Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2

The physiological roles of plasminogen activator inhibitor-a (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types, This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-P treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-S following proteolytic cleavage of the Arg(380)-Thr(381) bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu(368)) and P14 (Thr(367)) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).