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 Bbeta-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.
Highlights
Hemostasis requires a fine balance between coagulation and fibrinolysis [1]
Two point mutants had either the Ser-90 or Asp-87 of thrombin-activable fibrinolysis inhibitor (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
Construction and Expression of TAFI Mutants—To determine whether the amino acid residues adjacent to the cleavage site influence the thrombomodulin dependence of TAFI activation, we constructed four mutants of human TAFI with alterations in the P6-PЈ3 region
Summary
Materials—The synthetic carboxypeptidase substrate anisylazoformyllysine (AAFK) [16] was a generous gift from Dr William L. 10-l aliquots were removed and quenched with 190 l of AAFK (120 M final concentration) and PPAck (1 M final concentration) in a microtiter plate, and the initial rates of substrate hydrolysis were measured. To quantify the kinetics of TAFI activation, each TAFI mutant at various concentrations (0 –2.0 M) was incubated with Solulin at various concentrations (0 –50 nM) in the presence of CaCl2 (5 mM) and thrombin (0.25 nM or 0.5 nM). TAFI-deficient plasma was diluted 1:3 in HBS/Tween 0.01% before clotting was initiated by the addition of thrombin (5 nM), CaCl2 (10 mM), PCPS (20 M), tPA (0.3 nM), TAFI mutant zymogens (0 –90 nM), and Solulin (10 nM). Clot lysis was monitored by the change in turbidity of each reaction at 400 nm in a Spectramax Plus plate reader (Molecular Devices, Sunnyvale, CA), and the time to 50% lysis was determined graphically as the midpoint between maximum turbidity of a clot and the minimum absorbance when a clot was completely lysed
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