Regulation of thrombin activity by antithrombin and heparin.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
S T Olson
Publications and source records attributed to S T Olson.
High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: role for antithrombin in the regulation of kallikrein.
The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with heparin on the regulation of kallikrein by the heparin-activated inhibitor, antithrombin, were investigated. H-kininogen, at levels sufficient to fully complex kallikrein, greatly potentiated the acceleration of antithrombin inhibition of kallikrein produced by heparin with high affinity for antithrombin. At I = 0.15, pH 7.4, 25 degrees C, kininogen thus maximally increased the heparin enhancement of the second-order rate constant for the antithrombin-kallikrein reaction from 13-fold (1.6 x 10(2) M-1 s-1 to 2.1 x 10(3) M-1 s-1) to 1200-fold (1.9 x 10(5) M-1 s-1). In contrast, H-kininogen had no effect on the antithrombin-kallikrein reaction in the absence of heparin, nor did the protein enhance the rate constants of 1.7 x 10(4) and 3.4 x 10(4) M-1 s-1 for kallikrein reactions with its primary plasma inhibitors C1-inhibitor and alpha 2-macroglobulin, respectively, in the absence or presence of heparin. Consistent with these results, SDS gel electrophoresis of the 125I-labeled kallikrein-inhibitor complexes formed in a mixture of these kallikrein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha 2-macroglobulin for kallikrein, accounting for 54% of the total kallikrein complexes, only when both heparin and H-kininogen were present. Similarly, the presence of therapeutic levels of heparin (approximately 1 unit/mL) in normal, factor XII-deficient, and prekallikrein-deficient plasmas enhanced the rate of inactivation of added kallikrein by 2.3-fold and significantly altered the partitioning of radiolabeled kallikrein from predominantly C1-inhibitor and alpha 2-macroglobulin complexes (86-92%) to mostly antithrombin complexes (50-53%). Experiments in antithrombin-deficient and H-kininogen-deficient plasmas confirmed that the enhanced kallikrein inactivation rate and predominant formation of antithrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kallikrein inhibition in plasma remained significant (approximately 40-70%) at optimal concentrations of unfractionated or size- and antithrombin affinity-fractionated heparin, in the presence of plasma levels of calcium and zinc ions, at 37 degrees C, and with minimal plasma dilution. These results suggest that antithrombin and H-kininogen may play important roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.
Antithrombins↗
Parallel mechanisms of high molecular weight kininogen action as a cofactor in kallikrein inactivation and prekallikrein activation reactions.
The mechanism by which high molecular weight kininogen (H-kininogen) potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin [Olson, S. T., Sheffer, R., & Francis, A. M. (1993) Biochemistry (preceding paper in this issue)] was investigated at I = 0.15, pH 7.4, 25 degrees C. Single-chain, two-chain, and light-chain, but not heavy-chain, forms of H-kininogen were similarly effective in potentiating the heparin-accelerated antithrombin-kallikrein reaction, indicating that the light-chain region of the protein was responsible for promoting kallikrein inactivation and that cleavage of H-kininogen did not significantly affect this promoting activity. H-kininogen potentiation increased in a saturable manner with increasing kininogen concentration, reflecting a KD (23 +/- 8 nM) similar to that previously measured for H-kininogen binding to kallikrein by equilibrium methods. Both H-kininogen-stimulated and unstimulated heparin rate enhancements initially increased with increasing heparin concentration in a manner corresponding to the saturation of antithrombin with heparin (KD = 10-30 nM). However, at higher heparin concentrations, the stimulated but not the unstimulated heparin rate enhancement decreased in parallel with the saturation of a protein-heparin interaction with a KD (0.4 +/- 0.2 microM) comparable to that directly measured for the H-kininogen-heparin interaction (2.0 +/- 0.2 microM). These results implied that H-kininogen stimulation required the formation of a quaternary complex in which antithrombin and H-kininogen-kallikrein complex were bound to the same heparin chain. In keeping with this interpretation, a synthetic heparin pentasaccharide representing the antithrombin binding sequence accelerated the antithrombin-kallikrein reaction to an extent similar to that of full-length heparin chains containing this sequence, but the pentasaccharide acceleration was not stimulated by H-kininogen. The importance of H-kininogen-kallikrein complex binding to heparin for kininogen stimulation was further indicated from the marked salt dependence of the second-order rate constant for the association of H-kininogen-kallikrein complex but not free kallikrein with antithrombin-heparin complex, under conditions where saturation of the two binary complexes was maintained. Kinetic analyses of antithrombin-kallikrein reactions as a function of the inhibitor concentration indicated that the KD for an initial antithrombin-kallikrein encounter complex was decreased 20-fold by heparin binding to antithrombin and an additional 200-fold by H-kininogen also binding to kallikrein. By contrast, rate constants for the conversion of the encounter complex to a stable complex were comparable for all reactions.(ABSTRACT TRUNCATED AT 400 WORDS)
Heparin↗
Transmission of conformational change from the heparin binding site to the reactive center of antithrombin.
