Plasminogen localization within thrombi. Thrombi were formed by whole blood perfusion (1000 s⁻¹) over a collagen/TF-coated surface with or without hirudin (3 µg/ml). Platelets labeled with DiOC₆ (0.5 µg/ml) or fibrinogen-OG488 (75 µg/ml) was included. Thrombi were perfused with plasminogen–DL-633 (0.8 µM). (A) Representative overlays of plasminogen (blue) and fibrin(ogen) (red), or platelets (green). (B) Overlap coefficients (R) for plasminogen with fibrin(ogen) and/or platelets as determined using Zeiss Live 7 software. (C) Representative images of thrombi labeled for plasminogen-DL633 (blue) and fibrin(ogen)-OG488 (red). (D) Quantification of plasminogen and fibrinogen fluorescence intensity (AU). (E) Confocal z-stacks were recorded of labeled thrombi (16-bit images of 1024 × 1024 pixels; 106 × 106 μm; stack distance 0.5 μm; 50 slices). Representative images and overlays of plasminogen (blue) and platelets (green), or fibrin(ogen) (red) taken from z-stacks at the base (0 μm), center (10 μm), and top (20 μm) of thrombi before (top panel) or after (bottom panel) visible fibrin formation. (F) Thrombi were formed as above after pre-incubation with plasminogen–DL-633 (0.8 µM) and fibrinogen-OG488 (75 µg/ml) in the presence of 10 nM tPA. The fluorescent thrombi were perfused (t = 0) with buffer 1 (HEPES buffer, pH 7.45) for 8 minutes, followed by buffer 2 (HEPES buffer, pH 7.45 with 10 nM tPA) for up to 28 minutes. Shown are representative images of thrombi labeled for fibrin(ogen) (red) and plasminogen (blue). Scale bars represent 50 µm. *P < .05; ***P < .001. Data represented as mean ± SEM, n ≥ 3.