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

Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of alpha(2)-antiplasmin (alpha(2)AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-alpha(2)AP between -Pro12-Asn13- to yield Asn-alpha(2)AP, which is crosslinked to fibrin approximately 13x more rapidly than Met-alpha(2)AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-alpha(2)AP to Asn-alpha(2)AP 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 K-i of 5.7 nM vs. 6.1 mu M, indicating that an approximately 1000-fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K-i 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-alpha(2)AP 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.