Amino Acid Residues in the P6-P′3 Region of Thrombin-activable Fibrinolysis Inhibitor (TAFI) Do Not Determine the Thrombomodulin Dependence of TAFI Activation

Abstract

Thrombin bound to thrombomodulin activates thrombin-activable fibrinolysis inhibitor (TAFI) and protein C much more efficiently than thrombin alone. Although thrombomodulin has been proposed to alter the thrombin active site, the recently determined structure of the thrombin-thrombomodulin complex does not support this proposal. In this study, the contribution of amino acids near the activation site of TAFI toward thrombomodulin dependence was determined, utilizing four variants of TAFI with specific substitutions in the P6-P 3 region surrounding the Arg-92 cleavage site. Two point mutants had either the Ser-90 or Asp-87 of TAFI replaced with Ala, a third mutant had the thrombin activation site of the fibrinogen B␤-chain substituted into positions 91-95 of TAFI, and a fourth mutant had the thrombin activation site of protein C substituted into positions 90 -95 of TAFI. Each of these mutants was expressed, purified, and characterized with respect to activation kinetics and functional properties of the enzyme. Even though fibrinogen is poorly cleaved by thrombin-thrombomodulin, the fibrinogen activation site does not significantly alter the thrombomodulin dependence of TAFI activation. The TAFI variant with the protein C activation sequence is only slowly activated by thrombin-thrombomodulin, and not at all by free thrombin. Mutating Asp-87 to Ala increases the catalytic efficiency of activation 3-fold both in the presence and absence of thrombomodulin, whereas mutating Ser-90 to Ala effects only minor kinetic differences compared with wild type TAFI. The thermal stabilities and antifibrinolytic properties of the enzymes were not substantially altered by any of the mutations that allowed for efficient activation of the enzyme. We conclude that residues in the P6-P 3 region of TAFI do not determine the thrombomodulin dependence of activation, which lends support to the argument that the role of thrombomodulin is to optimally orient thrombin and its substrate, rather than to allosterically alter the specificity of the thrombin active site.Hemostasis requires a fine balance between coagulation and fibrinolysis (1). Inappropriately up-regulated coagulation results in a thrombotic tendency, whereas augmented fibrinolysis results in a bleeding tendency. A central protein in this process is thrombomodulin, which fundamentally alters the substrate specificity of thrombin (2). Free thrombin is a potent procoagulant enzyme, whereas thrombin bound to thrombomodulin exerts anticoagulant and antifibrinolytic effects. In response to a vascular injury, the coagulation cascade produces free thrombin that catalyzes the formation of a fibrin clot. Subsequently, the thrombin-thrombomodulin complex inhibits the coagulation cascade, while still protecting the formed clot from premature lysis. In this way clot formation is highly localized, and any clots formed presumably remain intact long enough to prevent excessive blood loss at a site of injury.Thrombin-thrombomodulin mediates its anticoagulant and antifibrinolytic effects through ...

Thrombomodulin Changes the Molecular Surface of Interaction and the Rate of Complex Formation between Thrombin and Protein C

“…Because this parameter changes with different substrates (Table I), it is quite likely that thrombomodulin changes the conformation of protein C rather than thrombin. This possibility was first voiced by Hayashi et al (42) and is consistent with a large body of kinetic, thermodynamic, and structural data (9,14,16,43) as well as with the recent observation that Ca 2ϩ and Na ϩ binding affect the conformation of the activation peptide of protein C (6). The flexibility of this domain documented by fluorescence spectroscopy suggests that thrombo- modulin may change the orientation of the scissile bond, which should cause the change in E 1 observed experimentally.…”

Extended Interactions with Prothrombinase Enforce Affinity and Specificity for Its Macromolecular Substrate

“…Exosite binding plays a role in the action of thrombin on its diverse protein substrates with different P 1 -P 3 sequences preceding the scissile bond (28). Recent results with activation site mutants of thrombin-activated fibrinolysis inhibitor (TAFI) suggest that it is exosite binding rather than specific recognition of sequences surrounding the scissile bond that likely explains the ability of thrombomodulin to specifically enhance TAFI and protein C activation by thrombin (29). Thus, exosite-dependent substrate recognition may represent a prevalent mechanism underlying the function of the trypsin-like enzymes of coagulation.…”

The Roles of Selected Arginine and Lysine Residues of TAFI (Pro-CPU) in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

“…In no instance did the TAFIa concentration at 10 min exceed 10% of the input concentration of TAFI. The activation kinetics then were analyzed by the enzyme central, parallel assembly model described previously (14,23,32). In this model, the enzyme (T) can interact with TM to form the T-TM complex with dissociation constant K d1 or with TAFI to form the T-TAFI complex with dissociation constant K m1 .…”

“…They also demonstrated that the mutation D349A in the peptide connecting EGF-like domains 3 and 4 eliminated the cofactor activity of TM for TAFI activation (16). In addition, Schneider et al (23) showed that the TM dependence of TAFI activation is not determined by residues occupying the P6-P3Ј positions of TAFI.…”