Effect of AZ3976 on clot lysis in human plasma with added PAI-1. Before addition of plasma, 1.7 nm PAI-1 in buffer was preincubated briefly at 37 °C with 0 to 0.22 mm AZ3976. The reaction was started by dilution with plasma containing tPA to final concentration of 50% plasma, 1.2 nm tPA and 0.65 nm PAI-1. Fibrin formation was monitored from the change in A405 in a microplate reader at 2-min intervals at 37 °C for up to 10 h. A, example of plasma clot lysis experiment: A405 is plotted versus time. Curves are shown for PAI-1 with 82.5 (b), 41.25 (c), 20.6 (d), 10.3 (e), 5.16 (f), and 0 μm (g) AZ3976 and for tPA only (a), without added PAI-1. B, the IC50 determination. The clot lysis time was defined as the time to halve the amplitude on the negative slope minus the time to halve the amplitude on the positive slope where the maximal effect (100%) was the time without added PAI-1 and minimal effect (0%) the time without added compound. The line depicts the best fit using Grafit IC50 fit. The mean of four experiments is plotted ± S.E.