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.
Highlights
The endothelial membrane protein thrombomodulin (TM)2 is a central regulator in the balance between coagulation and fibrinolysis [1, 2]
The results indicate that all mutations decrease activation efficiency of thrombin-activable fibrinolysis inhibitor (TAFI), but none influence the thermal stability of TAFIa
Previous studies revealed some of the elements of the structures of IIa, TAFI, and TM needed for efficient TAFI activation [16, 18, 20, 23]
Summary
Eaton et al [9] originally sequenced TAFI and showed that it consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain [9]. TM consists of 10 structural elements: a N-terminal domain homologous to the family of C-type lectins (residues 1–226), six tandem epidermal growth factor (EGF)-like domains joined by small interdomain peptides (residues 227– 462), a serine/threonine-rich domain (residues 463– 497), a transmembrane domain (residues 498 –521), and a cytoplasmic tail (residues 522–559) [1, 2, 17, 18]. It is predominantly expressed on the luminal surface of endothelial cells lining normal blood vessels [19]. The results indicate that all mutations decrease activation efficiency of TAFI, but none influence the thermal stability of TAFIa
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