THE COMPOSITION OF COMPLEXES BETWEEN PLASMINOGEN-ACTIVATOR INHIBITOR-1, VITRONECTIN AND EITHER THROMBIN OR TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Vitronectin (VN) is an obligatory cofactor for the inhibition of throm bin by plasminogen activator inhibitor 1 (PAI-1). It accelerates the r ate of association between thrombin and PAI-1 mars than two orders of magnitude. In contrast, VN does not accelerate the association between tissue-type plasminogen activator (t-PA) and PAI-1. Previously, we sp orted that the anti-PAI-1 monoclonal antibody (MoAb) CLB-2C8 binds to a short stretch of amino acids of PAI-1, located between residues 128 and 145, and prevents PAI-1 binding to VN. Furthermore, MoAb CLB-2C8 f ully blocks the inhibitory activity of PAI-1 towards t-PA, emphasizing the importance of this area for the interaction with t-PA. Here, we s how that this area is also required for the interaction between thromb in and PAI-1, since MoAb CLB-2C8 fully prevents inhibition of thrombin by PAI-1. In spite of similar structural require ments for the intera ction between t-PA, PAI-1 and VN and between thrombin, PAI-1 and VN, t he intermediate reaction products are clearly distinct. By employing s urface plasmon resonance (SPR), using the BIAcore equipment, and by im munoprecipitation we demonstrate that. in the presence of VN, t-PA and PAI-1 form exclusively equimolar binary t-PA/PAI-1 complexes. Thrombi n, PAI-1 and VN generate equimolar. binary thrombin/PAI-1 complexes an d in addition equimolar, ternary complexes and multimers.