Identification of a target site in plasminogen activator inhibitor-1 that allows neutralization of its inhibitory properties concomitant with an allosteric up-regulation of its antiadhesive properties

The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1 ) it plays an important role as a direct inhibitor of the plasminogen activ ation system, and 2) its interaction with the adhesive glycoprotein vitrone ctin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close as sociation between its conformational and functional properties. Indeed, PAI -1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (MA-124K1) th at inhibits rat PAI-1 activity and simultaneously increases the binding of inactive PAI-1 to vitronectin (the affinity constant of PAI-1 for vitronect in is 2 x 10(7) M-1 in the absence of MA-124K1 and 160 x 10(7) M-1 in the p resence of MA-124K1). To the best of our knowledge, this is the first monoc lonal antibody dissociating the proteinase inhibitory properties from the v itronectin binding properties in PAI-1. Mutation of Glu(212) and/or Glu(220 ) in rat PAI-1 to Ala results in a strongly reduced affinity or absence of binding to MA-124K1. The three-dimensional structure of PAI-1 reveals that these residues constitute a conformational epitope close to the reactive-si te loop and compatible with the effect of MA-124K1 on the inhibitory proper ties of PAI-1, However, the vitronectin binding site is localized at the op posite site of the molecule, indicating that the effect of MA-124K1 involve s an allosteric modulation of the vitronectin binding site. Cell culture ex periments revealed a significant reduction of cell attachment and migration in the presence of MA-124K1, providing evidence for the functional relevan ce of this antibody-mediated up-regulation of the vitronectin binding prope rties of PAI-1. In conclusion, a novel mechanism for interference with PAI- 1 functions has been identified and is of importance in the modulation of c ell migration and related events (e.g, tumor metastasis).