SUBSTRATE BEHAVIOR OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 IS NOT ASSOCIATED WITH A LACK OF INSERTION OF THE REACTIVE-SITE LOOP

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the se rpin superfamily. In the present study, we have evaluated the effect o f substitution, with a proline, at positions P5, P7, P14, P15, or P16, on the conformational flexibility and functional properties of PAI-1. These mutants (PAI-1-P5, Ile-->Pro at P5; PAI-1-P7, Ala-->Pro at P7; PAI-1-P14, Thr-->Pro at P14; PAI-1-P15, Gly-->Pro at P15; PAI-1-P16, S er-->Pro at P16) were purified and fully characterized. WtPAI-1 had a specific activity of 68 +/- 10% (mean +/- SD, n = 6) whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 had specific activities of 34 +/- 9.3%, 42 +/ - 10%, and 36 +/- 11%, respectively. PAI-1-P14 and PAI-1-P15 did not e xhibit significant inhibitory activity. Conformational analysis reveal ed that wtPAI-1 preparations contained 12 +/- 2.0% substrate, whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 were characterized with a significan tly (p < 0.001) increased substrate behavior (i.e., 43 +/- 6.1%, 42 +/ - 1.5% and 22 +/- 1.7%, respectively). The inactive variants PAI-1-P14 and PAI-1-P15 behaved exclusively as substrates toward various serine proteinases. Heat denaturation studies revealed that cleavage of any noninhibitory substrate form of PAI-1 resulted in an insertion of the NH2-terminal side of the reactive site loop. Incubation with plasmin s howed the presence of a unique plasmin cleavage site (Lys191-Ser192) e xclusively present in all latent forms studied. We conclude that (a) t he entire P5 to P16 region in PAI-1 plays an important role in the fun ctional and conformational properties of PAI-1; (b) the substrate beha vior of serpins is not associated with a lack of insertion of the reac tive site loop; (c) the identification of a plasmin cleavage site in l atent PAI-1 may provide new insights in the mechanisms for the inactiv ation of storage pools of PAI-1.