Fibrinolysis under physiological flow conditions. (A) Thrombi were formed from blood samples obtained from cardiothoracic patients pre- and post-tranexamic acid treatment (2 mg). Blood was preincubated with DiOC₆ (0.5 µg/ml) to label platelets and fibrinogen-AF647 (16.7 µg/ml), and perfused (1000 s⁻¹) over a collagen/TF-coated surface for 7 minutes in the presence of 75 nM tPA. HEPES buffer, pH 7.45 (t = 0) was then allowed to perfuse through thrombi at 1000 s⁻¹. The fluorescent thrombi were lysed by perfusion (t = 0) with buffer 1 (HEPES buffer, pH 7.45) for 10 minutes. Quantification of surface area covered with fibrin(ogen) as compared with t = 0. Data represent mean ± SD, n = 3. (B) Thrombi from blood of normal individuals were formed as described for (A) except with inclusion of 10 nM tPA, and during the lysis stage flow was alternated between 1000 s⁻¹ and stasis every 2 minutes. (C) Thrombi were formed as for (B) before perfusing at 1000 s⁻¹, 300 s⁻¹, or 150 s⁻¹ (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) until lysis was complete. Quantification is shown as time to 50% lysis (mean ± SD). (D) Thrombi were formed as above in the presence of 75 nM tPA. After formation (t = 0), thrombi were subsequently perfused with HEPES buffer, pH 7.45, whole blood, or heparinized whole blood containing tPA (75 nM) for up to 20 minutes. Representative images of thrombi labeled for fibrin(ogen) (red), platelets (green), and overlay (yellow). Scale bars represent 50 µm. Quantification shown of percentage of fibrin(ogen) lysed based on initial surface coverage from 0 to 3 minutes. AFU, average fluorescence units. Data represent mean ± SEM, n ≥ 3, P < .05.