Effect of suPAR on scuPA-mediated clot lysis: zymography. (A) Lysis of clots prepared from purified bovine fibrinogen. Clots were prepared by adding thrombin (0.2 NIH U/mL final concentration) to bovine fibrinogen (3 mg/mL) in PBS for 60 minutes at room temperature. scuPA (10 pmol in 10 μL PBS or equimolar concentrations of suPAR and scuPA were added for 2 hours at 37°C and the size of each lytic area was measured. The lytic areas generated by scuPA were 1.60 and 1.63 cm², respectively; the corresponding areas for scuPA/suPAR were 1.19 and 1.05 cm², respectively. In this and in each panel below, the experiment shown is representative of three so performed. (B) Lysis of clots prepared from human plasma. Clots were prepared by adding thrombin (0.4 NIH U/mL final concentration) to citrated human plasma for 60 minutes at room temperature. scuPA, scuPA/suPAR or suPAR (10 pmol in 10 μL) were added for 2 hours at 37°C. The size of the lytic areas generated by scuPA/suPAR were 0.94 and 0.90 cm². Effect of ATF on suPAR/scuPA-induced lysis of plasma clots. (C) scuPA/suPAR (10 pmol in 10 μL PBS) was added to the plasma clots for 2 hours at 37°C in the absence or presence of 500 pmol ATF.