Heparin greatly increases the rates at which antithrombin inhibits target proteinases. An important part of this rate acceleration is a heparin-induced conformational change in antithrombin. To answer the question of whether or not this change is transmitted to the reactive center, we have prepared a recombinant P1 mutant of antithrombin, R393C, labeled the cysteine with nitrobenzofuran (NBD) fluorophore, and examined the perturbation of NBD fluorescence intensity as a function of bound sulfated oligosaccharide. Two high-affinity heparins, low-affinity heparin, and dextran sulfate were used. We found (i) that binding to antithrombin of all these oligosaccharides resulted in transmission of conformational change to P1 in the reactive center, (ii) that these oligosaccharides all gave enhancements of the rate of inhibition of factor Xa beyond any contribution from surface approximation, and (iii) that the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approximation.
Antithrombins↗
Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.
Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.
Amino Acid Sequence↗
Surface-independent acceleration of factor XII activation by zinc ions. I. Kinetic characterization of the metal ion rate enhancement.
The effect of zinc ions (Zn(II)) on the activation of factor XII in the absence of a procoagulant surface was investigated by initial velocity kinetic studies at I = 0.15, pH 7.4, and 25 degrees C. Zinc ions at concentrations greater than 160 microM potentiated 99-fold the kcat/KM for the activation of factor XII by kallikrein and, at an optimum concentration of 110 microM, accelerated 140-fold the apparent kcat/KM for factor XII autoactivation. High molecular weight kininogen had no effect on either metal-potentiated reaction. Analysis of the factor XII concentration dependence of initial activation rates revealed that Zn(II), at levels that saturate the effect, accelerates kallikrein activation of factor XII by lowering KM (from 52 to 7.3 microM) and raising kcat (from 2.6 to 31 min-1). For the autocatalytic activation reaction of factor XII in the presence of optimal Zn(II), apparent KM and kcat values of 2.4 microM and 0.041 min-1, respectively, were determined, but these parameters were not resolvable in the absence of the metal ion. Zinc ions minimally affected kallikrein enzymatic activity and inhibited factor XIIa enzymatic activity with KI values of 20-40 microM, suggesting that the rate-enhancing effects of the metal ion are due to interactions with the substrate (factor XII) rather than with the enzyme. The Zn(II) inhibition of factor XIIa enzymatic activity accounted for a decreased Zn(II) enhancement of factor XII autoactivation at high metal ion concentrations (> 110 microM). The Zn(II) concentration dependence of the acceleration of factor XII activation reactions were sigmoid and characterized by Hill coefficients of 3.3-4.3, suggesting that cooperative binding of at least four zinc ions to factor XII was responsible for the Zn(II) potentiating effect. The Zn(II) enhancement of the rates of factor XII activation decreased both above and below pH 7.4 with midpoint pH values of 6.5-7.0 and 8.0, consistent with histidine and possibly water ligands mediating Zn(II) binding to the protein. Despite an apparent weaker binding of Zn(II) to factor XII at pH 6.5, indistinguishable maximum accelerating effects of the metal ion were observed at saturation at this pH, indicating that the increased positive charge of factor XII resulting from protonation at the lower pH did not mimic the effect of Zn(II) binding. These results imply that zinc ions induce a conformational change in factor XII that makes it a better substrate for its enzyme activators.
Factor XII↗
Surface-independent acceleration of factor XII activation by zinc ions. II. Direct binding and fluorescence studies.
To determine the role of Zn(II)-factor XII interactions in the rate-enhancing effect of Zn(II) on factor XII activation demonstrated in the preceding paper, equilibrium binding of zinc ions to factor XII, and the spectroscopic changes accompanying this binding were investigated. Equilibrium dialysis provided direct evidence for the binding of Zn(II) to factor XII. The binding data were consistent with 7.8 +/- 0.3 zinc ions binding with an indistinguishable Kd of 91 +/- 6 microM. Binding of Zn(II) was accompanied by a 10% quenching of the intrinsic protein fluorescence and a 2-nm red shift of the wavelength of maximum emission. These spectroscopic changes were specific for factor XII and were not observed with factor XIIa. The Zn(II) concentration dependence of factor XII fluorescence quenching was sigmoid and paralleled the Zn(II)-accelerating effect of factor XII activation by kallikrein and factor XIIa, indicating that the spectral change was reporting Zn(II)-factor XII interactions responsible for the enhanced activation rate. The apparent cooperativity of Zn(II) effects on factor XII fluorescence quenching and activation kinetics, and the apparent noncooperativity in Zn(II) binding to factor XII measured by equilibrium dialysis could be explained by a two-state model in which Zn(II) binding is linked to a conformational change in the protein. The Zn(II)-induced quenching of factor XII fluorescence exhibited a pH dependence consistent with the involvement of histidine residues in the binding of Zn(II). Dynamic quenching of factor XII protein fluorescence by iodide or acrylamide, in the absence and presence of Zn(II), revealed heterogeneity in the environment of the 13 tryptophan residues of factor XII that was markedly reduced by metal ion binding. Together, these results indicate that cooperative interactions of Zn(II) with factor XII induce structural changes in the zymogen that facilitate its proteolytic cleavage and activation.
