NEUTRALIZATION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 INHIBITORY PROPERTIES - IDENTIFICATION OF 2 DIFFERENT MECHANISMS

Plasminogen activator inhibitor-1 (PAI-1), a unique member of the serp in superfamily, plays an important role in fibrinolysis and is an esta blished risk factor for cardiovascular diseases. PAI-1 can occur in th ree interconvertible conformations: an active, a latent and a substrat e form. To study conformational and functional relationships in PAI-1, a wide variety of monoclonal antibodies were evaluated for their infl uence on PAI-1 activity. Out of 77 monoclonal antibodies, directed aga inst human PAI-1, six were selected for their strong inhibitory effect towards PAI-1 activity, i.e., 80 to 100% inhibition in the presence o f a 1- to 16-fold molar excess of monoclonal antibody. Detailed analys is of the reaction products formed during the interaction between PAI- 1 and its target proteinases tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activaro (u-PA), in the presence of thes e monoclonal antibodies, revealed two distinct mechanisms of PAI-1 ina ctivation. Incubation of PAI-1 with one series of monoclonal antibodie s resulted in the absence of any reaction indicative for direct intera ction with the reactive-site loop or a facilitated conversion to the l atent conformation. The loss of PAI-1 activity in the presence of the other group of monoclonal antibodies was associated with the concomita nt formation of a 41 kDa cleavage product after interaction with the t arget proteinase. The latter observation demonstrates that binding of these antibodies induced a conformational change thereby converting th e inhibitory, active conformation to the non-inhibitory substrate conf ormation. No conformational changes could be observed in latent PAI-1 under these conditions. Analysis of cross-reactivity revealed that som e of these functionally important epitopes were conserved throughout P AI-1 obtained from various species including rabbit, mouse and/or pig, resulting in similar functional and conformational effects induced by these antibodies, Thus, we have demonstrated the occurrence of two di stinct mechanisms by which the inhibitory activity of PAI-1 can be neu tralized. This may have implications for the design of therapeutic or preventive strategies to interfere with PAI-1 activity. Cross-reactivi ty of these inhibitory antibodies with PAI-1 from various species may also allow their application in experimental animal models studying th e in vivo role of PAI-1 in various diseases (e.g. atherosclerosis, thr ombosis, angiogenesis, ...).