Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor

We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify t he thrombin cleavage site in activated TAFI (TAFIa) and to determine the re lative contribution of proteolytic cleavage and thermal instability in regu lation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gin did not prevent thrombin-mediated cleavage nor did muta tion to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gin abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gin substitution s decreased the thermal stability of TAFIa, The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role i n regulating the activity if TAFIa, Incubation of TAFIa under conditions th at result in complete thermal inactivation of the enzyme accelerates subseq uent thrombin- and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFI a activity. Collectively, these studies point to a role for the thermal ins tability, but not for proteolytic cleavage, of TAFIa in regulation of its a ctivity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.