Generation of a stable activated thrombin activable fibrinolysis inhibitor variant.
Activated thrombin activable fibrinolysis inhibitor (TAFIa), generated upon activation of TAFI, exerts an antifibrinolytic effect. TAFIa is a thermolabile enzyme, inactivated through a conformational change. The objective of the current study was to generate a stable variant of human TAFIa. Using a site-directed as well as a random mutagenesis approach to generate a library of TAFI mutants, we identified two mutations that increase TAFIa stability, i.e. a Ser305 to Cys and a Thr329 to Ile mutation, respectively. Combining these mutations in TAFI-Ala147-Ile325, the most stable isoform of TAFIa (half-life of 9.4 +/- 0.4 min), revealed a TAFIa half-life of 70 +/- 3.1 min (i.e. an 11-fold increase versus 6.3 +/- 0.3 min for TAFIa-Ala147-Thr325, the most frequently occurring isoform of TAFI in humans) at 37 degrees C. Moreover, clot lysis (induced by tissue plasminogen activator) experiments in which TAFI-Ala147-Cys305-Ile325-Ile329 was added to TAFI-depleted plasma revealed a 50% clot lysis time of 313 +/- 77 min (i.e. a 3.0-fold increase versus 117 +/- 10 min for TAFI-Ala147-Thr325). The availability of a more stable TAFIa variant will facilitate the search for inhibitors and allow further structural analysis to elucidate the mechanisms of the instability of TAFIa.
Ceresa
Erik
E
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Van de Borne
Kirsten
K
Peeters
Miet
M
Lijnen
Henri Roger
HR
Declerck
Paul J
PJ
Gils
Ann
A
D001483:Base Sequence; D025901:Carboxypeptidase B2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D005342:Fibrinolysis; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D016296:Mutagenesis; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism
D017931:DNA Primers; D018180:Thrombomodulin; D025901:Carboxypeptidase B2; D013917:Thrombin