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 Ser(305) to Cys and a Thr(329) to Ile mutation, respectively. Combining these mutations in TAFI-Ala(147)-Ile(325), 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-Ala(147)-Thr(325), the most frequently occurring isoform of TAFI in humans) at 37 C. Moreover, clot lysis ( induced by tissue plasminogen activator) experiments in which TAFI-Ala(147)-Cys(305)-Ile(325)- Ile(329) 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-Ala(147)-Thr(325)). 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.