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.     

Interaction of S-protein of complement with thrombin and antithrombin III during coagulation. Protection of thrombin by S-protein from antithrombin III inactivation

S-protein, the inhibitor in plasma of the membrane attack complex of complement, appears to have a second function in coagulation. S-protein during clotting enters into a trimolecular complex with thrombin and antithrombin III (ATIII). Functionally, S-protein in the presence of low concentrations of heparin, protects thrombin from inactivation by ATIII. Complex formation between S-protein and thrombin, and between S-protein, thrombin, and ATIII, was demonstrated by agarose gel electrophoresis and by two-dimensional immunoelectrophoresis of purified proteins and in recalcified, clotted plasma. Formation of the trimolecular S-thrombin-ATIII complex was strictly dependent on the presence of thrombin. No association was detectable between S-protein and ATIII or between S-protein and prothrombin. Heparin was not required for the formation of the bimolecular S-protein-thrombin complex or the trimolecular S-protein-ATIII complex. The protective effect of S-protein on inactivation of thrombin by ATIII was demonstrated in functional assays with purified proteins and in plasma only in the presence of low concentrations of heparin. Thus, S-protein may mediate its effect by scavenging heparin required for ATIII activation. It is suggested that the protection of thrombin by S-protein from inactivation by ATIII may be of physiological importance.     

Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed.     

The molecular-mass dependence of dextran sulfate enhancement of inactivation of thrombin and fibrinogen and on factor Xa neutralization by antithrombin III

To study molecular-mass dependence of dextran sulfate (DS) for interactions with several plasma proteins, a commercial preparation of the sulfated polysaccharide was fractionated by gel filtration chromatography into six subfractions with relatively different molecular masses. Simple two-component systems were available to measure the interactions of the proteins with the subfractions of DS. These were done to determine the rates of time-dependent changes in intrinsic fluorescence of thrombin and fibrinogen, and the enzyme inactivation in the presence of DS. Their interactions were also confirmed in three-component systems, in which the interactions of DS with thrombin and fibrinogen were measured by the displaced binding by FTC-heparin, and DS-enhanced proteolysis by chymotrypsin, respectively. Moreover, the neutralization of factor Xa by antithrombin III (AT III) depended on the molecular mass of DS. All the results obtained indicate that most of the general interactions of thrombin, fibrinogen, and probably AT III increased with increasing molecular mass of DS.     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.     

Heparan sulphate with no affinity for antithrombin III and the control of haemostasis

Heparan sulphate with no affinity for antithrombin III (ATIII) was observed to cause acceleration of the factor Xa:ATIII interaction by 1100-fold (k2, 7 X 10(7) M-1.min-1) and the prothrombinase:ATIII interaction by 2900-fold (k2, 2.5 X 10(7) M-1.min-1). Although high-affinity heparan sulphate catalyzed higher acceleration and at lower concentration, in natural mixtures of the two forms the activity of the no affinity form predominated. Heparan sulphate had no significant effect on the thrombin:ATIII interaction but inhibited its potentiation by heparin (Kd 0.3 microM). From the estimated concentration of heparan sulphate on the endothelial cell surface it is proposed that the non-thrombogenic property of blood vessels is due to the acceleration of the factor Xa or prothrombinase:ATIII interaction by the greater mass of surface-bound heparan sulphate rather than by the much smaller proportion of heparin-like molecules (with high affinity for antithrombin III) which may be present.     

Structure-activity relationship in heparin: A synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity

The structures of the tetrasaccharide (β-D-glucuronic acid)1→4 (N-sulfate-3,6-di-O-sulfate-α-D-glucosamine)1→4(2-O-sulfate-α-L-iduronic acid)1→4(N-sulfate-6-O-sulfate-D-glucosamine) and of the pentasaccharide (N-sulfate-6-O-sulfate-α-D-glucosamine)1→4(β-D-glucuronic acid)1→4(N- sulfate-3,6-di-O-sulfate-α-D-glucosamine)1→4(2-O-sulfate-α-L-iduronic acid)1→4(N-sulfate-6-O-sulfate-D-glucosamine), both prepared for the first time, by chemical synthesis from D-glucose and D-glucosamine, have been confirmed by nuclear magnetic resonance. The synthetic tetrasaccharide neither binds to AT-III nor induces anti-factor Xa activity enhancement of this inhibitor. In contrast, the synthetic pentasaccharide strongly binds to AT-III (Ka : 7.10⁶M^?1) forming an equimolar complex and also enhances the AT-III inhibitory activity towards factor Xa. These results confirm that the synthetic pentasaccharide with the above structure corresponds to the actual minimal sequence required in heparin for binding to AT-III.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