Acrylamide↗
Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin.
Explore the source record for details and available documents.
Antithrombins↗
Kinetic characterization of the substrate reaction between a complex of antithrombin with a synthetic reactive-bond loop tetradecapeptide and four target proteinases of the inhibitor.
A tetradecapeptide corresponding to the P1 to P14 region of the reactive-bond loop of antithrombin (AT) binds to the inhibitor, presumably as a middle strand of the A beta-sheet, thereby converting AT from an inhibitor to a substrate of thrombin (Björk, I., Ylinenjärvi, K., Olson, S.T., and Bock, P. E. (1992) J. Biol. Chem. 267, 1976-1982). The kinetics of cleavage of the AT reactive bond in the AT-peptide complex by four target proteinases were quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. The kcat/Km values for thrombin and factor IXa were indistinguishable from the second-order rate constants for AT inhibition of these enzymes, whereas the values for factor Xa and plasmin were 10-17-fold higher than the inhibition rate constants. Heparin with high affinity for AT accelerated the substrate reaction with thrombin to an extent consistent with the reduced heparin affinity of the AT-peptide complex. These data show that blocking by the peptide of the putative intramolecular association of the P1 to P14 region of the AT reactive-bond loop with the A beta-sheet leads to AT functioning as a substrate of its target enzymes with an efficiency that equals or exceeds the action of uncomplexed AT as an inhibitor and with the expected heparin activation. The results thus suggest that a substrate-like attack of the proteinase on the inhibitor reactive bond in an exposed loop initiates the inhibition reaction. This attack presumably induces the subsequent trapping of the enzyme by the insertion of the reactive-bond loop into the A beta-sheet.
Amino Acid Sequence↗
Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation.
Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd approximately 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd approximately 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proportion of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.
Animals↗
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)
Antithrombin III↗
Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center.
Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically investigate the roles of the reactive center P1 and P1' residues in PAI-1 function, saturation mutagenesis was utilized to construct a library of PAI-1 variants. Examination of 177 unique recombinant proteins indicated that a basic residue was required at P1 for significant inhibitory activity toward uPA, whereas all substitutions except proline were tolerated at P1'. P1Lys variants exhibited lower inhibition rate constants and greater sensitivity to P1' substitutions than P1Arg variants. Alterations at either P1 or P1' generally had a larger effect on the inhibition of tPA. A number of variants that were relatively specific for either uPA or tPA were identified. P1Lys-P1'Ala reacted 40-fold more rapidly with uPA than tPA, whereas P1Lys-P1'Trp showed a 6.5-fold preference for tPA. P1-P1' variants containing additional mutations near the reactive center demonstrated only minor changes in activity, suggesting that specific amino acids in this region do not contribute significantly to PAI-1 function. These findings have important implications for the role of reactive center residues in determining serine protease inhibitor (serpin) function and target specificity.
Amino Acid Sequence↗
Conversion of antithrombin from an inhibitor of thrombin to a substrate with reduced heparin affinity and enhanced conformational stability by binding of a tetradecapeptide corresponding to the P1 to P14 region of the putative reactive bond loop of the inhibitor.
A synthetic tetradecapeptide having the sequence of the region of the antithrombin chain amino-terminal to the reactive bond, i.e. comprising residues P1 to P14, was shown to form a tight equimolar complex with antithrombin. A similar complex has previously been demonstrated between alpha 1-proteinase inhibitor and the analogous peptide of this inhibitor (Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R. and Laurell, C.-B. (1990) Eur. J. Biochem. 194, 51-56). The antithrombin-peptide complex had a conformation similar to that of reactive bond-cleaved antithrombin and, like the cleaved inhibitor, also had a higher conformational stability and lower heparin affinity than intact antithrombin. These properties suggest that the peptide bound to intact antithrombin at the same site that the P1 to P14 segment of the inhibitor occupies in reactive-bond-cleaved antithrombin, i.e. was incorporated as a sixth strand in the middle of the major beta-sheet, the A sheet. The extent of complex formation was reduced in the presence of heparin with high affinity for antithrombin, which is consistent with heparin binding and peptide incorporation being linked. Antithrombin in the complex with the tetradecapeptide had lost its ability to inactivate thrombin, but the reactive bond of the inhibitor was cleaved as in a normal substrate. These observations suggest a model, analogous to that proposed for alpha 1-proteinase inhibitor (Engh, R.A., Wright, H.T., and Huber, R. (1990) Protein Eng. 3, 469-477) for the structure of intact antithrombin, in which the A sheet contains only five strands and the P1 to P14 segment of the chain forms part of an exposed loop of the protein. The results further support a reaction model for serpins in which partial insertion of this loop into the A sheet is required for trapping of a proteinase in a stable complex, and complete insertion is responsible for the conformational change accompanying cleavage of the reactive bond of the inhibitor.
