IDENTIFICATION OF THE BINDING-SITE FOR A LOW-MOLECULAR-WEIGHT INHIBITOR OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 BY SITE-DIRECTED MUTAGENESIS

A novel low-molecular-weight inhibitor, AR-H029953XX, was developed fr om a known fibrinolytic compound, flufenamic acid, which prevented com plex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To ex plore the binding site for AR-H029953XX, mutants of human PAI-1 were c onstructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutatio ns in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stabilit y and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2- PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inh ibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2PAI-1 (Va l121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/ Arg118Glu), and C4-PAI-1 (Arg1 15Glu) were all comparable in activity and stability to wt-PAI-1. AR-H 029953XX (K-i = 25 mu M) prevented complex formation between tPA and a ctive wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4 -PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In co ntrast, AR-H029953XX had almost no inhibitory effect on the complex fo rmation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent P AI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus app ears to be located in the neighborhood of the postulated epitope for C LB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhi bitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in lat ent and cleaved PAI-1.