Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation

We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.     

Interaction of factor Xa with heparin does not contribute to the inhibition of factor Xa by antithrombin III-heparin

Factor Xa modified by reductive methylation (greater than 92%) loses the capacity to bind heparin as determined both by gel chromatography and by sedimentation equilibrium ultracentrifugation. The kinetic properties of methylated factor Xa differ, with respect to KM and Vmax for a synthetic tripeptide substrate and for antithrombin III inhibition rate constants, from those of the unmodified enzyme. The 10,000-fold rate enhancement elicited by the addition of heparin to the antithrombin III inhibition reaction, however, is the same. The observed second-order rate constants (k"obs) for antithrombin III inhibition of factor Xa and methylated factor Xa are 3000 and 340 M-1 s-1, respectively, whereas k"obs values for the inhibition of factor Xa or methylated factor Xa with antithrombin III-heparin are 4 X 10(7) and 3 X 10(6) M-1 s-1, respectively. These findings provide direct evidence that the interaction of factor Xa with heparin is not involved in the heparin-enhanced inhibition of this enzyme.     

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.     

The effects of phospholipid and factor Va on the inhibition of factor Xa by antithrombin III

The rate of inhibition of Factor Xa by antithrombin III was found to be influenced by either phospholipid or Factor Va combined with phospholipid. Our results confirmed that Factor Va and phospholipid could protect Factor Xa from inhibition. However, when antithrombin III levels were extrapolated to infinity, the protective effect of lipid and Factor Va were eliminated and the rate of inhibition was accelerated. This indicated that the protective effect that was observed at low antithrombin III concentrations in the presence of phospholipid and Factor Va was due to inhibition of binding of the inhibitor to Factor Xa.     

S protein modulates the heparin-catalyzed inhibition of thrombin by antithrombin III. Evidence for a direct interaction of S protein with heparin

The interference of S protein with the heparin-catalyzed inhibition of thrombin by antithrombin III was studied in a purified system and in plasma. The effect of S protein to counteract heparin activity was documented by kinetic analysis of the initial phase of the inhibition reaction. Addition of S protein induced a concentration-dependent reduction of the inhibition rate, reflected in a decrease of the apparent pseudo-first-order rate constant by a factor of 5-8 in the presence of a twofold molar excess of S protein over antithrombin III. A non-competitive interaction of S protein with the thrombin--antithrombin-III--heparin inhibition reaction with Ki = 0.6 microM was found. While the association constant of thrombin--antithrombin III in the presence of 0.05 U/ml heparin amounted to 2.5 X 10(8) M-1, an approximately 200-fold decrease of this value was observed in the presence of S protein. The fast formation of the covalent complex between thrombin and antithrombin III in the presence of heparin was impaired as a result of the presence of S protein, as was shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the absence of heparin the inhibition of thrombin by antithrombin III alone was not influenced by S protein. The heparin-counteracting activity of S protein was found to be mainly expressed in the range of 0.01-0.1 U/ml heparin, thereby shifting the point of 50% inhibition of thrombin from 0.003 U/ml to 0.1 U/ml heparin with a second-order rate constant of k2 = 1.4 X 10(6) M-1. A direct interaction of S protein with heparin was demonstrated by crossed immunoelectrophoresis with purified proteins as well as in plasma and serum. The analysis of plasma and serum by crossed immunoelectrophoresis against rabbit anti-(human S protein) serum revealed an additional cathodal peak in the serum sample, resulting from the interaction of S protein with serum components. These findings not only indicate a direct interaction of S protein with heparin in the onset of the inhibition of thrombin by antithrombin-III--heparin, but also a contribution of S protein during enzyme-inhibitor complex formation.     

Identification of basic residues in the heparin-binding exosite of factor Xa critical for heparin and factor Va binding

We recently demonstrated that a template mechanism makes a significant contribution to the heparin-accelerated inactivation of factor Xa (FXa) by antithrombin at physiologic Ca(2+), suggesting that FXa has a potential heparin-binding site. Structural data indicate that 7 of the 11 basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in FXa. These residues, Arg(93), Lys(96), Arg(125), Arg(165), Lys(169), Lys(236), and Arg(240) were substituted with Ala in separate constructs in Gla domainless forms. It was found that all derivatives cleave Spectrozyme FXa with similar catalytic efficiencies. Antithrombin inactivated FXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of heparin, however, k(2) with certain mutants were impaired up to 25-fold. Moreover, these mutants bound to heparin-Sepharose with lower affinities. Heparin concentration dependence of the inactivation revealed that only the template portion of the cofactor effect of heparin was affected by the mutagenesis. The order of importance of these residues for binding heparin was as follows: Arg(240) > Lys(236) > Lys(169) > Arg(165) > Lys(96) > Arg(93) >/= Arg(125). Interestingly, further study suggested that certain basic residues of this site, particularly Arg(165) and Lys(169), play key roles in factor Va and/or prothrombin recognition by FXa in prothrombinase.     

