The distal hinge of the reactive site loop and its proximity - A target tomodulate plasminogen activator inhibitor-1 activity

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulator y role in various physiological processes (e.g. fibrinolysis and pericellul ar proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclo nal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-rea ctivities of these monoclonals with PAI-1 from different species and sequen ce alignments between these PAI-1s, combined with the three-dimensional str ucture, revealed several charged residues as possible candidates to contrib ute to the respective epitopes. The production, characterization, and subse quent evaluation of a variety of alanine mutants using surface plasmon reso nance revealed that the residues His(185), Arg(186), and Arg(187) formed th e major sites of interaction for MA-44E4. In contrast, the epitopes of MA-4 2A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A 2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A 7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350) Asp(355) and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) 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. T he localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucid ates two previously unidentified molecular mechanisms to modulate PAI-1 act ivity and opens new perspectives for the rational development of PAI-1 neut ralizing compounds.