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 the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation 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 Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions 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 in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin- and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.
Boffa
M B
MB
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Bell
R
R
Stevens
W K
WK
Nesheim
M E
ME
D025901:Carboxypeptidase B2; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010949:Plasma / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors
D011994:Recombinant Proteins; D002268:Carboxypeptidases; D025901:Carboxypeptidase B2; D013917:Thrombin