The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type.

The effect of various well-characterized heparin preparations on the inactivation of human Factor XIa by human antithrombin III was studied. The heparin preparations used were unfractionated heparin and four heparin fractions obtained after anion-exchange chromatography. Inactivation of Factor XIa was monitored with S2366 as chromogenic substrate and followed pseudo-first-order reaction kinetics under all reaction conditions tested. Enhancement of the rate of inhibition of Factor XIa in the presence of unfractionated heparin correlated to the binding of antithrombin III to heparin. From the kinetic data a binding constant of 0.1 microM was inferred. The maximum rate enhancement, achieved at saturating heparin concentrations, was 30-fold. The rate enhancement achieved in the presence of each of the heparin fractions could also be correlated to the binding of antithrombin III to the heparin. The binding constant inferred from the kinetic data varied from 0.10 to 0.28 microM and the number of binding sites for antithrombin III varied from 0.06 to 0.74 site per heparin molecule. The maximum rate enhancements, achieved at saturating heparin concentrations, were strongly dependent on the type of heparin used and varied from 7-fold for fraction A to 41-fold for fraction D. Therefore, although the stimulation of Factor XIa inactivation by antithrombin III could be quantitatively correlated to the binding of antithrombin III to heparin, the heparin-catalysed inhibition of Factor XIa is dependent not only upon the degree of binding of antithrombin III to heparin but also upon the type of heparin to which antithrombin III is bound.     

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin.

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin's heparin-binding domain inhibited both beta(1) and non-beta(1) integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins alpha-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.     

Probing the heparin-binding domain of human antithrombin III with V8 protease

From structural analysis on genetically abnormal and chemically modified human antithrombin III [Koide, T., Odani, S., Takahashi, K., Ono, T. and Sakuragawa, N. (1984) Proc. Natl Acad. Sci. USA 81, 289-293; Chang, J.-Y. and Tran, T. H., (1986) J. Biol. Chem. 261, 1174-1176; Blackburn, M. N., Smith, R. L., Carson, J. and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941], the heparin-binding site of antithrombin III has been suggested to be in the region of Pro-41, Arg-47 and Trp-49. In this study the heparin-binding site was probed by preferential cleavage of V8 protease on heparin-treated and non-treated native antithrombin III. The study has been based on the presumption that the heparin-binding site of antithrombin III is situated at exposed surface domain and may be preferentially attacked during limited proteolytic digestion. Partially digested antithrombin III samples were monitored by quantitative amino-terminal analysis and amino acid sequencing to identify the preferential cleavage sites. 1-h-digested antithrombin III was separated on HPLC and peptide fragments were isolated and characterized both qualitatively and quantitatively. The results reveal that Glu-Gly (residues 34-35), Glu-Ala (residues 42-43) and Glu-Leu (residues 50-51) are three preferential cleavage sites for V8 protease and their cleavage, especially the Glu-Ala and the Glu-Leu sites, was drastically inhibited when antithrombin III was preincubated with heparin. Both high-affinity and low-affinity antithrombin-III-binding heparins were shown to inhibit the V8 protease digestion of native antithrombin III, but the high-affinity sample exhibited a higher inhibition activity than the low-affinity heparin. These findings (a) imply that the segment containing residues 34-51 is among the most exposed region of native antithrombin III and (b) support the previous conclusions that this region may play a pivotal role in the heparin binding.     

Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.     

Tryptophan residue at the heparin binding site in antithrombin III

Chemical modifications have demonstrated that the ultraviolet difference spectrum produced when heparin interacts with antithrombin III is due primarily to changes in the tryptophan environment. This is based on the observation that this spectrum could be abolished by treatment of antithrombin III with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide but not with tetranitromethane. The tryptophan-modified antithrombin III is still capable of binding to thrombin even when it has lost 85% of heparin cofactor activity. A marked decrease in reactivity of tryptophan residues is observed when modification is carried out in the presence of heparin. Evidence is presented that tryptophan is in the heparin binding site.     

