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-...

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serpin (serine proteinase inhibitor) superfamily (1-4), controls the plasminogen system at the level of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively). Because PAI-1 is the main physiological inhibitor of t-PA in plasma (5), increased levels of PAI-1 result in a hypofibrinolytic state and are correlated with various vascular disorders such as venous thrombo-embolism, coronary artery disease, myocardial infarction, and atherosclerosis (6 -9). The u-PA-inhibiting effect of PAI-1 has its main physiological implications in processes outside of the circulation (10, 11).PAI-1 is a unique serpin because of its functional and conformational flexibility (12). PAI-1, synthesized as an active molecule, converts spontaneously into a nonreactive, latent form, which can be partially reactivated by denaturing reagents (13). Additionally, a third distinct, noninhibitory form (substrate) is identified that is reactive toward its target proteinases without the formation of a stable complex (14 -16).Elucidation of the three-dimensional structure of active PAI-1 (17, 18) reveals that the N-terminal side of the reactive site loop (extended from P16 to P3Ј and including the bait peptide bond Arg 345 -Met 346 (P1-P1Ј)) is exposed and accessible for the target proteinase. The C-terminal side of the reactive site loop (P4Ј-P13Ј) forms strand s1C in ␤-sheet C.Conversion to the latent state implies the insertion of the N-terminal side of the reactive site loop into ␤-sheet A, the loss of strand s1C from ␤-sheet C, and the formation of an unusual extended loop by the C-terminal side of the reactive site loop, resulting in the distortion of the P1-P1Ј "bait" peptide bond (19). It is hypothesized that th...