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.     

Isolation and characterization of thrombomodulin from bovine lung

Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent Mr of 78,000 and 105,000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60 degrees C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin-induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier.     

Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism

The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III.

The relationship between thrombomodulin-associated O-linked glycosammoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high-Mr form containing GAGs and a low-Mr form lacking the majority of O-linked GAGs) of a recombinant human TM deletion mutant (rec-TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high-Mr rec-TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O-linked GAGs and the low-Mr form of rec-TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high-Mr form of rec-TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high-Mr rec-TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high-Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin-TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species-, organ-, or tissue-specific manner as a means to regulate TM function in diverse vasculatures.     

Studies on the mechanism of the rate-enhancing effect of heparin on the thrombin-antithrombin III reaction.

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticogulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.     

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.     

Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.     

Dependence of antithrombin III and thrombin binding stoichiometries and catalytic activity on the molecular weight of affinity-purified heparin

Heparin was fractionated by affinity chromatography on immobilized antithrombin III followed by gel filtration on Sephadex G-100. Eighteen fractions were obtained ranging in molecular weight from 9,700 to 34,300 as determined by sedimentation equilibrium. The binding stoichiometries of antithrombin III and thrombin interactions with the heparin of these fractions were measured, using changes in intrinsic and extrinsic fluorescence. Catalytic activity also was measured for each of the heparin fractions. As the molecular weight of heparin varied from about 10,000 to 30,000, the average number of antithrombin and thrombin sites/heparin molecule varied from 1.0 to 2.1 and 2.4 to 6.8. In addition, the molar specific activity increased 5.7-fold, an increase which correlated directly with the product of the number of antithrombin III and thrombin molecules bound. Thus as the number of bound molecules increased with increased molecular weight, the rate of reaction/bound antithrombin III increased in proportion to the number of bound thrombin molecules and vice versa. This can be explained by assuming that heparin functions as a template for both proteins, that all bound thrombin and antithrombin III molecules are accessible to each other, and that the rate at which a bound molecule reacts is proportional to the number of molecules of its interacting counterpart bound. These observations and conclusions are similar to those of Hoylaerts et al. (Hoylaerts, M., Owen, W. G., and Collen, D. (1984) J. Biol. Chem. 259, 5670-5677), who demonstrated that the rate at which single molecules of antithrombin III, covalently attached to heparin, react increases as the thrombin binding capacity (chain length) of heparin increases.     

Calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction by decreasing the apparent binding affinity of heparin for thrombin

The present study has shown that calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction. The initial rate of thrombin (4.0 nM) inhibition by antithrombin III (200 nM) in the presence of heparin (2.5 ng/ml) decreased from 3.6 nM/min (in the absence of calcium) to 0.12 nM/min in the presence of 10 mM calcium. In the absence of heparin, the initial rate of thrombin inhibition by antithrombin III was not affected by calcium. The heparin-catalyzed antithrombin III/thrombin reaction is described by the general rate equation for a random-order, bireactant, enzyme-catalyzed reaction (M. J. Griffith (1982) J. Biol. Chem. 257, 13899-13902). As such, the reaction is saturable with respect to both thrombin and antithrombin III. The apparent kinetic parameters for the heparin-catalyzed antithrombin III/thrombin reaction were determined in the presence and absence of calcium. The apparent heparin/antithrombin III dissociation constant values were not measurably different in the presence of 0, 1.0, and 3.0 mM calcium. The apparent heparin/thrombin dissociation constant value increased from 7.0 nM, in the absence of calcium, to 10 and 30 nM in the presence of 1.0 and 3.0 mM calcium, respectively. The maximum reaction velocity, at saturation with respect to both proteins, was not affected by calcium. It is concluded that calcium binds to functional groups within the heparin molecule which are required for thrombin binding.     

The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor.

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin.

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.     

Molecular weight analysis of antithrombin III-heparin and antithrombin III-thrombin-heparin complexes

The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.     

Isolation and characterization of thrombomodulin from human placenta

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

Purification and properties of guinea pig antithrombin III

Guinea pig antithrombin III has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography. The final product was homogeneous as judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of antithrombin III was determined by progressive inactivation of thrombin coagulant and amidolytic activity. Heparin cofactor activity was demonstrated by immediate inactivation of thrombin by antithrombin III in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin III occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities of heparin.     

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.     

Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin.

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.     

The catalysis by heparin of the reaction between thrombin and antithrombin

Fluorescence polarization has been used to study the kinetics of the combination of thrombin with antithrombin and its catalysis by the polysaccharide heparin. The heparin-catalysed combination of thrombin and antithrombin is saturable with respect to both thrombin and antithrombin. The rate-determining step of the reaction is approximately 1.7 s-1. The kinetics observed can be explained by proposing that the catalyst of the reaction is not heparin alone but a complex of heparin and antithrombin (bound at the high-affinity site). The temperature dependence of the heparin-catalysed reaction is indistinguishable from that of the uncatalysed reaction. This coincidence is consistent with the rate-limiting step being the same in both cases.     

Kinetic analysis of the role of zinc in the interaction of domain 5 of high-molecular weight kininogen (HK) with heparin

Previous investigations have shown that HK and its light chain bind heparin, preventing the enhancement of antithrombin inhibition of thrombin and potentiating the inhibition of plasma kallikrein by antithrombin. We found that both HK and HKa bound heparin, but HK exhibited a greater affinity. We therefore localized the binding sites for heparin on HK. HK domains 5 and 6 of the light chain as well as domain 3 from the heavy chain, expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli, were tested for binding to immobilized heparin by surface plasmon resonance using a BiaCore 2000 instrument. GST-D5, but not GST-D3, GST-D6, or GST, bound to heparin when the recombinant domains were present at a concentration of 70 nM. To localize more precisely the amino acid sequences on D5, both of the subdomains, histidine-glycine-rich GST-(K420-D474) and histidine-glycine-lysine-rich GST-(H475-S626), were expressed and tested for binding to immobilized heparin. The K(d) was much lower for GST-(K420-D474) than for GST-(H475-S626) in the presence or absence of Zn(2+). GST-(K420-D474) was effective in decreasing the rate of inactivation of thrombin by antithrombin in the presence of heparin and Zn(2+), while GST-(H475-S626) had no effect. We conclude that the binding of heparin to HK is a complex function of Zn(2+) interacting with histidines in the sequence K420-D474 to create high-affinity binding sites. HK has the potential to be an important modulator of heparin therapy.