Inactivation of Active Thrombin-activable Fibrinolysis Inhibitor Takes Place by a Process That Involves Conformational Instability Rather Than Proteolytic Cleavage

Marx, Pauline F.; Hackeng, Tilman M.; Dawson, Philip E.; Griffin, John H.; Meijers, Joost C. M.; Bouma, Bonno N.

doi:10.1074/jbc.275.17.12410

Cited by 93 publications

(131 citation statements)

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“…Regulated by an intrinsic temperature-dependent instability (i.e., half-life of 10 min at 37°C, 45 min at 30°C, and several hours at 22°C, and stable at 0°C) [4], TAFIa is converted into an inactive conformation (TAFIai; 36 kDa). Furthermore, it has been shown that this intrinsic instability of TAFIa is of major importance for the in vivo downregulation of its activity [5][6][7]. The underlying structural mechanisms of this rapid and spontaneous loss of activity are still unclear, and this has complicated the study of the effect of TAFIa on fibrinolysis.…”

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“…It could thus be expected that a 2 -AP deficiency would promote adipose tissue development. a 2 -AP is a 70 kDa single-chain glycoprotein that occurs in murine plasma at a concentration of about 90 lg mL )1 [6], and inhibits plasmin at a very high rate, similar to the human system [7]. Mice with inactivation of the a 2 -AP gene display a higher endogenous fibrinolytic potential than wild-type littermates [6].…”

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“…a 2 -AP is a 70 kDa single-chain glycoprotein that occurs in murine plasma at a concentration of about 90 lg mL )1 [6], and inhibits plasmin at a very high rate, similar to the human system [7]. Mice with inactivation of the a 2 -AP gene display a higher endogenous fibrinolytic potential than wild-type littermates [6]. To test the hypothesis that a 2 -AP may affect adipogenesis and adipose tissue development, we kept 5-week-old male a 2 -AP-deficient (a 2 -AP )/) ) mice and wild-type (a 2 -AP +/+ ) littermates (genetic background 50% C57Bl/6 and 50% 129 SVj) on a standard fat diet (SFD, KM -04-k12; Muracon, Oud Turnhout, Belgium; containing 13% kcal as fat, and a caloric value of 10.9 kJ g )1 ) or on a high-fat diet (HFD, Harlan TD 88137; Zeist, the Netherlands; containing 42% kcal as fat, and a caloric value of 20.1 kJ g )1 ) [8].…”

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Announcing a TAFIa mutant with a 180‐fold increased half‐life and concomitantly a strongly increased antifibrinolytic potential

Ceresa

Peeters

Declerck

et al. 2007

Journal of Thrombosis and Haemostasis

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Announcing a TAFIa mutant with a 180-fold increased half-life and concomitantly a strongly increased antifibrinolytic potential Activated thrombin-activatable fibrinolysis inhibitor (TAFIa; 36 kDa), formed upon activation of TAFI (56 kDa) by thrombin-thrombomodulin, plays a pivotal role in fibrinolysis and inflammation [1][2][3]. Regulated by an intrinsic temperature-dependent instability (i.e., half-life of 10 min at 37°C, 45 min at 30°C, and several hours at 22°C, and stable at 0°C) [4], TAFIa is converted into an inactive conformation (TAFIai; 36 kDa). Furthermore, it has been shown that this intrinsic instability of TAFIa is of major importance for the in vivo downregulation of its activity [5][6][7]. The underlying structural mechanisms of this rapid and spontaneous loss of activity are still unclear, and this has complicated the study of the effect of TAFIa on fibrinolysis. In addition, attempts to crystallize human TAFI or TAFIa have been unsuccessful so far, probably due to glycosylation in the prosegment or to the intrinsic instability of the enzyme. Attempts have been made to increase the stability of TAFIa by mutagenesis [7] or by construction of chimeric variants [8].Although some variants exhibit an increased TAFIa stability [8], this effect is not associated with an increased antifibrinolytic effect. Recently, we reported the generation of the TAFI-S305C-T325I-T329I variant, harboring the naturally occurring Ile isoform at position 325 and two stabilizing mutations i.e., S305C and T329I [9]. We determined a half-life of 70 ± 3.0 min at 37°C for the TAFIa-S305C-T325I-T329I variant vs. 6.3 ± 0.3 min for wild-type TAFIa (TAFIa-wt) (harboring residue Thr325) (elevenfold increase) and observed a 3.0-fold increased antifibrinolytic potential vs. TAFIa-wt. Simultaneously, Knecht et al. [10] reported that the H333Y and H335Q mutations also increase TAFIa stability. TAFIa-H333Y-H335Q had a half-life of 90 min at 37°C vs. 12 min for TAFIa-wt (7.5-fold increase) and a concomitantly 7-fold increased antifibrinolytic potential vs. TAFIa-wt.In the current study, we combined all five previously reported stabilizing mutations, generating TAFI-S305C-T325I-T329I-H333Y-H335Q. The functional activity and stability, activation and enzyme kinetics and antifibrinolytic potential were determined and compared to those of TAFIa-wt.TAFIa activity and stability were determined as described previously [9], and similar activities of 32 ± 2.1 U mg )1 and 26 ± 3.3 U mg )1 (Table 1, P ¼ 0.06) for TAFIa-wt and TAFIa-S305C-T325I-T329I-H333Y-H335Q, respectively, were found. Both variants could be equally well inhibited by potato

