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 physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C−protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1−P14 of the reactive center loop) into β-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1−P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in β-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in β-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 localized to the loop-inserted part of β-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.