The Crystal Structure of Thrombin-activable Fibrinolysis Inhibitor (TAFI) Provides the Structural Basis for Its Intrinsic Activity and the Short Half-life of TAFIa

Abstract

Mature thrombin-activable fibrinolysis inhibitor (TAFIa) is a highly unstable metallocarboxypeptidase that stabilizes blood clots by clipping C-terminal lysine residues from partially degraded fibrin. In accordance with its in vitro antifibrinolytic activity, animal studies have reported that inhibition of mature TAFI aids in the prevention of thrombosis. The level of TAFI activity is stringently regulated through (i) controlled proteolytic truncation of the zymogen (TAFI), generating the mature enzyme, TAFIa, and (ii) the short half-life of TAFIa. TAFI itself exhibits an intrinsic enzymatic activity, which is likely required to provide a baseline level of antifibrinolytic activity. The novel crystal structure presented here reveals that the active site of TAFI is accessible, providing the structural explanation for the its intrinsic activity. It also supports the notion that an "instability region" exists, in agreement with site-directed mutagenesis studies. Sulfate ions, bound to this region, point toward a potential heparin-binding site and could explain how heparin stabilizes TAFIa.Thrombin-activable fibrinolysis inhibitor (TAFI) 6 (1), also known as plasma carboxypeptidase B (CPB), carboxypeptidase B2, carboxypeptidase R, or procarboxypeptidase U (2-4), is a member of the M14A subfamily of metallocarboxypeptidases (MEROPS data base; Ref. 5). It has been described as a fibrinolysis inhibitor that circulates in plasma as a plasminogen-binding protein. TAFI is involved in the fibrinolytic pathway and therefore seems to be highly regulated (6). It is a 60-kDa glycoprotein secreted by the liver with an isoelectric point of 5.0 (2). The glycans are mainly located on the pro-domain. They account for around 20% of the human TAFI mass and are assumed to play a role in stability and solubility (7). The prodomain consists of a 92-residue N-terminal peptide, which can be removed by proteolysis, generating a 304-residue catalytic region called TAFIa (1-3).The removal of this peptide takes place by proteolytic cleavage of a specific Arg-Ala peptide bond at the loop between the pro-domain and the catalytic moiety. These two residues are Arg-92-Ala-93, numbered according to the N terminus of TAFI. The scission and dissociation of the two moieties causes the pI to shift from ϳ5 to 8 for mature TAFIa, rendering the protein more basic and less soluble than TAFI (7). Several proteases, including trypsin, thrombin, and plasmin, are able to cleave the Arg-92-Ala-93 bond. In vivo, the thrombin/thrombomodulin complex is most likely responsible for generation of TAFIa, although plasmin may also be able to function in this manner (8). TAFIa down-regulates fibrinolysis by removing C-terminal lysine residues from fibrin that has already been partially degraded by plasmin. Those lysine residues act as ligands for the lysine-binding sites of plasminogen and tissuetype plasminogen activator (9). Removal of the lysine residues attenuates the fibrin cofactor function in the activation of plasminogen by tissue-type plasminogen activator, re...

The Roles of Selected Arginine and Lysine Residues of TAFI (Pro-CPU) in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

“…Recent studies have shown that the structure of TAFI is indeed similar to that of procarboxypeptidase B, thus supporting the model (36,37).…”

Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

“…Crystal Structure Solution of the TAFI-TCI Complex-The structure of TAFI-TCI was solved by Patterson search techniques, assaying several strategies and combination of searching models, which corresponded to 100% identical chemical species: bovine TAFIa, TCI, and the pro-domain of bovine TAFI (45,52). Unambiguous solutions were found for the mature enzyme moiety and the inhibitor, but not for the prodomain.…”

“…The crystal structures of human and bovine TAFI revealed the molecular basis for the thermodynamic instability of the mature protease as compared with its related, but more robust pancreatic counterparts (44,45). Furthermore, the incentive for the intrinsic proteolytic activity of the protein was revealed.…”

“…The TAFI pro-domain was partially rotated away from the active site, exposing the catalytic residues to the solvent. Additionally, the corresponding salt-bridge in pro-CPB, located between the pro-domain and the substrate binding site in the catalytic moiety, was missing in TAFI, thus unlocking the active site (44,45). Protein inhibitors of MCPs described to date include: potato carboxypeptidase inhibitor (PCI), leech carboxypeptidase inhibitor (LCI), tick carboxypeptidase inhibitor (TCI), Ascaris carboxypeptidase inhibitor (ACI), and latexin (46 -50).…”

Biochemical Characterization of Bovine Plasma Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)

“…Significantly, the position of the pro-peptide is rotated 12° away from the active site, exposing access to the catalytic residues. Another significant distinction is the lack of the corresponding salt bridge between Asp 41 and Arg 145 in TAFI [42]. These distinctions might explain the intrinsic activity of TAFI [11,12].…”