J Thromb Haemost. 2003 May; 1(5): 1028-33.

Elucidation of a novel epitope of a substrate-inducing monoclonal antibody against the serpin PAI-1.

Naessens D, Gils A, Compernolle G, Declerck PJ.

Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, K.U. Leuven, Belgium.

Plasminogen activator inhibitor-1 (PAI-1) is the most important physiological inhibitor of plasminogen activators. Inhibition of PAI-1 constitutes a putative strategy for the prevention of cardiovascular disease. The monoclonal antibody MA-8H9D4 inhibits PAI-1 activity by inducing a substrate behavior in PAI-1. To identify the epitope, a rational approach was used to design various PAI-1 alanine mutants (n = 16) for evaluation of their affinity. PAI-1-R300A, PAI-1-Q303A and PAI-1-D305A had affinities for MA-8H9D4 of < 10(5) M(-1), 2.0 x 10(8) M(-1) and 2.5 x 10(8) M(-1), respectively, whereas the affinity of wtPAI-1 is 3.3 x 10(9) M(-1). The epitope on the axis of arginine 300, glutamine 303 and aspartic acid 305, located on the loop between alpha-helix I and beta-strand 5A, demonstrates that MA-8H9D4 interferes with the final locking step in the serpin/proteinase interaction, thereby explaining its substrate inducing properties. The location of the epitope as well as the proposed mechanism of action is clearly different from that of other substrate inducing monoclonal antibodies against PAI-1. Elucidation of this novel epitope and the previously unidentified molecular mechanism opens new perspectives for the rational development of PAI-1-neutralizing compounds, as well as for the further exploration of synergistic effects between different PAI-1-inhibiting compounds.