Refolding properties of antithrombin III. Mechanism of binding to heparin

The presence of two unfolding domains in antithrombin III during its denaturation in guanidinium chloride has previously been reported (Villanueva, G. B., and Allen, N. (1983) J. Biol. Chem. 258, 11010-11013). In the present work, we report the results of refolding studies on antithrombin III. Circular dichroism and intrinsic fluorescence studies have demonstrated that the first unfolding domain of low stability (midpoint at 0.7 M guanidinium chloride) is irreversible upon renaturation, whereas the second unfolding domain (midpoint at 2.3 M guanidinium chloride) is reversible. The intermediate form of antithrombin III, termed AT-IIIR, which has lost the structural features of the first domain was investigated. Clotting assays and electrophoretic analyses showed that AT-IIIR had lost 60% of heparin cofactor activity but was still capable of forming sodium dodecyl sulfate-stable complexes with thrombin. Although certain regions of this molecule do not refold to the conformation of native antithrombin III, the tryptophan residues refold to a conformation identical with the native state. This was demonstrated by fluorescence quenching, solvent perturbation, and chemical modification studies. However, the tryptophan-ascribed fluorescence enhancement and absorption difference spectrum which occur when heparin binds to antithrombin III are reduced by 70%. On the basis of these data, the binding of heparin to antithrombin III is interpreted in terms of a two-step mechanism. The primary binding occurs in the region without tryptophan and is followed by a secondary conformational rearrangement which affects the tryptophan environment. The mechanism of the binding of heparin and antithrombin III has been previously studied by kinetic methods, and the data also support a two-step mechanism. The agreement of these two studies employing entirely different approaches to the same problem lends support to the validity of this postulated mechanism.     

A peptide model for the heparin binding site of antithrombin III.

A peptide model for the heparin binding site of antithrombin III (ATIII) was synthesized to elucidate the structural consequences of heparin binding. This peptide [ATIII(123-139)] and a sequence-permuted analogue (ATIII random) showed similar conformational behavior (as analyzed by circular dichroism spectroscopy) in aqueous and organic media. In the presence of heparin, however, the peptide ATIII(123-139) assumed a stable conformation, whereas peptide ATIII random did not. Complex formation was saturable and sensitive to salt. The ATIII(123-139)-heparin complex contained beta-structure, rather than helical structure. This finding is incompatible with current models of heparin binding and suggests that heparin binding may induce nonnative structures at the binding site which could, in turn, lead to activation of ATIII. The peptide ATIII(123-139) was able to inhibit the binding of ATIII by heparin, consistent with the notion that this peptide may be a model for the heparin binding site.     

Contribution of monosaccharide residues in heparin binding to antithrombin III

The importance of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide iduronic acid(1)----N-acetylglucosamine 6-O-sulfate(2)----glucuronic acid(3)----N-sulfated glucosamine 3,6-di-O-sulfate(4)----iduronic acid 2-O-sulfate(5)----N-sulfated glucosamine 6-O-sulfate(6)----iduronic acid 2-O-sulfate(7)----anhydromannitol 6-O-sulfate(8) was determined by comparing with synthetic tetra- and penta-saccharides its ability to bind human antithrombin. The octasaccharide had an affinity for antithrombin of 1 X 10(-8) M (10.2 kcal/mol) measured by intrinsic fluorescence enhancement at 6 degrees C. The synthetic pentasaccharide, consisting of residues 2-6, had an affinity of 3 X 10(-8) M (9.6 kcal/mol). The same pentasaccharide, except lacking the 3-O-sulfate on residue 4, had an affinity of 5 X 10(-4) M (4.5 kcal/mol) measured by equilibrium dialysis. The tetrasaccharide, consisting of residues 2-5, bound antithrombin with an affinity of 5 X 10(-6) M (6.8 kcal/mol). The tetrasaccharide, consisting of residues 3-6, had an affinity of 5 X 10(-5) M (5.5 kcal/mol). Since the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 4-5 kcal/mol or a 40-50% loss in binding energy of the pentasaccharide, these two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.     

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.     

Arg-129 plays a specific role in the confirmation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharode sequence of heparin

Small amounts of a variant antithrombin (AT) bearing an Arg-129 to Gln mutation were purified from plasma by means of affinity chromatography on insolubilized herapin at very low ionic strength. As a control, two variant antithrombins, one bearing on Pro-41 to Leu mutation and the other an Arg-47 to His mutation, were purified in the same way. The biochemical characterization of the variants and the kinetic study of thrombin and activated factor X (F Xa) inhibition in the presence of heparin and heparin derivatives suggest that Arg-129 plays a specific role in AT conformation and F Xa inhibition enhancement. Indeed, the purified variant adopted the locked conformation described ,for AT submitted to mild denaturing conditions (Carrell, R.W., Evans, D.Li and Stein, P.E. (1991) Nature 353, 576–578) and resembling the latent form of plasminogen activator inhibitor (PAI) (Mottonen J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geohegan, K.F., Gerard, R.D. and Goldsmith, E.J. (1992) Nature 355, 270–273). Moreover, the mutant AT was partially reactivated by heparin for thrombin inhibition, but did not respond to the specific pentasaccharide domain of heparin for F Xa inhibition.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.     

Interactions between heparin and factor Xa inhibition of prothrombin activation

The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles.