Biochemical Importance of Glycosylation in Thrombin Activatable Fibrinolysis Inhibitor

Buelens, K.; Hillmayer, Kerstin; Compernolle, Griet; Declerck, Paul; Gils, Ann

doi:10.1161/circresaha.107.157099

Cited by 23 publications

(42 citation statements)

References 21 publications

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“…The specific activity (expressed as units per milligram) of TAFIa was evaluated at 22°C using the chromogenic assay and a standard of hippuric acid, spanning a concentration range from 16M to 2mM to quantify the amount of hippuric acid formed during the substrate reaction. 21 …”

Section: Ma-tck26d6mentioning

confidence: 99%

Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model

Vercauteren¹,

Emmerechts

Peeters³

et al. 2011

Blood

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The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Activated thrombin activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis and is an attractive target to develop profibrinolytic drugs. TAFI can be activated by thrombin, thrombin/thrombomodulin, or plasmin, but the in vivo physiologic TAFI activator(s) are unknown. Here, we generated and characterized MA-TCK26D6, a monoclonal antibody raised against human TAFI, and examined its profibrinolytic properties in vitro and in vivo. In vitro, MA-TCK26D6 showed a strong profibrinolytic effect caused by inhibition of the plasmin-mediated TAFI activation. In vivo, MA-TCK26D6 significantly decreased fibrin deposition in the lungs of thromboembolism-induced mice. Moreover, in the presence of MA-TCK26D6, plasmin-␣ 2 -antiplasmin complexes in plasma of thromboembolism-induced mice were significantly increased compared with a control antibody, indicative of an acceleration of fibrinolysis through MA-TCK26D6. In this study, we show that plasmin is an important TAFI activator that hampers in vitro clot lysis. Furthermore, this is the first report on an anti-TAFI monoclonal antibody that demonstrates a strong profibrinolytic effect in a mouse thromboembolism model. (Blood. 2011;117(17): 4615-4622)

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Section: Ma-tck26d6mentioning

confidence: 99%

Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model

Vercauteren¹,

Emmerechts

Peeters³

et al. 2011

Blood

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“…Inhibition of Intrinsic TAFI Activity by Protein InhibitorsThe ability of PCI, LCI, and TCI to inhibit the intrinsic TAFI activity was analyzed as described previously (19,21). Briefly, 15 l of TAFI (0.37 M, final concentration) in 50 mM Tris-HCl, pH 7.5 was titrated using PCI, LCI, or TCI (ranging from 0 to 50 M, final concentration).…”

Section: Methodsmentioning

confidence: 99%

“…This is the only MCP pro-domain that is glycosylated, thus suggesting biological significance. In this context, it has been speculated that the carbohydrates aid in stabilization and solubility of the enzyme and may affect the intrinsic proteolytic activity (15,19). In addition, the majority of the identified transglutaminase amine acceptor sites are also located in the pro-domain, thus enabling TAFI to be cross-linked to other proteins during coagulation/fibrinolysis (18,20,21).…”

mentioning

confidence: 99%

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

Valnickova

Sanglas

Arolas

et al. 2010

Journal of Biological Chemistry

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We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the prodomain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the prodomain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.Pancreatic carboxypeptidase A1 is the archetype of the M14 metallocarboxypeptidase (MCP) 3 family (1), also referred to as the funnelin tribe of metallopeptidases (2). The members of this family are subdivided into A/B or N/E MCPs on the basis of primary structure and structural homologies (3). They are also classified according to their substrate preference for C-terminal hydrophobic (A-type) or basic (B-type) amino acid residues. Thrombin-activatable fibrinolysis inhibitor (TAFI) (EC 3.4.17.20; UniProt Q96IY4) is a paralogue of pancreatic procarboxypeptidase B1 (pro-CPB1), pro-carboxypeptidases A1 (pro-CPA1), and A2 (pro-CPA2) and is also known as plasma pro-carboxypeptidase B2, R, or U (4 -9). Similarly to other B-type MCPs, TAFI possesses an N-terminal pro-domain that functions as a gatekeeper preventing substrate access to a preformed active site (for a review, see Refs. 2, 3). In human and porcine pancreatic pro-CPB1, access to the active site is completely shielded, resulting in no detectable intrinsic enzymatic activity of the zymogen (10). This is in contrast to human pro-CPA1 and pro-CPA2, which allow cleavage of small peptide substrates (11). Equally, pro-carboxypeptidase B from Pacific dogfish and African lungfish exhibit intrinsic activity toward small substrate molecules (12, 13). Likewise, TAFI interferes with carboxypetidase N assays (14), and is capable of cleaving larger peptide substrates due to its low but continuous carboxypeptidase activity...

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“…In the article published by Buelens et al 14 in this issue of Circulation Research, the authors report the construction, expression, and characterization of several mutants of TAFI missing 1 or more N-linked glycosylation sites. This type of posttranslational modification may also influence many of the biochemical properties of the proteins, such as stability, dynamics, and ligand binding.…”

mentioning

confidence: 99%

“…20 Moreover, N-glycosylation is crucial during the normal processing of human coagulation factor VII. 21 Finally, N-linked glycosylation is important in determining molecular weight, thrombin cleavage, and functional activity of human protein S. 22 Using an elegant approach, Buelens et al 14 have determined the biochemical importance of the glycosylation of TAFI. From their data it can be concluded that it is mainly the glycosylation at Asn86 that contributes to the biochemical properties of TAFI.…”

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confidence: 99%

Glycosylation of Thrombin Activatable Fibrinolysis Inhibitor

Biscetti

2008

Circulation Research

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Biochemical Importance of Glycosylation in Thrombin Activatable Fibrinolysis Inhibitor

Cited by 23 publications

References 21 publications

Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model

Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model

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

Glycosylation of Thrombin Activatable Fibrinolysis Inhibitor

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