Localization of thrombomodulin-binding site within human thrombin

A binding site for thrombomodulin on human thrombin (alpha-thrombin) was elucidated by identifying an epitope for a monoclonal antibody for thrombin (MT-6) which inhibited the activation of protein C by the thrombin-thrombomodulin complex by directly inhibiting the binding of thrombin to thrombomodulin. An 8.5-kDa fragment isolated by digestion of thrombin with Staphylococcus aureus V8 protease followed by reversed-phase high performance liquid chromatography (HPLC) and a peptide isolated by reversed-phase HPLC after reduction of the 8.5-kDa fragment, which was composed of three peptides linked by disulfide-bonds, bound directly to MT-6 and thrombomodulin. The amino acid sequence of the peptide coincided with the sequence of residues Thr-147 to Asp-175 of the B-chain of thrombin. A synthetic peptide corresponding to Thr-147 to Ser-158 of the B-chain inhibited the binding of thrombin to thrombomodulin. Elastase-digested thrombin, which was cleaved between Ala-150 and Asn-151, lost its binding affinity for both MT-6 and thrombomodulin. These findings indicate that the binding site for thrombomodulin is located within the sequence between Thr-147 and Ser-158 of the B-chain.     

The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.     

The effect of bovine thrombomodulin on the specificity of bovine thrombin

Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.     

Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated to TAFIa 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 degrees 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.     

Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.     

Macromolecular specificity determinants on thrombin for fibrinogen and thrombomodulin

The endothelial cell surface membrane protein thrombomodulin binds thrombin with high affinity and acts as both a cofactor for protein C activation and an inhibitor of fibrinogen hydrolysis. We have previously shown that bovine thrombomodulin is a competitive inhibitor of fibrinogen binding to thrombin but has no effect on thrombin activity toward tripeptide substrates or antithrombin III. Hence, thrombomodulin and fibrinogen may share macromolecular specificity sites on thrombin which are distinct from the active site. In this investigation, we have studied the interaction of thrombin-thrombomodulin with fibrinogen and various thrombin derivatives. We show that fibrinogen is a competitive inhibitor of thrombomodulin binding to thrombin, with a Kis = 10 microM. Thrombin derivatives (bovine (pyridoxal phosphate)4-thrombin and human thrombin Quick I), which bind fibrinogen with much reduced affinity, are shown to also interact with thrombomodulin with greatly reduced affinity. These results are consistent with the hypothesis that thrombomodulin and fibrinogen share macromolecular specificity sites on thrombin.     

A thrombin-based peptide corresponding to the sequence of the thrombomodulin-binding site blocks the procoagulant activities of thrombin.

Thrombomodulin, a cofactor in the thrombin-catalyzed activation of protein C, blocks the procoagulant activities of thrombin such as fibrinogen clotting, Factor V activation, and platelet activation. The binding site for thrombomodulin within human thrombin has been localized at a region comprising residues Thr147-Ser158 of the B-chain of thrombin. The dodecapeptide sequence, TWTANVGKGQPS, corresponding to these residues inhibits thrombin binding to thrombomodulin with an apparent Ki = 94 microM (Suzuki, K., Nishioka, J., and Hayashi, T. (1990) J. Biol. Chem. 265, 13263-13267). We have found that the inhibitory effect of the dodecapeptide on the thrombin-thrombomodulin interaction is sequence-specific, and that residues Asn151, Lys154, and Gln156 are essential for thrombomodulin binding. The dodecapeptide was also found to directly block thrombin procoagulant activities, fibrinogen clotting (concentration for half-maximum inhibition, 385 microM). Factor V activation (concentration for half-maximum inhibition, 33 microM), and platelet activation (concentration for half-maximum inhibition, 645 microM). This peptide did not block thrombin inhibition by antithrombin III, but blocked thrombin inhibition by hirudin. These findings suggest that the binding site for thrombomodulin in thrombin is shared with the sites for fibrinogen, Factor V, platelets, and hirudin, and that, therefore, the inhibition of thrombin procoagulant activities by thrombomodulin in part results from blocking of the interaction between thrombin and the procoagulant protein substrates by thrombomodulin.     

The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin

We have prepared a monoclonal antibody directed against human thrombomodulin. We used the antibody to measure thrombomodulin molecules in cultured human endothelial cells from umbilical vein and in a human lung cancer cell line (A549). Endothelial cells contain approximately 30,000-55,000 molecules of thrombomodulin/cell while the A549 cell has about 1/4 of this number. About 50-60% of thrombin binding sites on endothelial cells are thrombomodulin, while about 90% of thrombin binding sites on A549 cells are thrombomodulin. Exposure of these cells to thrombin decreased thrombomodulin on the cell surface suggesting that internalization of thrombin-thrombomodulin occurred. The internalized 125I-thrombin was degraded in the cells and thrombomodulin reappeared on the cell surface after 30 min, suggesting the recycling of thrombomodulin. The rate of protein C activation correlated with the presence of the thrombin-thrombomodulin complex on the cell surface. The binding of thrombin to cell-surface thrombomodulin accelerates protein C activation; the subsequent internalization of the thrombin-thrombomodulin complex is associated with cessation of protein C activation. Therefore, endocytosis of thrombin-thrombomodulin may serve to control protein C activation. The uptake and degradation of thrombin bound to thrombomodulin may provide a mechanism for clearance of thrombin from the circulation.     

Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.     

Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation

Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombin-activable 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 interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains.

