Substrate Behavior of Plasminogen Activator Inhibitor-1 Is Not Associated with a Lack of Insertion of the Reactive Site Loop

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Abstract
Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. In the present study, we have evaluated the effect of 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, Ser→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 exhibit significant inhibitory activity. Conformational analysis revealed that wtPAI-1 preparations contained 12 ± 2.0% substrate, whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 were characterized with a significantly (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 showed the presence of a unique plasmin cleavage site (Lys191-Ser192) exclusively present in all latent forms studied. We conclude that (a) the entire P5 to P16 region in PAI-1 plays an important role in the functional and conformational properties of PAI-1; (b) the substrate behavior of serpins is not associated with a lack of insertion of the reactive site loop; (c) the identification of a plasmin cleavage site in latent PAI-1 may provide new insights in the mechanisms for the inactivation of storage pools of PAI-1.