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°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.

Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation

“… and Wu et al . , demonstrating that several positively charged residues (Arg12 and Lys42–Lys44 ) within the globular structure of the activation peptide might be important for the activation of TAFI by thrombin–thrombomodulin. Indeed, lack of the globular structure in the deletion mutant prevents efficient interaction with thrombomodulin, thereby abolishing the cofactor function.…”

Solulin increases clot stability in whole blood from humans and dogs with hemophilia

“…Figure 4 shows that the thrombin-Solulin complex activated TAFI much more efficiently than it did protein C. As described previously, 29 TAFI activation by the thrombinSolulin complex can be described using the Michaelis-Menten model. Using the Michaelis-Menten model to fit the data, a K m of 0.71M and a k cat of 1.09/s were determined, which implies a catalytic efficiency of 1.53/M/s.…”

Selective modulation of thrombin‐activatable fibrinolysis inhibitor (TAFI) activation by thrombin or the thrombin‐thrombomodulin complex using TAFI‐derived peptides

“…Thrombin binding to Arg12‐Glu28, however, seems less likely because the TAFI‐thrombin‐thrombomodulin model published by Wu et al . shows that this area of TAFI already interacts with thrombomodulin. Binding to the first site may result in TAFI activation by means of cleavage at Arg92, whereas binding to the second site may result in Arg12 cleavage in TAFI.…”

“…Because TAFI contains two different thrombin cleavage sites (Arg92 and Arg12, although the relevance of cleavage at Arg12 is still under investigation) [17] it is tempting to speculate that thrombin can bind to TAFI at two separate locations: one site consisting of Gly205-Glu232 (peptides 18 and 19) and a second site consisting of either Arg12-Glu28 (peptide 2) or Cys383-Val401 (peptide 34). Thrombin binding to Arg12-Glu28, however, seems less likely because the TAFIthrombin-thrombomodulin model published by Wu et al [23] shows that this area of TAFI already interacts with thrombomodulin. Binding to the first site may result in TAFI activation by means of cleavage at Arg92, whereas binding to the second site may result in Arg12 cleavage in TAFI.…”