TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg 92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu-form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin. TAFI (thrombin-activable fibrinolysis inhibitor)1 is a recently discovered 60-kDa single-chain plasma protein that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis (1, 2). It is present in plasma at a concentration of about 75 nM (3). TAFI was discovered independently in several different laboratories and consequently has acquired several aliases. It was described initially as an unstable carboxypeptidase in human serum and was named carboxypeptidase U (4, 5). It also was named plasma carboxypeptidase B by Eaton et al. (6) and subsequently plasma procarboxypeptidase B (pro-plasma carboxypeptidase B) by Tan and Eaton (7). In addition, on the basis of its instability in serum, it probably can be identified as carboxypeptidase R described by Campbell and Okada (8).Recent studies show that the thrombin-thrombomodulin complex, rather than free thrombin, is probably the physiologic activator of TAFI (2). In addition, activated TAFI (TAFIa) down-regulates tissue plasminogen activator (t-PA)-induced fibrinolysis half-maximally at a concentration of 1.0 nM (2). Because this is only about 1.3% of the level of the zymogen in plasma, ample TAFIa could be generated to modulate fibrinolysis very significantly in vivo (2). Bajzar et al. (3) showed that the apparent profibrinolytic effect of activated protein C is absent in TAFI-deficient plasma or when plasma is supplemented with an anti-TAFI monoclonal antibody. They also showed in plasma systems supplemented with soluble thrombomodulin or in systems utilizing cultured endothelial cells as a source o...

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...

We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions decreased the thermal stability of TAFIa. The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin-and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.The balance between the activities of the coagulation and fibrinolytic cascades is essential to prevent excessive blood loss upon injury to the vasculature while maintaining the blood in a fluid state at sites distant from the injury. The recent identification of thrombin-activable fibrinolysis inhibitor (TAFI 1 (1)) (also known as plasma procarboxypeptidase B (2) and carboxypeptidase U (3)) has illuminated a novel molecular linkage between these cascades. The production of thrombin by the coagulation cascade results in the generation of the active form of TAFI (TAFIa) that down-regulates fibrinolysis by removal of the carboxyl-terminal lysine residues from partially degraded fibrin that are important for the development of positive feedback in the fibrinolytic cascade (4).The M r ϳ60,000 TAFI zymogen is cleaved after Arg-92 by thrombin to yield an activation peptide and a M r ϳ35,000 enzyme (TAFIa) that possesses basic carboxypeptidase activity (1, 2). The catalytic efficiency of thrombin activation of TAFI is enhanced 1250-fold in the presence of the cofactor thrombomodulin (5). The enzymatic activity of TAFIa is intrinsically unstable, decaying with a half-life of about 8 -9 min at body temperature in the absence of additional proteolytic cleavage (6). Previous studies from our laboratory have revealed that the instability of TAFIa activity is highly sensitive to temperature; the half-life of TAFIa is increased approximately 15-fold by decreasing from 37 to 25°C the temperature at which the enzym...

Background: Plasma lipoprotein(a) (Lp(a)) levels can be reduced through proprotein convertase subtilisin/kexin type 9 (PCSK9) through an unknown mechanism. Results: Lp(a) catabolism in hepatoma cells and primary fibroblasts is inhibited by PCSK9 via the low density lipoprotein receptor (LDLR). Conclusion: LDLR mediates the effects of PCSK9 on Lp(a) internalization. Significance: Our results provide a mechanistic explanation for the effects of PCSK9 inhibitors on plasma Lp(a) levels.

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Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B. Cleavage of the zymogen by thrombin/ thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo. In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with k cat ‫؍‬ 0.55 s ؊1 , K m ‫؍‬ 0.54 M, and K d ‫؍‬ 6.0 nM for rTAFI and k cat ‫؍‬ 0.61 s ؊1 , K m ‫؍‬ 0.55 M, and K d ‫؍‬ 6.6 nM for pTAFI. The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis. The half-life of both enzymes at 37°C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to ϳ62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme. Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (⌬H o ‡ Х 45 kcal/mol and ⌬S o ‡ Х 80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and ⑀-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.The balance between the activities of the coagulation and fibrinolytic cascades is essential to protect the organism from excessive blood loss upon injury as well as to maintain the fluidity of blood within the vasculature. Imbalances lead to a tendency toward either bleeding or thrombosis, the latter of which is manifested as heart attacks and strokes.The coagulation and fibrinolytic cascades consist of a series of zymogen to enzyme conversions, terminating in the proteolytic enzymes thrombin and plasmin, which, respectively, catalyze the deposition and removal of fibrin. However, when in a complex with the endothelial cell-surface cofactor thrombomodulin, the specificity of thrombin is changed from fibrinogen to protein C (1), thus changing thrombin to an anticoagulant rather than a procoagulant enzyme.When formed in the context of a fibrin clot, activated protein C was fou...

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Background: Lipoprotein(a) [Lp(a)] levels predict the risk of myocardial infarction (MI) in populations of European ancestry; however, few data are available for other ethnic groups. Furthermore, differences in isoform size distribution and the associated Lp(a) concentrations have not fully been characterized between ethnic groups. Methods: We studied 6086 cases of first MI and 6857 controls from the INTERHEART study that were stratified by ethnicity and adjusted for age and sex. A total of 775 Africans, 4443 Chinese, 1352 Arabs, 1856 Europeans, 1469 Latin Americans, 1829 South Asians, and 1221 Southeast Asians were included in the study. Lp(a) concentration was measured in each participant using an assay that was insensitive to isoform size, with isoform size being assessed by Western blot in a subset of 4219 participants. Results: Variations in Lp(a) concentrations and isoform size distributions were observed between populations, with Africans having the highest Lp(a) concentration (median=27.2 mg/dL) and smallest isoform size (median=24 kringle IV repeats). Chinese samples had the lowest concentration (median=7.8 mg/dL) and largest isoform sizes (median=28). Overall, high Lp(a) concentrations (>50 mg/dL) were associated with an increased risk of MI (odds ratio, 1.48; 95% CI, 1.32–1.67; P <0.001). The association was independent of established MI risk factors, including diabetes mellitus, smoking, high blood pressure, and apolipoprotein B and A ratio. An inverse association was observed between isoform size and Lp(a) concentration, which was consistent across ethnic groups. Larger isoforms tended to be associated with a lower risk of MI, but this relationship was not present after adjustment for concentration. Consistent with variations in Lp(a) concentration across populations, the population-attributable risk of high Lp(a) for MI varied from 0% in Africans to 9.5% in South Asians. Conclusions: Lp(a) concentration and isoform size varied markedly between ethnic groups. Higher Lp(a) concentrations were associated with an increased risk of MI and carried an especially high population burden in South Asians and Latin Americans. Isoform size was inversely associated with Lp(a) concentration, but did not significantly contribute to risk.