A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.     

The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface

The location of the active site of the membrane-bound anticoagulant complex of thrombin and thrombomodulin has been determined relative to the membrane surface using fluorescence energy transfer. Thrombin was reacted with 5-(dimethylamino)-1-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (DEGR-CK) to yield DEGR-thrombin, an analogue of thrombin with a fluorescent dye covalently attached to its active site. When DEGR-thrombin was titrated with thrombomodulin that had been reconstituted into phospholipid vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes, each in an active site of a DEGR-thrombin bound to thrombomodulin, and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer reached a maximum when DEGR-thrombin and thrombomodulin were equimolar in the sample, as expected for the formation of a 1:1 complex between thrombin and thrombomodulin. This energy transfer was dependent upon the binding of DEGR-thrombin to thrombomodulin because no energy transfer was observed with vesicles that lacked thrombomodulin, and the extent of energy transfer was reduced greatly by the addition of excess unmodified nonfluorescent thrombin to compete with DEGR-thrombin for binding to the thrombomodulin. From the dependence of the energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between a dye in the active site of the thrombin-thrombomodulin complex and a dye at the membrane surface was determined to average 66 A (65 +/- 3 A for phosphatidylcholine vesicles without and 67 +/- 5 A for those with 20% phosphatidylserine). This distance was also insensitive to the presence or absence of Ca2+. These direct measurements indicate that the active site of the membrane-bound thrombin-thrombomodulin complex is located far above the phospholipid surface, that the peptide bond cleaved during the activation of protein C is situated about 66 A above the membrane, that the thrombin binding site on thrombomodulin is positioned more than 45 A above the membrane, ant that thrombin, with a diameter near 40 A, is not positioned alongside thrombomodulin near the membrane to form the thrombin-thrombomodulin complex but is instead bound "on top" of thrombomodulin.     

Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation.

The association of thrombin with thrombomodulin, a non-enzymatic endothelial cell surface receptor, alters the substrate specificity of thrombin. Complex formation converts thrombin from a procoagulant to an anticoagulant enzyme. Structure-function analysis of this change in specificity is facilitated by the availability of two soluble proteolytic derivatives of thrombomodulin, one consisting of the six repeated growth factor-like domains of thrombomodulin (GF1-6) and the other containing only the fifth and sixth such domains (GF5-6). Both derivatives can bind to thrombin and block fibrinogen clotting activity, though only the larger GF1-6 can stimulate the activation of protein C. To ascertain whether the substrate specificity change from fibrinogen to protein C is accompanied by structural changes in the active site of the enzyme, fluorescent dyes were positioned at different locations within the active site. A 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached to the active site serine to form dansyl-thrombin, while either a fluorescein or an anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg linkage. The environment of the dansyl dye was altered in a similar fashion when either GF1-6 or GF5-6 bound to thrombin, since a similar reduction in dansyl emission intensity was elicited by these two thrombomodulin derivatives (25 and 32%, respectively). These spectral changes, and all others in this study, were saturable and reached a maximum when the ratio of thrombomodulin derivative to thrombin was close to 1. The environments of the fluorescein and ANS dyes were also altered when GF1-6 bound to thrombin because binding resulted in emission intensity changes of -13% and +18%, respectively. In contrast, no fluorescence changes were observed when the fluorescein and ANS thrombin derivatives were titrated with GF5-6. Thus, the structure of the active site was altered by thrombomodulin both immediately adjacent to the active site serine and also more than 15 A away from it. However, the structural change far from Ser-195 was only elicited by thrombomodulin species that stimulate thrombin-dependent activation of protein C.     

Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors.

Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.     

Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage

Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperature-dependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg(302). Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg(302).     

Inactivation of chicken liver pyruvate carboxylase by 1,10-phenanthroline.

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.     

Combined segregation-linkage analysis of plasma thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels with TAFI gene polymorphisms

By decreasing plasminogen binding to fibrin surface, the thrombin activatable fibrinolysis inhibitor (TAFI) has been hypothesized to constitute an early marker for atherothrombotic diseases. Previous studies have shown that plasma TAFI levels exhibit a high interindividual variability that is only poorly explained by lifestyle factors. Several polymorphisms of the TAFI gene have been described, and a combination of a C+1542G substitution in the 3' untranslated region and an Ala147Thr amino acid change has been shown to explain 60% of TAFI variability in a sample of unrelated individuals. A segregation-linkage analysis was performed to determine whether these polymorphisms are directly involved in the genetic regulation of TAFI levels, or whether they are only markers in linkage disequilibrium (LD) with unmeasured TAFI-linked quantitative trait loci (QTLs). The sample consisted of 97 healthy nuclear families from the Stanislas Cohort. The C+1542G and Ala147Thr polymorphisms were in complete negative LD, with minor allele frequencies of 0.27 and 0.28, respectively. Results of the segregation-linkage analysis provided evidence of two TAFI-linked QTLs in LD with the two measured polymorphisms, which would explain 78% of the TAFI variance, as compared with 55% explained by the C+1542G and the Ala147Thr polymorphisms combined. The two putative QTLs would have minor allele frequencies of 0.45 and 0.32, respectively. The hypothesis that one of the measured polymorphisms is one of the QTLs was rejected. The putative QTLs also did not seem compatible with the other TAFI gene polymorphisms that we have previously described. More extensive sequencing of the TAFI gene is necessary to identify the functional variants.     

Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.