The effect of recombinant PAI-1 on membrane surface–retained tPA. (A) T_F1/2 values of tPA-GFP (abscissa) before (■: 53 granules) and after (□: 55 granules) addition of 40 nM recombinant PAI-1 (6 cells, 4 independent experiments) shown as percentages of total occurrences (ordinate). Exogenous PAI-1 increased the frequency of rapidly disappearing fluorescence having short T_F1/2. (B) Fibrin autography of the supernatants of the cells incubated with exogenous rPAI-1 at 0, 10, and 40 nM for 60 minutes. The amounts of tPA increased as a function of the amounts of added PAI-1, in the form of a tPA-PAI-1 complex in the supernatant. A representative image from 5 independent experiments is shown. (C) T_F1/2 values of tPA-GFP (abscissa) before (■: 89 granules), after incubation with 400 nM RAP (: 80 granules), and after further addition of 40 nM recombinant PAI-1 (□, 76 granules) from 6 independent experiments shown as percentages of total occurrences (ordinate). The presence of RAP did not change the effect of PAI-1 as shown in panel A.