Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated toTAFIa by plasmin, thrombin, or the thrombin-thrombomodulin complex. The enzyme TAFIa attenuates clot lysis by removing lysine residues from a fibrin clot. Screening of nine human cDNA libraries indicated a common variation in TAFI at position 325 (Ile-325 or Thr-325). This is in addition to the variation at amino acid position 147 (Ala-147 or Thr-147) characterized previously. Thus, four variants of TAFI having either Ala or Thr at position 147 and either Thr or Ile at position 325 were stably expressed in baby hamster kidney cells and purified to homogeneity. The kinetics of activation of TAFI by thrombin/thrombomodulin were identical for all four variants; however, Ile at position 325 extended the half-life of TAFIa from 8 to 15 min at 37°C, regardless of the residue at position 147. In clot lysis assays with thrombomodulin and the TAFI variants, or with pre-activated TAFI variants, the Ile-325 variants exhibited an antifibrinolytic effect that was 60% greater than the Thr-325 variants. Similarly, in the absence of thrombomodulin, the Ile-325 variants exhibited an antifibrinolytic effect that was 30 -50% greater than the Thr-325 variants. In contrast, the variation at position 147 had little if any effect on the antifibrinolytic potential of TAFIa. The increased antifibrinolytic potential of the Ile-325-containing TAFI variants reflects the fact that these variants have an increased ability to mediate the release of lysine from partially degraded fibrin and suppress plasminogen activation. These findings imply that individuals homozygous for the Ile-325 variant of TAFI would likely have a longer lived and more potent TAFIa enzyme than those homozygous for the Thr-325 variant. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a zymogen found in human plasma (1), which is also known as plasma procarboxypeptidase B (2) and procarboxypeptidase U (3). It can be activated by thrombin (1), plasmin (4), or the thrombinthrombomodulin complex (5) to the carboxypeptidase B-like enzyme, TAFIa. When exposed to a fibrin clot, TAFIa catalyzes the removal of carboxyl-terminal lysines, thereby diminishing the cofactor activity for plasminogen activation (6). Less efficient plasminogen activation on the fibrin clot corresponds to prolongation of fibrinolysis, and in this way TAFIa can serve as a potent antifibrinolytic enzyme. Studies performed using an in vitro human plasma model have found that clot lysis times can be attenuated up to 3-fold in the presence of TAFIa as compared with clots lysed in the absence of TAFIa (5).Activation of TAFI is catalyzed only slowly by thrombin alone; however, in the presence of thrombin/thrombomodulin, the efficiency of activation is increased 1000-fold. Despite the large thrombomodulin dependence of TAFI activation, in vitro clot lysis assays done in the absence of thrombomodulin still exhibit prolonged clot lysis times as compared with similar assays performed with TAFI-depleted plas...

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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...

Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 ؋ 10 6 M ؊1 s ؊1 measured in a soluble buffer system to 3.2 ؋ 10 6 M ؊1 s ؊1 in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis.As a fibrin clot forms in response to a vascular injury, it regulates its own degradation by serving as cofactor for plasminogen (Pgn) 1 activation to plasmin (Pn) (1). Once formed, Pn directly catalyzes clot breakdown by cleaving fibrin. In the process, carboxyl-terminal lysine residues are exposed that serve to enhance Pgn activation through a positive feedback mechanism for Pn formation (2). Once formed, Pn is quickly inhibited by the potent serine protease inhibitor antiplasmin (AP), which forms a tight nearly irreversible complex with Pn. Therefore, Pn levels and consequently the rate of fibrinolysis are determined by the balance between the kinetics of Pn formation and inhibition.Thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently identified plasma zymogen (3-5) that can be activated to the carboxypeptidase B-like enzyme TAFIa by thrombin/ thrombomodulin (6), free thrombin (3, 7), or plasmin (3,8). In the context of a fibrin clot, TAFIa removes carboxyl-terminal lysine residues from Pn-modified fibrin (9). Although no naturally occurring inhibitors of TAFIa have been identified in human plasma, TAFIa is thermally unstable and this instability is probably how TAFIa is regulated once it is activated (10, 11). Previously, a naturally occurring polymorphism at amino acid position 325 (Thr/Ile) was identi...

Summary. The plasma carboxypeptidase activated thrombinactivable fibrinolysis inhibitor (TAFIa), is thermally unstable at 37 8C, with a half-life of 8 or 15 min depending on the isoform. The arginine analog, 2-guanidinoethylmercaptosuccinate (GEMSA), not only inhibits TAFIa but also slows the spontaneous inactivation of the enzyme, thereby reducing the activity of TAFIa, while extending its apparent half-life. Because, as shown in previous work, the ability of TAFIa to prolong clot lysis can be more dependent on its half-life than its concentration, in this study we determined whether reversible inhibitors of TAFIa could paradoxically prolong clot lysis. Potato tuber carboxypeptidase inhibitor (PTCI) or GEMSA were titrated into normal pooled human plasma, in the presence of soluble thrombomodulin. Both inhibitors mediate a biphasic antifibrinolytic effect, prolonging clot lysis at lower concentrations and enhancing clot lysis at higher concentrations. The antifibrinolytic effect of GEMSA is maximized at 1 mmol L À1 , increasing clot lysis time from 100 min to 350 min. The antifibrinolytic effect of PTCI is maximized at 100 nmol L À1 , increasing clot lysis time from 100 min to 240 min. To further characterize the nature of this biphasic effect, TAFI at various concentrations was added to TAFI-immunodepleted human plasma in the presence of PTCI or GEMSA. The magnitude of the effect depends on the concentration of TAFIa, the concentration of inhibitor, and the potency of the inhibitor. We propose that the biphasic antifibrinolytic effect is mediated by the dynamic equilibrium of free TAFIa that inactivates quickly, and TAFIa bound to inhibitor that inactivates slowly. TAFIa inhibitors used as therapeutic agents might not only enhance lysis at higher concentrations, but also stabilize fibrin clots at intermediate concentrations.