Reversible inhibitors of TAFIa can both promote and inhibit fibrinolysis

Schneider, M.; Nesheim, Michael E.

doi:10.1046/j.1538-7836.2003.00028.x

Cited by 44 publications

(44 citation statements)

References 17 publications

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“…rPCI concentrations of Ն200 nM are required to accelerate fibrinolysis, and a 2.0 -2.5-fold acceleration is achieved at concentrations of Ն1000 nM (clot lysis time, 170 Ϯ 7 and 80 Ϯ 6 min in the absence and presence of 1500 nM rPCI and 1.2 nM tPA, respectively). These results are in agreement with previous studies performed using commercial PCI (39,40). Compared with rPCI, the effect of rTCI differs in three aspects: 1) rTCI has only a minor pro-fibrinolytic effect at low concentrations; 2) ϳ10-fold lower concentrations (Ն15 nM) are sufficient to accelerate fibrinolysis; and 3) the maximal pro-fibinolytic effect is more pronounced (2.8-fold acceleration at 500 nM).…”

Section: H Nmr Analysis Of Rtci In H 2 O and D 2 Osupporting

confidence: 91%

A Carboxypeptidase Inhibitor from the Tick Rhipicephalus bursa

Arolas

Lorenzo

Rovira

et al. 2005

Journal of Biological Chemistry

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A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.

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Section: H Nmr Analysis Of Rtci In H 2 O and D 2 Osupporting

confidence: 91%

A Carboxypeptidase Inhibitor from the Tick Rhipicephalus bursa

Arolas

Lorenzo

Rovira

et al. 2005

Journal of Biological Chemistry

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“…The stabilizing potency of different carboxypeptidase inhibitors depends on their K i , as it appears that only the free enzyme inactivates. 16 We decided to soak TAFI crystals with the inhibitor GEMSA, which has a K i of 11 M. The TAFI-GEMSA complex diffracted to 3.4 Å. GEMSA was found bound in the catalytic cleft S1Ј pocket where the carboxy-terminal arginine or lysine residue of the substrate would bind. The observed binding mode is consistent with that of GEMSA inhibited carboxypeptidase D 35 (PDB entry 1H8L).…”

Section: Inhibitor Binding Reduces Flap Dynamicsmentioning

confidence: 99%

“…13,14 Thermal stability of TAFIa is increased by binding of small peptide substrates, like hippuryl-arginine, 15 or carboxypeptidase inhibitors such as 2-guanidino-ethyl-mercaptosuccinic acid (GEMSA). 16,17 In addition, extensive mutagenesis studies have identified 5 mutations (S305C, T325I, T329I, H333Y, and H335Q) which, in combination, stabilize TAFIa activity 180-fold. [18][19][20] In this paper we present crystal structures of wild-type TAFI, a TAFI-inhibitor complex, and a TAFI mutant with a 70-fold more stable active enzyme.…”

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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

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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...

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“…The fi rst compared the extended-release formulation of AZD0837 (in doses of 150, 300, and 450 mg once daily and 200 mg bid) with warfarin (dose-adjusted to achieve an INR between 2.0 and 3.0) in 955 patients with atrial fi bril- agents is paradoxical enhancement of TAFIa activity at low doses. [186][187][188] Presumably, this refl ects allosteric modulation at the active site of the enzyme. If this phenomenon is common to all TAFIa inhibitors, optimal dosing of these agents will be problematic.…”

Section: Azd0837mentioning

confidence: 99%