PAI-1. the physiological inhibitor of tissue-type and urokinase-type plasminogen activator, is a unique member of the serpins as it exists in three distinct conformations: an active inhibitory conformation, a non-inhibitory substrate conformation, and a non-reactive latent conformation. Proline substitution of single residues in the P16-P20 region (situated at the proximal hinge of the reactive site loop) of wild-type PAI-1 (wtPAI-1) and a stabilized PAI-1-variant (PAI-1-stab; N150H, K154T, Q301P, Q319L, and M3541, t(1/2) = 150), respectively, resulted in two series of PAI-1-variants with different properties. In wtPAI-1 only substitution at P IS resulted in a pronounced u-PA specificity and substrate behaviour towards t-PA. In contrast, in PAI-I-stab substitution at either P18, P19 or P20 resulted in a u-PA specificity and a significantly increased substrate behaviour towards t-PA and u-PA. Importantly, analysis of the kinetics of inhibition did not reveal any differences in the second-order rate constants of inhibition (k similar to 10(7)M(-1) s(-1)). The pronounced differences observed for identical mutations in wtPAI-1 vs PAI-1-stab demonstrate that not merely the sequence of the reactive loop but also intramolecular interactions between the hF/s3A-loop and the main part of the molecule govern the functional and conformational behaviour of PAI-1. (C) 2004 Elsevier Inc. All rights reserved.