The intrinsic enzymatic activity of plasma procarboxypeptidase U (TAFI) can interfere with plasma carboxypeptidase N assays

Willemse, Johan; Polla, Magnus; Hendriks, Dirk

doi:10.1016/j.ab.2006.05.020

Cited by 23 publications

(27 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…They are in agreement with a recent report that indicates that human TAFI displays a surprisingly high intrinsic activity toward standard substrates of B-like enzymes and large peptides and a significant fibrinolytic activity (20,30). Since TAFIa has a half-life in blood of only minutes, it seems quite reasonable to consider the possibility that TAFI, which is much more stable and soluble (7), plays a complementary (or even a direct) role in the antifibrinolytic function assigned to the enzyme.…”

Section: Discussionsupporting

confidence: 81%

“…Considering that the TAFI pro-domain is ϳ1.2 Å farther away from the catalytic domain as compared with proCPB due to the 12°rotation described above, it may be feasible that TAFI (zymogen) is able to accommodate substrates. Biochemical evidences (20,30) indicate that, in contrast to other B forms, TAFI possesses a significant intrinsic proteolytic activity (20). This experimental observation can now be rationalized in view of the structural determinants described here.…”

Section: Crystallization Structure Determination and Overallmentioning

confidence: 73%

See 1 more Smart Citation

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

Anand

Pallarès

Valnickova

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 81%

Section: Crystallization Structure Determination and Overallmentioning

confidence: 73%

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

Anand

Pallarès

Valnickova

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Like proCPA2 and in contrast to proCPB, the catalytic site residues of TAFI are in the "active" conformation, 13 which is consistent with the reported activity of TAFI for small peptide substrates. 32 The main difference between the 3 structures is a rotation of the activation peptide of TAFI away from the catalytic domain by 16°and 12°compared with proCPA2 and proCPB, respectively ( Figure 1B,C). In addition there is a difference in the length of the ␣-helix in the connecting linker, which is longer in pro-carboxypeptidases of the A type compared with CPB's, whereas in TAFI this helix is of an intermediate length.…”

Section: Comparison With Other Pro-carboxypeptidasesmentioning

confidence: 99%

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Marx

Brondijk²,

Plug

et al. 2008

Blood

115

View full text Add to dashboard Cite

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

show abstract

“…Previously, the carboxypeptidase activity of TAFI zymogen has been assigned to small synthetic substrates such as hippuryllinked amino acids (20,21) and N-(4-methoxyphenylazoformyl)-Lys-OH (22). Recently, TAFI zymogen has been shown to have carboxypeptidase activity toward the tetrapeptide PFGK and larger fibrin peptides (20).…”

mentioning

confidence: 99%

Thrombin-activable Fibrinolysis Inhibitor Zymogen Does Not Play a Significant Role in the Attenuation of Fibrinolysis

Foley¹,

Kim²,

Nesheim

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products. Thrombin-activable fibrinolysis inhibitor (TAFI)3 is a 60-kDa carboxypeptidase-like protein that circulates in plasma at a concentration of ϳ75 nM (1). TAFI (also known as procarboxypeptidase U, plasma carboxypeptidase B, and carboxypeptidase R) was discovered independently by several groups (2-6), and its role in fibrinolysis was subsequently characterized (6). Thrombin in complex with thrombomodulin activates TAFI with a catalytic efficiency of 1.2 M Ϫ1 s Ϫ1, which is ϳ1250-fold greater than that with thrombin alone (7). Plasmin has also been shown to activate TAFI (0.008 M Ϫ1 s Ϫ1 ), and the second-order rate constant of this reaction increases by 16-fold in the presence of unfractionated heparin (8). Activated TAFI (TAFIa) plays a significant role in attenuating fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which can then solubilize the fibrin clot by cleaving fibrin after specific arginine and lysine residues (9). TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues on fibrin (6, 10), thereby decreasing the tissue-type plasminogen activator (tPA) cofactor activity of plasmin-modified fibrin (11). Removal of these C-terminal lysine residues suppresses plasminogen activation (12, 13) and down-regulates the conversion of Glu-plasminogen to Lysplasminogen, which is a 20-fold better substrate for tPA than . TAFIa is intrinsically unstable, with its inactivation being highly temperature-dependent (15). The inactivation of TAFIa is thought to be accompanied by a large structural change (16). The existence of a TAFIa concentration threshold has been demonstrated, such that TAFIa at a concentration above the threshold inhibits fibrinolysis. On...

show abstract

The intrinsic enzymatic activity of plasma procarboxypeptidase U (TAFI) can interfere with plasma carboxypeptidase N assays

Cited by 23 publications

References 16 publications

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

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

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Thrombin-activable Fibrinolysis Inhibitor Zymogen Does Not Play a Significant Role in the Attenuation of Fibrinolysis

Contact Info

Product

Resources

About