Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage

Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulat ion as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B- like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inact ivated by both proteolysis by thrombin and spontaneous temperature-dependen t loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thromb in, In this study we followed TAFI activation and TAFIa inactivation by thr ombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry, Mass matching of the fragments revealed that TAFIa was cleaved at Ar-302. Studie s of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on it s conformational instability rather than proteolytic cleavage at Arg(302).