Figure 2.

Human plasminogen activator inhibitor–1 (hPAI-1) in pleural lavages and rabbit pleural mesothelial cells (RPMCs) harvested from rabbits intrapleurally injected with AdPAI-1/AdlacZ. (A) hPAI-1 was detected in pleural lavages (top; Lavage, hPAI-1) and lysates (middle; lysates and hPAI-1) of RPMCs from rabbits injected with a 3:1 mixture of AdPAI-1/AdlacZ, but was not present in lavages or RPMCs of control (PBS or AdEV/AdlacZ) animals. β-actin was used as a loading control for lysates (bottom). (B) A significant fraction (20–25%) of hPAI-1 in pleural lavages was active. Pleural lavages from two animals (Lanes 1 and 2) were supplemented with exogenous human tc urokinase-type plasminogen activator (tcuPA) and incubated for 5 minutes at 4°C, and the reaction mixtures were subjected to SDS-PAGE followed by Western blot analysis of the hPAI-1 antigen. The upper band corresponds to the inhibitory PAI-1/uPA complex, which forms because of the mechanism-based inhibition of uPA by active PAI-1. The lower band corresponds to uncomplexed, latent PAI-1. (C) High levels of PAI-1 activity were detected in the pleural lavages of animals transduced with AdPAI-1/AdlacZ. Aliquots of pleural lavages were titrated with human tcuPA, as described in Materials and Methods. The concentration of active PAI-1 in each sample was calculated, assuming a stoichiometry of inhibition close to unity. A statistically significant difference (P > 0.05) between AdPAI-1/AdlacZ and the two control groups (P = 0.029 for both) is indicated by an asterisk.