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Announcing a TAFIa mutant with a 180‐fold increased half‐life and concomitantly a strongly increased antifibrinolytic potential

Ceresa

Peeters

Declerck

et al. 2007

Journal of Thrombosis and Haemostasis

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“…Instead, TAFIa is highly unstable, with reported halflives at 37°C of 8 -9 (7,8) and 15 min (9). Whereas TAFIa can also be cleaved by thrombin at Arg-302, this cleavage is subsequent to the spontaneous structural changes corresponding to inactivation of the enzyme (8,10) and is therefore probably not relevant to the regulation of the enzyme.…”

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confidence: 99%

Two Naturally Occurring Variants of TAFI (Thr-325 and Ile-325) Differ Substantially with Respect to Thermal Stability and Antifibrinolytic Activity of the Enzyme

Schneider¹,

Boffa²,

Stewart³

et al. 2002

Journal of Biological Chemistry

161

201

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Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated toTAFIa by plasmin, thrombin, or the thrombin-thrombomodulin complex. The enzyme TAFIa attenuates clot lysis by removing lysine residues from a fibrin clot. Screening of nine human cDNA libraries indicated a common variation in TAFI at position 325 (Ile-325 or Thr-325). This is in addition to the variation at amino acid position 147 (Ala-147 or Thr-147) characterized previously. Thus, four variants of TAFI having either Ala or Thr at position 147 and either Thr or Ile at position 325 were stably expressed in baby hamster kidney cells and purified to homogeneity. The kinetics of activation of TAFI by thrombin/thrombomodulin were identical for all four variants; however, Ile at position 325 extended the half-life of TAFIa from 8 to 15 min at 37°C, regardless of the residue at position 147. In clot lysis assays with thrombomodulin and the TAFI variants, or with pre-activated TAFI variants, the Ile-325 variants exhibited an antifibrinolytic effect that was 60% greater than the Thr-325 variants. Similarly, in the absence of thrombomodulin, the Ile-325 variants exhibited an antifibrinolytic effect that was 30 -50% greater than the Thr-325 variants. In contrast, the variation at position 147 had little if any effect on the antifibrinolytic potential of TAFIa. The increased antifibrinolytic potential of the Ile-325-containing TAFI variants reflects the fact that these variants have an increased ability to mediate the release of lysine from partially degraded fibrin and suppress plasminogen activation. These findings imply that individuals homozygous for the Ile-325 variant of TAFI would likely have a longer lived and more potent TAFIa enzyme than those homozygous for the Thr-325 variant. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a zymogen found in human plasma (1), which is also known as plasma procarboxypeptidase B (2) and procarboxypeptidase U (3). It can be activated by thrombin (1), plasmin (4), or the thrombinthrombomodulin complex (5) to the carboxypeptidase B-like enzyme, TAFIa. When exposed to a fibrin clot, TAFIa catalyzes the removal of carboxyl-terminal lysines, thereby diminishing the cofactor activity for plasminogen activation (6). Less efficient plasminogen activation on the fibrin clot corresponds to prolongation of fibrinolysis, and in this way TAFIa can serve as a potent antifibrinolytic enzyme. Studies performed using an in vitro human plasma model have found that clot lysis times can be attenuated up to 3-fold in the presence of TAFIa as compared with clots lysed in the absence of TAFIa (5).Activation of TAFI is catalyzed only slowly by thrombin alone; however, in the presence of thrombin/thrombomodulin, the efficiency of activation is increased 1000-fold. Despite the large thrombomodulin dependence of TAFI activation, in vitro clot lysis assays done in the absence of thrombomodulin still exhibit prolonged clot lysis times as compared with similar assays performed with TAFI-depleted plas...

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“…TAFIa is a labile enzyme, with a half-life of Ͻ10 min at 37°C (7)(8)(9). TAFIa is inactivated by conformational instability, after which it becomes more susceptible to proteolytic degradation by thrombin (4,(7)(8)(9)(10)(11). Plasmin can inactivate TAFIa by proteolysis of the catalytic domain (12).…”

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Generation and Characterization of a Highly Stable Form of Activated Thrombin-activable Fibrinolysis Inhibitor

Marx

Havik

Marquart

et al. 2004

Journal of Biological Chemistry

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Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is ϳ40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293-333) and TAFI-CPB-(293-401). TAFI-CPB-(293-333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFIdeficient plasma. Binding of TAFI-CPB-(293-333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293-401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293-401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.

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Inactivation of Active Thrombin-activable Fibrinolysis Inhibitor Takes Place by a Process That Involves Conformational Instability Rather Than Proteolytic Cleavage

Cited by 93 publications

References 21 publications

Announcing a TAFIa mutant with a 180‐fold increased half‐life and concomitantly a strongly increased antifibrinolytic potential

Announcing a TAFIa mutant with a 180‐fold increased half‐life and concomitantly a strongly increased antifibrinolytic potential

Two Naturally Occurring Variants of TAFI (Thr-325 and Ile-325) Differ Substantially with Respect to Thermal Stability and Antifibrinolytic Activity of the Enzyme

Generation and Characterization of a Highly Stable Form of Activated Thrombin-activable Fibrinolysis Inhibitor

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