Amino Acid Sequence↗
Role of protein conformational changes, surface approximation and protein cofactors in heparin-accelerated antithrombin-proteinase reactions.
Explore the source record for details and available documents.
Animals↗
Parallel procoagulant and anticoagulant pathways for high molecular weight kininogen coagulant function.
High molecular weight kininogen (HK) or its procoagulant light-chain but not the heavy chain potentiated the heparin enhancement of antithrombin III inactivation of plasma kallikrein and factor XIa from 10-50-fold to approximately 1000-fold at I 0.15, pH 7.4, 25 degrees C. This potentiation resulted in antithrombin becoming a predominant inhibitor of kallikrein and factor XIa in heparinized normal but not HK-deficient plasmas. The heparin chain-length and salt dependence of this potentiation suggested an anticoagulant action of HK analogous to its procoagulant action.
Antithrombin III↗
Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations.
Papain was labelled by attachment of the fluorescent groups 2-(4'-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.
Animals↗
Quantitative evaluation of solution equilibrium binding interactions by affinity partitioning: application to specific and nonspecific protein-heparin interactions.
A variation of the quantitative affinity chromatography (QAC) method of Winzor, Chaiken, and co-workers for the analysis of protein-ligand interactions has been developed and used to characterize sequence-specific and nonspecific protein-heparin interactions relevant to blood coagulation. The method allows quantitation of the binding of two components, A and B, from the competitive effect of one component, B, on the partitioning of the other component, A, between an immobilized acceptor phase and solution phase at equilibrium. Under the conditions employed, the differences in total A concentrations yielding an equivalent degree of saturation of the immobilized acceptor in the absence and presence of B defines the concentration of A bound to B in solution, thereby enabling conventional Scatchard or nonlinear least-squares analysis of the A-B equilibrium interaction. Like the QAC method, quantitation of the competitor interaction does not depend on the nature of the affinity matrix interaction, which need only be described empirically. The additional advantage of the difference method is that only the total rather than the free competitor ligand concentration need be known. The method requires that the partitioning component A be univalent, but allows for multivalency in the competitor, B, and can in principle be used to study binding interactions involving nonidentical, interacting, or nonspecific overlapping sites. Both the binding constant and the stoichiometry for the specific antithrombin-heparin interaction as well as the apparent binding constant for the nonspecific thrombin-heparin interaction at low thrombin binding densities obtained using this technique were in excellent agreement with values determined using spectroscopic probes.
Antithrombin III↗
Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific binding models.
Equilibrium binding of human alpha-thrombin to heparin was investigated at pH 7.4 as a function of thrombin and heparin concentrations, NaCl concentration, temperature, and heparin chain length with the extrinsic fluorescence probe, p-aminobenzamidine, or by quantitative affinity chromatography, in order to distinguish between sequence-specific and nonspecific electrostatic modes of binding. Analysis of binding data by a nonspecific binding model developed for protein-nucleic acid interactions, or by the discrete binding site model previously used to analyze the thrombin-heparin interaction, indicated that both models described the binding interaction equally well over the range of thrombin binding densities accessible to measurement. However, the strong dependence of the thrombin-heparin binding interaction on NaCl concentration, its minimal dependence on temperature, and the increase in apparent binding affinity with increasing heparin oligosaccharide chain length were best accounted for by a nonspecific electrostatic association of thrombin with 5 to 6 anionic residues contained in a 3-disaccharide binding site of heparin. This interaction was characterized by an intrinsic dissociation constant (KD,obs) of 6-10 microM at physiological ionic strength. Although the nonspecific binding model satisfactorily described the binding of thrombin to heparin chains ranging in size from 3 to approximately 13 disaccharides in terms of a single intrinsic KD,obs, deviations from this model were apparent with longer heparin chains (approximately 22 to approximately 35 disaccharides) from a progressive decrease in the intrinsic KD,obs of up to 4-fold. Sedimentation equilibrium analyses of thrombin-heparin complexes suggested a second weaker binding site on thrombin for heparin, which accounted for these deviations as well as the observed insolubility of thrombin-heparin complexes at high thrombin binding densities.
Binding Sites↗