THE USE OF FLUORESCENT-PROBES TO CHARACTERIZE CONFORMATIONAL-CHANGES IN THE INTERACTION BETWEEN VITRONECTIN AND PLASMINOGEN-ACTIVATOR INHIBITOR-1

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of ti ssue-type plasminogen activator and urokinase, is known to convert rea dily to a latent form by insertion of the reactive center loop into a central P-sheet. Interaction with vitronectin stabilizes PAI-1 and dec reases the rate of conversion to the latent form, but conformational e ffects of vitronectin on the reactive center loop of PAI-1 have not be en documented. Mutant forms of PAI-1 were designed with a cysteine sub stitution at either position P1' or P9 of the reactive center loop. La beling of the unique cysteine with a sulfhydryl-reactive fluorophore p rovides a probe that is sensitive to vitronectin binding. Results indi cate that the scissile P1-P1' bond of PAI-1 is more solvent exposed up on interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environ ment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation co nstants of approximately 100 nM are calculated for the vitronectin/PAI -1 interaction from titrations using both fluorescent probes. Furtherm ore, experiments in which PAI-1 failed to compete with heparin for bin ding to vitronectin argue for separate binding sites for the two ligan ds on vitronectin.