Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin.
OBJECTIVE
Resistance of thrombi to plasmin digestion depends primarily on the amount of α(2)-antiplasmin (α(2)AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-α(2)AP between -Pro12-Asn13- to yield Asn-α(2)AP, which is crosslinked to fibrin approximately 13× more rapidly than Met-α(2)AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-α(2)AP to Asn-α(2)AP and thereby enhance endogenous fibrinolysis.


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 μm, 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-α(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 IC(50) value in plasma remaining comparable to that in phosphate buffer.


CONCLUSIONS
These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.
William K. Warren Medical Research Center and Department of Medicine, University of Oklahoma College of Medicine, Oklahoma City, OK 73126, USA. kyung-lee@ouhsc.edu
K N
KN
Lee
K W
KW
Jackson
V J
VJ
Christiansen
E K
EK
Dolence
P A
PA
McKee
D010450:Endopeptidases; D005342:Fibrinolysis; D018093:Gelatinases / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D000085822:Prolyl Oligopeptidases; D011498:Protein Precursors / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D012995:Solubility; D015912:Thrombolytic Therapy; D013997:Time Factors; D000979:alpha-2-Antiplasmin / Q000737:chemistry / Q000378:metabolism*; D000099167:Fibroblast Activation Protein Alpha
D010450:Endopeptidases; D018093:Gelatinases; D008565:Membrane Proteins; D010455:Peptides; D000085822:Prolyl Oligopeptidases; D011498:Protein Precursors; D012697:Serine Endopeptidases; D000979:alpha-2-Antiplasmin; D000099167:Fibroblast Activation Protein Alpha; C062388:pro-alpha-2-plasmin inhibitor; C000705287:PREPL protein, human
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