Activated protein C - Protein C inhibitor complex formation: Characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop

Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiol ogically most important inhibitor of activated protein C. We have made a mo noclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved l oop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tet radecapeptide (corresponding to residues P1-P14 of the reactive center loop ) into beta -sheet A of the uncleaved inhibitor also exposed the epitope. T he antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substi tuted to the residues found in mouse protein C inhibitor (Thr and Ile, resp ectively), were also inserted in beta -sheet A. The Arg P13/Thr substitutio n led to a greatly impaired reactivity with the antibody, whereas the Ala P 9/Ile mutation resulted in a modest loss of reactivity with the antibody. T hese results indicate that complex formation leads to insertion of the reac tive center loop in beta -sheet A from Arg P14 and presumably beyond Ala P9 . Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been loca lized to the loop-inserted part of beta -sheet A, the part of the serpin wh ere the complexation-induced conformational change is most conspicuous.