The inner workings of thrombin-activatable fibrinolysis inhibitor: a study of the TM-mediated activation of TAFI and inactivation of TAFIa

Haley D N Marier, The University of Western Ontario

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a key regulator of hemostasis and inflammation. TAFI is activated by thrombin proteolysis, most effectively when in complex with cofactor thrombomodulin (TM). Soluble TM, encompassing EGF-like domains 3 through 6 is sufficient to promote TAFI activation, though the role of EGF 4 has not been assessed. Through rearrangement of the soluble TM domains, we determined that EGF 4 was acting as a spacer to promote TAFI activation. Activated TAFI (TAFIa) is a metastable enzyme, with a half-life of only 8-15 min at 37^oC. The mobile loop of TAFIa was expected to modulate stability; however, mutations in this region mimicking the stable pancreatic carboxypeptidase B did not increase half-life. To identify regions responsible for the instability of TAFIa, Trp-to-Phe mutations were made to monitor the decay in intrinsic fluorescence of TAFIa associated with the loss of enzymatic activity. No Trp residues were deemed independently responsible for the decay.