Fibrinolysis is delayed in fibrin immediately proximal to the platelet surface. Thrombi were formed by whole blood perfusion (1000 s⁻¹) over a collagen/TF-coated surface for 7 minutes in the presence of 10 nM tPA. Thrombi were subsequently perfused (t = 0) with HEPES buffer, pH 7.45 for 8 minutes, and then HEPES buffer containing 10 nM tPA. (A) Representative image of fibrin(ogen)-AF647 staining on thrombi at the start and images subjected to subtraction analysis of fibrinogen fluorescence (n = 3). (B) Blood samples were pre-incubated with DiOC₆ (0.5 µg/ml) to label platelets and fibrinogen-AF647 (16.7 µg/ml). Thrombi were allowed to form as above in the presence of 10 nM or 75 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 indicated concentration tPA) until lysis was complete. Representative images and overlays of platelets (green) and fibrin(ogen) (red) during lysis with 10 nM or 75 nM tPA. Images are representative of n = 8. Scale bars represent 50 µm.