A Fluorescent Probe Study of Plasminogen Activator Inhibitor-1

Abstract

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.

The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

“…The fluorescence change accompanying the reaction of NBD-labeled PAI-1 variants with at least a 5-fold molar excess of each proteinase was monitored at 480 nm, using an emission filter (Oriel 51300) with a cut-off below 515 nm (21,22,27). As found previously, all stopped-flow traces fit best to a double exponential function, yielding pseudo-first-order rate constants k obs1 and k obs2 (27). Of the two rate constants, the faster component (k obs1 ) yielded the major fluorescence change and was analyzed.…”

“…For the reaction with elastase, PAI-1 was incubated with 0.04 M proteinase for 1 h prior to data acquisition. Fluorescence enhancements were measured as previously described (27).…”

“…Labeling of single Cys-containing PAI-1 variants (i.e. P9C, E350A/P9C, E351A/P9C, and E350A/E351A/P9C) with iodoacetamido-NBD (Molecular Probes, Inc., Eugene, OR) were performed as described, with labeling efficiencies in the range of 1.0 -1.3 mol of NBD/mol of PAI-1 variant (27). Any latent PAI-1 that was generated from the labeling reaction was separated from its active counterpart by chromatography on immobilized ␤-anhydrotrypsin as described (23).…”

“…tion of k obs2 to the fluorescence change has been accounted for and discussed elsewhere (21,27). The plot of k obs versus proteinase concentration was fitted to an equation that assumes a two-step binding mechanism and is best described by a rectangular hyperbola function:…”

Defining the Native Disulfide Topology in the Somatomedin B Domain of Human Vitronectin

“…PAI-1 plays important roles in thrombosis and fibrinolysis and has been implicated in hemostasis, angiogenesis, and tumor metastasis (7)(8)(9)(10)(11). Low abundant PAI-1 circulates in blood complexed with vitronectin (12); unliganded PAI-1 undergoes rapid "selfinactivation" into an inactive "latent" form in which the inhibitory reactive-site loop inserts as a new strand into the main ␤-sheet of the molecule (13)(14)(15). Ample evidence suggests that vitronectin regulates the activity of PAI-1 and PAI-1-mediated cellular events by stabilizing the active form of the inhibitor and delaying its conformational transition to the latent state (16 -18).…”