogen-DL633 (0.8 µM) or fibrinogen-AF647 (16.7 µg/ml) was added for 5 minutes. (A) Spread PS-negative platelets showing plasminogen-DL633 binding. (B) Plasminogen-DL633 binding on PS-positive platelets (left) ± tirofiban (middle), or GPRP (right). Annexin A5–FITC (1/20 dilution) and 2 mM CaCl₂ were added immediately prior to imaging. (C) Representative quantification of plasminogen-DL633 binding expressed as mean intensity ± SEM determined using Bitplane’s Imaris ×64 software. (D) Platelets on collagen + thrombin surface stained with PAC-1 FITC showing plasminogen-DL633 (top panel) and fibrinogen-AF647 (bottom panel) binding on spread- and balloon-shaped subpopulations. (E) Platelets on collagen + thrombin surface stained with PAC-1 FITC and showing plasminogen-DL633 binding. Arrows indicate a platelet with plasminogen bound that tethers to a spread plasminogen-positive platelet, which subsequently expresses active α_IIbβ₃, represented by positive PAC-1 staining. Images were recorded every minute for 60 minutes and selected images are shown. (F) Staining for platelet-derived PAI-1 in collagen + thrombin-stimulated platelets using an anti–PAI-1 DyLight 488 antibody and PS (Annexin A5-AF647) (top). PAI-1 co-localization with plasminogen-DL633 in balloon-shaped (middle) and spread platelets (bottom). Scale bars represent 5 µm. Images are representative of n = 3.