A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was created (Ser-338-->Cys) in which cysteine was placed at the P9 position of the reactive center loop. Labeling this mutant with N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) provided a molecule with a fluorescent probe at that position. The NBD-labeled mutant was almost as reactive as wild type but was considerably more stable. Complex formation with tissue or urokinase type plasminogen activator (tPA or uPA), and cleavage between P3 and P4 with a catalytic concentration of elastase, all resulted in identical 13-nm blue shifts of the peak fluorescence emission wavelength and 6.2-fold fluorescence enhancements. Formation of latent PAI showed the same 13-nm spectral shift with a 6.7-fold fluorescence emission increase, indicating that the NBD probe is in a slightly more hydrophobic milieu. These changes can be attributed to insertion of the reactive center loop into the beta sheet A of the inhibitor in a manner that exposes the NBD probe to a more hydrophobic milieu. The rate of loop insertion due to tPA complexation was followed using stopped flow fluorimetry. This rate showed a hyperbolic dependence on tPA concentration, with a half-saturation concentration of 0.96 microM and a maximum rate constant of 3.4 s-1. These results demonstrate experimentally that complexation with proteases is presumably associated with loop insertion. The identical fluorescence changes obtained with tPa.PAI-1 and uPA.PAI-1 complexes and elastase-cleaved PAI-1 strongly suggest that in the stable protease-PAI-1 complex the reactive center loop is cleaved and inserted into beta sheet A and that this process is central to the inhibition mechanism.

and ‡ ‡Henry Ford Health System, Detroit, Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into ␤-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with ␤-anhydrotrypsin, which does not undergo rearrangement of ␤-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to ␤-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1⅐plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.Plasminogen activators (PAs) 1 are specific serine proteinases that activate the proenzyme plasminogen to the broad specificity enzyme plasmin (1). There are only two known physiologic activators of plasminogen, tissue-type PA (tPA) and urokinasetype PA (uPA) (2). In addition to their role in vascular fibrinolysis (3), PAs are thought to critically influence many other biological processes involving cell migration or tissue remodeling (4). These include ovulation (5), inflammation (6), tumor metastasis (7), and angiogenesis (8). The serpin PAI-1, the most efficient inhibitor known of both tPA and uPA, is thus a critical regulator of the PA system (9). PAI-1 is present in plasma at nM concentrations (10) and in platelets at a much higher concentration (11). This latter pool has been shown to contribute to clot stabilization in vivo (12). In plasma and the extracellular matrix, PAI-1 is associated with Vn (13-15), and this association may be involved in maintaining the integrity of the cell substratum in vivo. PAI-1 exists in at least three distinct conformational forms: active, latent, and cleaved (16,17). Active PAI-1 decays to the latent form with a half-life of approximately 1-2 h at 37°C (18). After treatment with denaturants, latent PAI-1 can be partially returned to the active form (19). Although the biologic significance of the latent conformation remains unknown, it may contribute to the regulation of PA...

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