title: The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex.
abstract: Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Chengliang
authors.initials: C
authors.lastname: Wu
authors.forename: Paul Y
authors.initials: PY
authors.lastname: Kim
authors.forename: Reg
authors.initials: R
authors.lastname: Manuel
authors.forename: Marian
authors.initials: M
authors.lastname: Seto
authors.forename: Marc
authors.initials: M
authors.lastname: Whitlow
authors.forename: Mariko
authors.initials: M
authors.lastname: Nagashima
authors.forename: John
authors.initials: J
authors.lastname: Morser
authors.forename: Ann
authors.initials: A
authors.lastname: Gils
authors.forename: Paul
authors.initials: P
authors.lastname: Declerck
authors.forename: Michael E
authors.initials: ME
authors.lastname: Nesheim
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025901:Carboxypeptidase B2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation / Q000502:physiology; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D018180:Thrombomodulin; D001120:Arginine; D025901:Carboxypeptidase B2; D013917:Thrombin; D008239:Lysine