Evaluation of the mechanism of inactivation of plasminogen activator inhibitor-1 by monoclonal antibodies using a stable variant

Abstract

A number of studies have shown that plasminogen activator inhibitor-1 (PAI-1) can be inactivated through different mechanisms. In the current study we have carried out a comparative analysis of the effects of various PAI-1 neutralizing antibodies on wild-type PAI-1 (wtPAI-1, t1/2 ≈ 2 h) and a stable PAI-1 mutant (PAI-1-stab, t1/2 ≈ 145 h). MA8H9D4, MA-33H1 and MA-55F4, switching active wtPAI-1 to substrate PAI-1, exerted qualitatively similar effects on PAI-1-stab. Yet, the effects observed with MA-33H1 and MA-55F4 were much less pronounced on PAI-1-stab. MA-33B8 and MA-35A5 appear to exert their neutralizing properties through an acceleration of the conversion of active to latent PAI-1, thereby reducing the half-life of wtPAI-1 400-fold, whereas the half-life of PAI-1-stab was reduced up to 7000-fold. Consequently, in the presence of MA-33B8 or MA-35A5, PAI-1-stab was only slightly more stable (i.e. 4- to 6-fold) than wtPAI-1. MA-56A7C10, converting active wtPAI-1 to the latent form, also inactivates PAI-1-stab even though in the latter no plasmin cleavable site, typically present in the latent conformation of wtPAI-1, was generated. This suggests that the non-reactive conformation induced in PAI-1-stab by MA-56A7C10 is not identical to the latent conformation induced in wtPAI-1. Thus, in spite of the highly increased stability of PAI-1-stab compared to wtPAI-1, PAI-1 neutralizing antibodies may inactivate this stable PAI-1 variant with an unexpected high efficiency. This indicates that the intramolecular interactions responsible for the increased stability of PAI-1-stab do contribute only marginally to its stability after interaction with the monoclonal antibodies.