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Keywords:

  • antiplasmin-cleaving enzyme inhibitor;
  • fibrinolysis;
  • fibroblast activation protein;
  • prolyl oligopeptidase;
  • Z-Pro-prolinal-insensitive peptidase;
  • α2-antiplasmin

Summary.  Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α2-antiplasmin (α2AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-α2AP between -Pro12-Asn13- to yield Asn-α2AP, which is crosslinked to fibrin approximately 13× more rapidly than Met-α2AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-α2AP to Asn-α2AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl-Arg-(8-amino-3,6-dioxaoctanoic acid)-d-Ala-l-boroPro selectively inhibited APCE vs. DPPIV, with an apparent Ki of 5.7 nm vs. 6.1 μm, indicating that an approximately 1000-fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent Ki of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose-dependent decrease of APCE-mediated Met-α2AP cleavage, which ultimately shortened plasminogen activator-induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC50 value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.