A substrate‐like form of plasminogen‐activator‐inhibitor type 1

Abstract

Recombinant plasminogen-activator-inhibitor type 1 (PAI-1) purified in an activc form from Escherichiu coli and eucaryotic cells was found to contain a mixture of three functionally distinct forms: an active form that forms coinplexcs with plasminogen activators (PAS), an inactive (latent) form that remains intact after incubation with PAS, and a substrate-like form which is easily cleaved by PAS. Since active PAL-1 purified from bacteria (rpPAI-1) contains only trace amounts of the inactive latent and the substrate-like forms, this material was used to study the effect of sodium dodecyl sulphate (SDS) on the structure and function of active PAI-1. After treatment with 0.01 % SDS, active rpPAI-1 was converted to an inactive form that did not form complexes with PAS, but exhibited characteristics similar to those of latent PAI-1. After treatment with 0.1% SDS, PAI-1 lost its inhibitory activity and was cleaved as a substrate in the reactive center. Circular dichroism spectral analysis reveals that SDS changed the conformation of PAI-1 dramatically, mainly by increasing its a-helical content.Plasminogen activation provides an important source of proteolytic activity in a variety of physiological processes [I -61. In mammals there are two types of plasminogen activators (PAS), tissue-type PA ( P A ) and urokinase-type PA (uPA), that both convert the proenzyme plasminogen into the active protease plasmin [2]. The PA system is regulated at many different levels including the direct blockage of PA activity by specific PA inhibitors (PAIs) [7 -151. Among these inhibitors only PA-inhibitor type 1 (PAI-1) efficiently neutralizes all forms of PAS including the single chain of tPA [3, 36, 171. Like the PAS, the synthesis of PAI-1 is highly regulated in many tissues [is] and increased levels of PAI-1 in patients, as well as in transgenic animals are correlated to thrombotic disease [19, 201, suggesting their target proteascs by providing a so-callcd 'bait' residue (P1 residue), that mimics the normal substrate of the target protease [24,25, 271. The mature form of PAI-1 is composed of 379 amino acids and contains no cysteine residues [22, 281. An interesting feature of PAI-1 is that it exists in two forms. An activc form, produced by cells and secreted into the culture media, and an inactive or latent form, with an apparently smaller Stokes' radius [29, 301. PAI-1 coiiverts spontaneously to the latent form [29, 301 and can subsequently be reactivated by treatment with denaturants or negatively charged phospholipids [31, 321. Because active PAT-1 rapidly converts to the latcnt form in culture media, most of the biochemical and functional studies of this inhibitor have so far been performed on inhibitor preparations which have been purified in the latent form and subsequently activated by denaturants like guanidinium-HCl, urea or SDS [29-3331. The fraction of active inhibitor in such preparations has varied greatly from less than 5% up to 55% [29-341. In one study it was found that PAL-1 activated by SDS did not function a...

The Profibrinolytic Enzyme Subtilisin NAT Purified fromBacillus subtilis Cleaves and Inactivates Plasminogen Activator Inhibitor Type 1

“…ogen activators through cleavage of its reactive site (21,27). Similar cleavage was observed even in the presence of vitronectin, a protein cofactor known to maintain PAI-1 activity (data not shown).…”

“…Purification of Active Recombinant Prokaryotic PAI-1 (rpPAI-1)-The cultivation of bacteria expressing PAI-1 and the purification of active non-glycosylated rpPAI-1 have been previously described (20,21). Purified active rpPAI-1 was stored at Ϫ80°C before use.…”

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

“…In addition to active and latent forms of PAI-1 mentioned above, a further conformation has recently been described, which acts as a non-inhibitory substrate for t-PA Urano et al, 1992;Munch et al, 1993). Unlike the latent conformation which does not bind to the active site of t-PA, the substrate conformation reacts with t-PA resulting in cleavage of the P 1 ± P 1 ' bond and regeneration of active t-PA Audenaert et al, 1994).…”

A Regulatory Hydrophobic Area in the Flexible Joint Region of Plasminogen Activator Inhibitor-1, Defined with Fluorescent Activity-neutralizing Ligands

“…Among these are some organochemicals, including 1-dodecyl sulfuric acid (42,43), the diketopiperazine derivative XR5118 (49), and the anthranilic acid derivative AR-H029953XX (50). The flexible joint region was implicated in the PAI-1 neutralizing activity of the latter, by the demonstration that Glu substitutions of three basic residues in hD and hE (see Fig.…”