Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Abstract

Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombinactivable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no loss of function in protein C activation for mutations within the c-loop of EGF3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reductions at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C. Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the rate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca 2؉ -dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mM Ca 2؉ . Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a 60-kDa plasma protein that circulates in plasma at concentration of about 75 nM (1). It is a zymogen that is activated to a carboxypeptidase B-like enzyme by a single thrombin-catalyzed cleavage at arginine 92 (2-4). The enzyme, designated TAFIa, catalyzes removal of carboxyl-terminal arginine and lysine residues in fibrin as it undergoes fibrinolysis (5). As a consequence, feedback up-regulation of plasminogen activation is eliminated, and the process of fibrinolysis is suppressed. The activation of TAFI by thrombin and subsequent actions of TAFIa define a molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses activity in the latter (6). Although thrombin at the high levels generated after the clotting of fibrin occurs can activate sufficient TAFI to suppress fibrinolysis, thrombin by itself is a relatively weak activator (2, 7). Thrombin bound to thrombomodulin, however, activates TAFI with a catalytic efficiency 1250-fold greater than that of thrombin alone (6). Thus, the thrombin-thrombomodulin complex is probably the physiologic ac...

Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

“…9,10,56 However, it currently appears unlikely that mutations in the lectinlike domain can appreciably influence TAFI activation, as this appears to be determined predominantly by EGF-like domains 3-5 of TM. 57,58 Mutation Asp486Tyr lies in the Ser/Thr-rich domain of TM. Although this domain has an important spacer function in positioning the thrombin biding site above the cell surface, 31 the mutation does not appear to influence this role.…”

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

“…These authors present evidence pointing to polymorphonucleur neutrophil-derived NADPH oxidase as a probable source of the biological oxidants that cause the TM inactivation typically observed in inflamed tissues. Oxidation of Met-388 leading to lessening of TM functions has also been the subject of studies by Wood et al (181,182) and Wang et al (175), while ROS-dependent inactivation of TM has been reported in a mouse model of vascular dysfunction and thrombosis (2). Furthermore, a relatively recent article by Stites and Froude (148) proposes that the elevated oxidative stress associated with smoking and diabetes is responsible for the prothrombotic state of these conditions by virtue of increased TM Met-388 oxidation and the subsequent decrease in circulating activated protein C levels.…”

Fluorescence Properties and Functional Roles of Tryptophan Residues 60d, 96, 148, 207, and 215 of Thrombin

“…This is not sufficient for TAFI activation, however. Further elements of EGF3 are required (54,55), comprising minimally the disulfide-bonded c-loop of EGF3. A recent study by Hall et al (56), involving replacement of surface residues with alanine, identified three mutants with decreased ability to activate TAFI, but not protein C, and another three that had the opposite characteristics.…”