Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms

Abstract

Plasminogen activator inhibitor-1 (PAI-1), a unique member of the serpin superfamily, plays an important role in fibrinolysis and is an established risk factor for cardiovascular diseases. PAI-1 can occur in three interconvertible conformations: an active, a latent and a substrate form. To study conformational and functional relationships in PAI-1, a wide variety of monoclonal antibodies were evaluated for their influence on PAI-1 activity. Out of 77 monoclonal antibodies, directed against human PAI-1, six were selected for their strong inhibitory effect towards PAI-1 activity, i.e., 80 to 100% inhibition in the presence of a 1- to 16-fold molar excess of monoclonal antibody. Detailed analysis 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 activator (u-PA), in the presence of these monoclonal antibodies, revealed two distinct mechanisms of PAI-1 inactivation. Incubation of PAI-1 with one series of monoclonal antibodies resulted in the absence of any reaction indicative for direct interaction with the reactive-site loop or a facilitated conversion to the latent conformation. The loss of PAI-1 activity in the presence of the other group of monoclonal antibodies was associated with the concomitant formation of a 41 kDa cleavage product after interaction with the target proteinase. The latter observation demonstrates that binding of these antibodies induced a conformational change thereby converting the inhibitory, active conformation to the non-inhibitory substrate conformation. No conformational changes could be observed in latent PAI-1 under these conditions. Analysis of cross-reactivity revealed that some of these functionally important epitopes were conserved throughout PAI-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 distinct mechanisms by which the inhibitory activity of PAI-1 can be neutralized. This may have implications for the design of therapeutic or preventive strategies to interfere with PAI-1 activity. Cross-reactivity of these inhibitory antibodies with PAI-1 from various species may also allow their application in experimental animal models studying the in vivo role of PAI-1 in various diseases (e.g. atherosclerosis, thrombosis, angiogenesis,...).