Abstract Number: LPB0018
Meeting: ISTH 2021 Congress
Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen implicated in regulating the balance between coagulation and fibrinolysis and is a molecular link between coagulation and inflammation. TAFIa, formed by cleavage by thrombin-thrombomodulin, has the unusual property of being intrinsically unstable, with a half-life at body temperature of 8 – 15 minutes; spontaneous TAFIa inactivation is a key mechanism regulating TAFIa activity. While TAFIa inactivation involves a conformational change, the nature of this change is unknown. Two regions of the enzyme have been implicated in the intrinsic instability: (1) the “dynamic flap” (residues 297-350) whose mobility is restricted by the activation peptide or by competitive inhibitors of TAFIa; and (2) the “mobile loop” (residues 142-150) whose mobility (in the bovine enzyme) is reduced by binding of tick carboxypeptidase inhibitor.
Aims: Whether the mobile loop impacts TAFI instability is unknown. We therefore constructed recombinant variants of human TAFI in which the mobile loop residues were mutated individually to their cognate residues in the stable pancreatic carboxypeptidase B (five variants in total); additionally, we deleted the extra residue in TAFI (∆142) or replaced the entire mobile loop with the corresponding region from the pancreatic enzyme (∆ML).
Methods: Recombinant TAFI variants were expressed in baby hamster kidney cells and purified by immunoaffinity chromatography. TAFIa was formed by incubation with thrombin-thrombomodulin, and then incubated at 37°C; timed aliquots were removed and residual TAFIa activity was measured using the substrate N-(4-methoxyphenylaxoformyl)-Arg (AAFR).
Results: All TAFI variants were activated at similar rates by thrombin-thrombomodulin and cleaved AAFR at similar rates, although the ∆142 and ∆ML variants showed approximately 75% and 50% activity, respectively. No differences in the half-lives of the variant enzymes at 37°C were observed compared to wild-type TAFIa.
Conclusions: The mobile loop (residues 142-150) in human TAFIa does not impact the intrinsic instability of the enzyme.
To cite this abstract in AMA style:Marier H, Boffa M. Role of the “Mobile Loop” (Residues 142-150) in the Intrinsic Instability of TAFIa [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/role-of-the-mobile-loop-residues-142-150-in-the-intrinsic-instability-of-tafia/. Accessed September 25, 2021.
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