Role of isoleucine residues 182 and 183 in thrombin-activatable fibrinolysis inhibitor.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, once activated, can attenuate fibrinolysis. The active form, TAFIa, is a labile enzyme, with a half-life of a few minutes at 37 degrees C. Understanding the molecular mechanisms of TAFIa inactivation will allow the development of compounds that modulate TAFIa activity. Based on their three-dimensional model of TAFI, Barbosa Pereira et al. [J Mol Biol (2002), vol. 321, pp. 537-547] suggested that Ile182 and Ile183 were involved in the instability of TAFIa. However, these carboxypeptidases are, unlike TAFIa, stable proteases. Therefore, we constructed, expressed and characterized a TAFI mutant in which Ile182 and Ile183 were changed into the residues found in pancreas carboxypeptidase B at corresponding positions, Arg and Glu. The active form of the mutant, TAFIa-I182R-I183E, had a similar half-life as wild-type TAFIa, showing that Ile182 and Ile183 were not involved in the regulation of TAFIa stability. Remarkably, however, TAFI-I182R-I183E was activated at a lower rate by thrombin-thrombomodulin (mutant: 45 +/- 2 U L(-1) s(-1) and wild type: 103 +/- 3 U L(-1) s(-1)), thrombin (mutant: 1 +/-0.1 U L(-1) s(-1) and wild type 3 +/- 0.2 U L(-1) s(-1)) and plasmin (mutant: 0.8 +/- 0.04 U L(-1) s(-1) and wild type: 5.0 +/-0.2 U L(-1) s(-1)) compared with wild-type TAFI. Accordingly, it had a sixfold reduced antifibrinolytic potential. In conclusion, analysis of TAFI-I182R-I183E showed that I182 and I183 are not involved in TAFIa inactivation by conformational instability but that these residues may be involved in the activation of TAFI and stabilization of the fibrin clot.
Marx
P F
PF
Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. p.f.marx@amc.uva.nl
Havik
S R
SR
Bouma
B N
BN
Meijers
J C M
JC
D019943:Amino Acid Substitution; D000818:Animals; D025901:Carboxypeptidase B2 / Q000737:chemistry / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D005342:Fibrinolysis / Q000235:genetics; D006207:Half-Life; D006801:Humans; D007532:Isoleucine; D007700:Kinetics; D016297:Mutagenesis, Site-Directed
D007532:Isoleucine; D025901:Carboxypeptidase B2