The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important protein in the regulation of fibrinolysis and inhibits its target proteinases through formation of a covalent complex. In the present study, we have identified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of these monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we predicted that the residues Glu Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serine proteinase inhibitor (serpin) superfamily (1-4) is an important protein in the regulation of fibrinolysis. PAI-1 is the most important physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma (5).PAI-1 is unique among the serpins because of its functional and conformational flexibility. The active conformation of PAI-1 inhibits its target proteinases by the formation of a stable, inactive complex. After the formation of an initial, reversible Michaelis-like complex, the proteinase cleaves the active site of PAI-1 and forms a stable, covalent complex resulting in the inactivation of the proteinase (6, 7). The structure of a covalent complex between PAI-1 and t-PA in particular, or between a serpin and its target proteinase in general, is presently unknown. However, two different models have been proposed. According to one model, the proteinase moves after the initial attack to the opposite pole of the serpin, thereby resulting in a complete insertion of the N-terminal side of the reactive-site loop (7-9). Alternatively, it was hypothesized that movement of the proteinase following the initial proteinase/ serpin interaction is less extended, yielding a complex in which the N-terminal side of the reactive-site loop is only partially inserted (10, 11), accompanied by a distortion of the catalytic triad of the proteinase (6) and stabilized by multiple interactions between serpin and proteinase.Although PAI-1 is synthesized as an active molecule, it converts spontaneously to an inactive, latent form that can be partially reactivated by denaturing agents (12). In this latent conformation, the active site is inaccessible for the target proteinases as a result of the insertion of the N-terminal side of the reactive-site loop in ␤-sheet A of the PAI-1 molecule (13). In addition, a third conformation with substrate properties has been identified (14 -16). This form of PAI-1 reacts with t-PA or u-PA, resulting in a cleavage of the active site of PAI-1 but without the formation of a covalent complex (17).Previously, we have characterized a panel of monoclonal antibodies that neutralize PAI-1 activity by converting the active pathway into the non-inhibitory substrate pathway (18). For two of these antibodies, MA-55F4C12 and MA-33H1F7, the binding region was found to be located remote of the reactivesite loop, within a region comprising residues at positions 128 -156 (19). Within the three-...

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