Plasminogen activator inhibitor type 1 (PAI-1), the fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase (u-PA), is a member of the serpin superfamily of proteins. Both in plasma and in the growth substratum of cultured endothelial cells, PAI-1 is associated with its binding protein vitronectin, resulting in a stabilization of active PAI-1. Recently, it has been demonstrated that the PAI-1-binding site on vitronectin is adjacent to a heparin-binding site (Preissner et al., 1990). Furthermore, it can be deduced that the amino acid residues, proposed to mediate heparin binding in the serpins antithrombin III and heparin cofactor II, are conserved in PAI-1. Consequently, here we have investigated whether PAI-1 also interacts with heparin. At pH 7.4, PAI-1 quantitatively binds to heparin-Sepharose and can be eluted with increasing [NaCl]. Binding of PAI-1 to heparin-Sepharose can be efficiently competed with heparin in solution (IC50, 7 microM). In the presence of heparin, the protease specificity of PAI-1 toward thrombin is substantially increased. This is shown by (i) quenching of thrombin activity of PAI-1 in the presence of heparin and (ii) induction of the formation of SDS-stable complexes between thrombin and PAI-1 by heparin. In a dose response curve, both effects reached a maximum at approximately 1 unit/mL and then diminished again upon further increasing the heparin concentration, strongly suggesting a template mechanism as an explanation for the observed effect. In contrast to vitronectin, heparin does not stabilize the active conformation of PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

. Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinasetype plasminogen activator (u-PA), with additional thrombin inhibitory properties . In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment . Upon incubating 'III-labeled a-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant . Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM . Metabolic labeling of endothelial cell proteins, followed by incubation of T HE activation of plasminogen is a key step both in the fibrinolytic system and in various physiological and pathophysiological processes, involving extracellular matrix degradation (e.g ., inflammatory reactions, malignant invasion and metastasis, ovulation, nidation, cell migration and tissue remodeling) (for review see Dano et al., 1985) . Therefore, theprecise regulation ofplasminogen activation with respect to time and location is of critical importance for the organism . A major physiological mechanism for the control of plasminogen activation is provided by the action of plasminogen activator inhibitor 1 (PAI-1)

Vitronectin is the carrier protein of plasminogen activator inhibitor 1 (PAI-1). We used a well-characterized panel of anti-human PAImonoclonal antibodies (MoAbs) to localize the vitronectin-binding site on PAI-I. By employing a direct vitronectin/PAI-1 binding assay and two vitronectin-dependent inhibition assays, we demonstrate that the anti-PAI-MoAbs CLB-5, CLB-10, CLB-2C8 and 11, directed against different epitopes in the region between amino acids 110 and 145, prevent the interaction of PAI-with vitronectin. We conclude that the region between amino acids 110 and 145 of PAI-harbours an important determinant for the interaction with vitronectin.

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