PURIFICATION AND CHARACTERIZATION OF PAI-1 FROM HUMAN ENDOTHELIAL CELLS

Platelet α-granules and endothelial cells contain an inhibitor of plasminogen activator, which inhibits both t-PA and u-PA. The inhibitor (PAI-1) is detectable after SDS-PAGE and zymography on fibrin/plasmin-ogen/u-PA detector gels. We have purified endothelial PAI-1 by a simple two-step procedure. Serum-free conditioned medium from human umbilical vein endothelial cells, grown in microcarrier culture, was fractionated on Sephadex CM-50, a cation exchanger, followed by gel filtration on Sephacryl S-200. Aprotinin was included throughout the procedure to maintain the activity of the inhibitor. The PAI-1 was purified 2000-fold with a recovery of about 7%. The purified protein had a specific activity of 8500 U/mg protein and the activity could be stimulated 14-fold by 4M guanidine. The purified PAI-1, of M 48000, was a single-chain glycoprotein.The ptoduct wasapparently homogeneous on a silver-stained SDS-polyacrylamide gel, the protein band co-migrating with PAI activity. Further, a rabbit antiserum raised against the purified PAI-1 revealed only a single band on immunoblots of material from each stage of the purification. The immunoglobulin fraction ofthe antiserum, incorporated into the detector gel for zymographic analysis, neutralised the inhibitor from plasma, platelets and endothelial cells, confirming their identity. Preincubation of PAI-1 from these sources with the immunoglobulin prevented formation of a complex with t-PA or u-PA. This purification procedure, in which no denaturants are employed, provides a homogeneous preparation of PAI-1 that is useful for studies on the stimulatory effects of denaturants. The antiserum raised has allowed the development of a sensitive ELISA, specific for PAI-1.