THE EXTRACELLULAR MATRIX (ECM) OF CULTURED BOVINE AORTIC ENDOTHELIAL CELLS (BAEs) CONTAINS FUNCTIONALLY ACTIVE TYPE I PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1)

The ECM of cultured BAEs was analyzed by immunoblotting and reverse fibrin autography and shown to contain PAI-1. PAI-1 containing ECM was incubated with tissue plasminogen activator (tPA) to determine whether the bound PAI-1 was functionally active or latent. The majority of the detectable PAI-1 in the ECM formed complexes with tPA indicating that it was active. The resulting tPA/PAI-1 complexes were released from ECM and recovered in the reaction solution indicating that the PAI-1 in such complexes had lost its ECM binding site. The PAI-1 could not be removed from ECM by incubating it in high salt (2M NaCl), sugar (galactose, mannose, 1 M), heparin (10 mg/ml) or lysine (1 M). However, PAI-1 could be extracted from ECM by treatment with arginine (0.5 M), potassium thiocyanate (2 M), or by incubation at acid pH (2.5). These treatments did not remove appreciable amounts of fibronectin or von willebrand's factor from ECM. ECM depleted of PAI-1 by acid extraction was able to bind all forms of exogenously added PAI-1 including both the active and the latent molecules. The majority of the bound PAI-1 could not form complexes with exogenously added tPA suggesting that the latent form was not activated as a consequence of this interaction. Although the PAI-1 activity in conditioned medium was unstable and decayed with a half life of less than 3 hrs, the half-life of ECM associated PAI-1 was greater than 24 h. These data suggest that PAI-1 is produced by cultured BAEs in an active form, and either released into the medium where it is rapidly inactivated, or bound to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.