A role for arginine‐12 in thrombin–thrombomodulin‐mediated activation of thrombin‐activatable fibrinolysis inhibitor

Abstract

To cite this article: Plug T, Kramer G, Meijers JCM. A role for arginine-12 in thrombin-thrombomodulin-mediated activation of thrombinactivatable fibrinolysis inhibitor. J Thromb Haemost 2014; 12: 1717-25.Summary. Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a proenzyme that links coagulation and fibrinolysis. TAFI can be activated by thrombin, the thrombin-thrombomodulin complex and plasmin through cleavage of the first 92 amino acids from the enzyme. In silico analysis of the TAFI sequence revealed a potential thrombin cleavage site at Arg12. The aim of this study was to determine whether TAFI can be cleaved at Arg12 and whether this cleavage plays a role in TAFI activation. Methods: A peptide based on the first 18 amino acids of TAFI was used to determine whether thrombin was able to cleave at Arg12. Mass spectrometry was performed to determine whether the Arg12-cleaved peptide was released from full-length TAFI. Furthermore, a TAFI mutant in which Arg12 was replaced by a glutamine (TAFI-R12Q) was constructed and characterized with respect to its activation kinetics. Results: The peptide and mass spectrometry data showed that thrombin was able to cleave TAFI at Arg12, but with low efficiency in full-length TAFI. Characterization of TAFI-R12Q showed no difference in thrombin-mediated activation from wild-type TAFI. However, there was an approximately 60-fold impairment in activation of TAFI-R12Q by the thrombin-thrombomodulin complex. Conclusions: Arg12 of TAFI plays an important role in thrombomodulin-mediated TAFI activation by thrombin. Thrombin is able to cleave TAFI at Arg12, but it remains to be determined whether Arg12 is part of an exosite for thrombomodulin or whether cleavage at Arg12 accelerates thrombomodulin-mediated TAFI activation.

Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation

“…1B, lanes 2-7). These results are in line with the data reported by Plug et al [18] and Wu et al [19], demonstrating that several positively charged residues (Arg12 [18] and Lys42-Lys44 [19]) within the globular structure of the activation peptide might be important for the activation of TAFI by thrombin-thrombomodulin. Indeed, lack of the globular structure in the deletion mutant prevents efficient interaction with thrombomodulin, thereby abolishing the cofactor function.…”

“…These results are in line with the data reported by Plug et al . and Wu et al . , demonstrating that several positively charged residues (Arg12 and Lys42–Lys44 ) within the globular structure of the activation peptide might be important for the activation of TAFI by thrombin–thrombomodulin.…”

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin‐activatable fibrinolysis inhibitor activation and activated thrombin‐activatable fibrinolysis inhibitor activity

“…In the current study, a complete TAFI fragmentation pattern analysis in the presence of different TAFI activators demonstrated that VHH-i83 only inhibits T/TM-mediated TAFI activation, indicating that VHH-i83 interferes with the cofactor function of thrombomodulin. The TAFI-VHH-i83 structure suggests that VHH-i83 sterically interferes with thrombomodulin binding by interacting with Arg12 in the AP, which substantiates findings in previous mutagenesis studies that Arg12 is a critical residue for thrombomodulin-dependent TAFI activation by thrombin [31,32]. We also observed that VHH-i83 did not inhibit thrombin-mediated cleavage of the deletion mutant TAFI-ACIIYQ-Δ 1-73 (data not shown).…”

“…This result is in agreement with the structure of TAFI-VHH-i83. Specifically, the interaction interface on the AP is in close proximity to Arg12 and Lys42-Lys44 (shortest distance: 3.6 A between atoms of Arg12 and Lys42), previously shown to constitute the thrombomodulin-binding site [30][31][32], thus indicating that VHH-i83 prevents the interaction with thrombomodulin.…”

Selective modulation of thrombin‐activatable fibrinolysis inhibitor (TAFI) activation by thrombin or the thrombin‐thrombomodulin complex using TAFI‐derived peptides

“…Recently, we published a study that showed that Arg12 of TAFI plays an important role in thrombin‐thrombomodulin‐mediated TAFI activation, but that it is not relevant for TAFI activation by thrombin alone . In this study we have not determined whether cleavage at Arg12 or binding to Arg12 is important for thrombomodulin‐mediated TAFI activation.…”