Lysine-binding-site–dependent and plasmin-activity–dependent amplification of cell surface–associated plasminogen activation by surface-retained tPA. (A) The increasing values in plg-568 fluorescence intensity at the most basal focal image in tPA-GFP–expressing cells were calculated from each initial value until that at 30 minutes, which represents the amount of accumulated plg-568 and is referred as dF_{30 min}. Bars show the mean ± SE of dF_{30 min} from 9, 4, 3, and 5 independent experiments in control (C), 480nM nonlabeled miniplasminogen plus 20nM Alexa Fluor 568–labeled miniplasminogen (mini), epsilon-aminocaproic acid (EACA), and carboxypeptidase B (cpB), respectively. All materials examined significantly reduced the accumulation of plg-568 (P < .001 vs control) except for 0.01mM EACA (P = .033 vs control). (B) Bars indicate mean ± SE of dF_{30 min} from 9, 4, 3, 6, 5, and 5 independent experiments in control (C), 1667 U/mL aprotinin (APR), 0.3μM α2-antiplasmin (α2AP), 40nM human recombinant PA inhibitor-1 (PAI-1), S478A, and CD, respectively. Supplementation of PAI-1 or α2AP reduced the accumulation of plg-568 (P = .012 and P = .0028, respectively, vs control), whereas the others significantly reduced the accumulation of plg-568 (P < .001 vs control). S478A indicates tPA-S478A-GFP–expressing cells; CD, tPA-CD-GFP–expressing cells (a heavy chain–deleted mutant of tPA, which is composed of signal peptide and catalytic domain). (C) Representative images during fibrin clot lysis on tPA-GFP–expressing cells (left) or tPA-CD-GFP–expressing cells (right) at 3 μm above and along the z axis at 50 minutes after fibrin clot formation. Green indicates tPA-GFP or tPA-CD-GFP; red, plg-568; and white, fbg-647. (D) Lysis area at 3 μm above the z axis was calculated every 10 minutes after fibrin network formation over a single tPA-GFP–expressing cell ● or tPA-CD-GFP–expressing cell ○. Results of 5 independent experiments are shown as mean ± SE.