Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin

Abstract

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 5.7nm vs. 6.1μm, indicating that an approximately 1000-fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent Ki of 7.4nm was found for POP inhibition, which is similar to 5.7nm 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 22h 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. © 2011 International Society on Thrombosis and Haemostasis.