Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Zuzana Valnickova,Ida B Thøgersen,Jan Potempa,Jan J Enghild

doi:10.1074/jbc.m606559200

Abstract

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.

Highlights

TAFI Zymogen Cleaves the Substrates Hipp-Arg and Hipp-Lys—When TAFI zymogen was incubated with the carboxypeptidase substrates hippuryl-Lys (Fig. 1A) or hippurylArg, a low but significant activity was apparent
The separation of the product and the substrate by reverse phase HPLC was critical for detection of the zymogen activity using this system. This activity was eliminated in the presence of 1,10-phenanthroline (Fig. 1B), supporting the hypothesis that TAFI zymogen exhibits authentic carboxypeptidase activity
The glycosylation of the TAFI activation peptide is unique among related pro-carboxypeptidases, which do not carry activation peptide-associated carbohydrate moieties [3]

Summary

EXPERIMENTAL PROCEDURES

Materials—Bovine trypsin, o-phenylenediamine dihydrochloride, 1,10-phenanthroline, 4,7-phenanthroline, phenylmethylsulfonyl fluoride, and chromogenic substrates for carboxypeptidases, N-benzoyl-Gly-Arg (hippuryl-Arg), Nbenzoyl-Gly-Lys (hippuryl-Lys), 3-(2-furyl)acryloyl-AlaArg-OH (FAAR) and 3-(2-furyl)acryloyl-Ala-Lys-OH (FAAK), were all obtained from Sigma. Serial dilutions of TAFI stopped by acidification (0.5% trifluoroacetic acid final con- zymogen or porcine CPB were added to the coated wells in centration), and samples were applied to a reverse phase 100 ␮l of TBS/well, and the plates were incubated for 1 h at column (Nucleosil 5 C18, 300A, 250 ϫ 2 mm, Phenomenex) 37 °C. Following the TAFI zymogen or CPB treatment of the sponding peak and converted to substrate turnover rates wells, a fixed concentration of human plasminogen A synthetic fibrin peptide (RGDSTFESKSYK, 1403.67 Da) was incubated with TAFI zymogen for 60 min at 37 °C at an enzyme:substrate molar ratio 1:26 (B). A random peptide (ERREKEAREASHRQKRSCEAGK, 2640.34 Da) containing a C-terminal lysine was incubated with TAFI zymogen for 10 min at 37 °C at an enzyme:substrate molar ratio of 1:4 and analyzed by MALDI-TOF MS (C). The data represent the enzyme-catalyzed reactions for 0.17 ␮M TAFI zymogen, 0.025 ␮M TAFIa, and 0.0025 ␮M CPB

TAFIa Km

RESULTS

DISCUSSION