Clot retraction coincides with reduced rtPA-mediated lysis and penetration. (A) rtPA was administered during different stages of clot retraction onto: (i) nascent unretracted thrombi, (ii) partly retracted thrombi that were 20 minutes old, and (iii) retracted thrombi that were 2 hours old. Schematics summarize the experimental design (left column). 3D surface-rendered micrographs (right columns) show representative thrombi just before rtPA delivery (0 min) and 60 minutes (+60 min) after rtPA delivery. (iv) Collated quantification of thrombus size 60 minutes after rtPA delivery. Results depict mean + SEM where data are expressed as a percentage of thrombus size just before rtPA delivery. (i) n = 4, (ii) n = 8, and (iii) n = 8 mice. *P < .05 and **P < .01 by 1-way ANOVA with Tukey correction. (B) Inactive rtPA⁴⁸⁸ was administered onto (i) nascent thrombi or (ii) 2-hour-old retracted thrombi and the binding/penetration of inactive rtPA⁴⁸⁸ to fibrin (detected via Alexa Fluor 546 anti-fibrin antibody) was imaged via confocal micrograph 20 minutes later. Micrographs depict confocal sections through representative thrombi. The direction of blood flow (arrow) and scale bars (10 μm) are shown.