BACKGROUND

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated to form activated TAFI (TAFIa) (Ala93-Val401) through removal of the N-terminal activation peptide (Phe1-Arg92). TAFIa is thermally unstable, and the role of the activation peptide in the activity and stability of TAFI zymogen remains unclear.

OBJECTIVES

To better understand the role of the activation peptide in the activity and stability of TAFI.

METHODS

We constructed a deletion mutant, TAFI-CIIYQ-∆1-73 , in which the first 73 amino acids of the activation peptide are absent. The intrinsic activity and functional stability were determined with a chromogenic assay. The activation of TAFI-CIIYQ-∆1-73 by TAFI activators was evaluated with western blot analysis.

RESULTS

In comparison with TAFI-CIIYQ, the deletion mutant exerted high intrinsic activity ('full' apparent TAFIa activity) without cleavage by TAFI activators. TAFI-CIIYQ-∆1-73 was cleavable by thrombin. However, in the presence of thrombomodulin, the thrombin-mediated cleavage of TAFI-CIIYQ-∆1-73 was not accelerated. TAFI-CIIYQ-∆1-73 showed a similar functional stability profile to that of TAFI-CIIYQ. Full cleavage by thrombin did not affect the apparent carboxypeptidase activity of TAFI-CIIYQ-∆1-73 , but resulted in a significant loss of functional stability.

CONCLUSIONS

A stable deletion mutant of TAFI with full carboxypeptidase activity without activation is described. The segment Ala74-Arg92 in the activation peptide contributes significantly to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen.