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 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.

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 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.

Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.

Fig. 1

Analysis of prolyl oligopeptidase (POP) activity in human plasma. A POP-specific substrate (S), acetyl-KLRP-AMC (51 μM) was incubated with 0.1 μg POP – supplemented or non-supplemented normal human plasma for selected times. (A) Comparison of POP activity in a single human plasma sample vs. phosphate buffer only. (B) Substrate (S) concentration dependency of POP activity in a four-person pooled plasma sample.

Fig. 2

Analysis of prolyl oligopeptidase (POP) activity in human plasma using a POP-specific inhibitor. Pooled plasma from four donors was assayed in the presence and absence of supplemental POP (0.05 μg); a POP-specific inhibitor, acetyl (Ac)-KLR-L-boroPro (10 μM) or Inhibitor no. 6 (10 μM) that inhibits both antiplasmin-cleaving enzyme (APCE) and POP. MEPLGRQLTSGP-AMC(100 μM) served as a substrate for APCE and POP. Each bar represents the mean ± SD of three experiments.

Fig. 3

Incubation of Met-α₂AP with antiplasmin-cleaving enzyme (APCE), dipeptidyl peptidase IV (DPPIV) or prolyl oligopeptidase (POP). Each enzyme was incubated with Met-α₂AP and analyzed by SDS-PAGE and immunoblot. Top panel: Coomassie-stained reduced SDS-PAGE analyses. Bottom panel: Immunoblot with antibody specific for the N-terminal peptide of Met-α₂AP and non-reactive with Asn-α₂AP. Only APCE cleaves Met-α₂AP.

Fig. 4

Inhibition of antiplasmin-cleaving enzyme (APCE)-catalyzed Met-α₂AP cleavage by Inhibitor no. 6. Met-α₂AP (12 μg) was incubated for 6 h with APCE (0.2 μg) and Inhibitor no. 6. Controls are indicated as either ‘No Inhibitor’ or ‘No Enzyme’. (A) Representative immunoblot detected by antibody specific for the N-terminal peptide of Met-α₂AP and which does not react with Asn-α₂AP. (B) Densitometric analysis of immunoblot (A) for percent inhibition of Met-α₂AP cleavage by Inhibitor no. 6. Percent inhibition was expressed as [(densitometric value when Inhibitor no. 6 was present – densitometric value for No Inhibitor)/(densitometric value for No Enzyme – densitometric value for No Inhibitor)] × (100). Each bar represents the mean ± SE of three experiments.

Fig. 5

Enhancement of plasma clot lysis by Inhibitor no. 6. After incubating Met-α₂AP (8.4 μg) and antiplasmin-cleaving enzyme (APCE) (1.2 μg) with selected concentrations of Inhibitor no. 6 in α₂AP-depleted plasma for 5 h, fibrin clot formation, α₂AP crosslinking to fibrin, and fibrinolysis were initiated by adding a mixture of thrombin, CaCl₂, and uPA. (A) Effect of inhibitor no. 6 on clot lysis. Controls are indicated as either ‘No Inhibitor’ or ‘No Met-α₂AP’. (B) Effect of Inhibitor no. 6 on plasma clot lysis time (PCLT) with Asn-α₂AP (8.4 μg) and APCE (1.2 μg). As expected, the curve for ‘Met-α₂AP, 50 μM Inhibitor no. 6’ is virtually identical to that for ‘Met-α₂AP, 50 μM Inhibitor no. 6’ in (A). The data in (B) show that Inhibitor no. 6 only affects APCE activity and none of the other fibrinolysis reactions. PCLT was defined as the midpoint between the highest and lowest absorbances in each clot lysis profile. Each data point for (A) and (B) is the mean ± SE of four and three experiments, respectively.

Fig. 6

α₂AP activity in normal plasma. (A) Percent Met-α₂AP and Asn-α₂AP as determined by Edman amino terminal analyses with time during incubation of normal plasma at 29 °C. (B) Enhancement of plasma clot lysis by Inhibitor no. 6. Normal plasma with neither added α₂AP nor antiplasmin-cleaving enzyme (APCE) was incubated in the absence or presence of Inhibitor no. 6 for 72 h at 29 °C after which clot formation and lysis were initiated by adding thrombin, CaCl₂, and uPA. Each data point is the mean ± SE of three determinations. The difference between dashed vertical lines indicates change in time for 50% lysis of inhibited vs. noninhibited control plasma while that between solid vertical lines is change in time for total lysis of inhibited vs. non-inhibited control plasma.

Fig. 7

Stability of inhibitors in plasma. After incubating Inhibitor no. 6 or Val-boroPro in plasma for selected times, antiplasmin-cleaving enzyme (APCE) inhibitory activity was assayed by adding APCE and the enzyme substrate, MEPLGRQLTSGP-AMC. Control plasma contained neither inhibitor. Percent inhibition was expressed as [(fluorescence for control – fluorescence with Inhibitor no. 6)/(fluorescence for control)] × (100).