Sequence variation in heparin octasaccharides with high affinity for antithrombin III

We have isolated from nitrous acid cleavage products of heparin two major octasaccharide fragments which bind with high affinity to human antithrombin. Octasaccharide S, with the predominant structure iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid-----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is sensitive to cleavage by Flavobacterium heparinase as well as platelet heparitinase and binds to antithrombin with a dissociation constant of (5-15) X 10(-8) M. Octasaccharide R, with the predominant structure iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is resistant to degradation by both enzymes and binds antithrombin with a dissociation constant of (4-18) X 10(-7) M. The occurrence of a 15-17% replacement of N-sulfated glucosamine 3,6-di-O-sulfate with N-sulfated glucosamine 3-O-sulfate and a 10-12% replacement of iduronic acid with glucuronic acid in both octasaccharides indicates that these substitutions have little or no effect on the binding of the oligosaccharides to the protease inhibitor. When bound to antithrombin, both octasaccharides produce a 40% enhancement in the intrinsic fluorescence of the protease inhibitor and a rate of human factor Xa inhibition of 5 X 10(5) M-1 s-1 as monitored by stopped-flow fluorometry. This suggests that the conformation of antithrombin in the region of the factor Xa binding site is similar when the protease inhibitor is complexed with either octasaccharide.     

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 X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.     

Localization of the disulfide bond in human antithrombin III required for heparin-accelerated thrombin inactivation

Heparin accelerates the rate of inhibition of thrombin by antithrombin III. Reduction of one of the three antithrombin disulfide bonds with dithiothreitol under mild conditions abolishes this rate-enhancing effect without affecting the rate of reaction in the absence of heparin. Alkylation of mildly reduced antithrombin III with [³H]iodacetic acid followed by digestion with cyanogen bromide yielded two major labeled peptides. The smaller peptide, containing Cys-422, was identified as extending from Gly-414 to the C-terminus, Lys-424. Our data are consistent with the larger labeled peptide being the one extending from Glu-104 to Met-243 and containing Cys-239. Cys-422 has been shown by others to be linked to Cys-239. These data indicate that the sensitive disulfide bond in antithrombin III extends between Cys-239 and Cys-422; the site at which thrombin cleaves the antithrombin III is between these two half-cystines.     

Effect of polyanetholesulphonic acid and xylan sulphate on antithrombin III activity.

Both polyanetholesulphonic acid and xylan sulphate prolonged the partial thromboplastin clotting time of plasma. The anticoagulant effect of both compounds was reduced following pre-incubation of plasma with antiserum specific for antithrombin III. Polyanetholesulphonic acid was more effective than xylan sulphate in inhibiting thrombin-initiated clotting of plasma, and potentiated antithrombin III inhibition of both thrombin and Xa. Xylan sulphate was more effective in potentiating antithrombin III inhibition of Xa than of thrombin. These differential effects of xylan sulphate on different blood serine proteases are discussed in terms of the antithrombin III-mediated anticoagulant activity of heparin.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

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.     

Elimination of P1 arginine 393 interaction with underlying glutamic acid 255 partially activates antithrombin III for thrombin inhibition but not factor Xa inhibition

The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu(255) on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg(393)-Glu(255) hydrogen bond and ionic interactions were eliminated by converting Glu(255) to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu(255)-mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wild-type parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000- and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although "approximation" is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all.     

Mechanisms responsible for catalysis of the inhibition of factor Xa or thrombin by antithrombin using a covalent antithrombin-heparin complex

Covalent antithrombin-heparin (ATH) complexes, formed spontaneously between antithrombin (AT) and unfractionated standard heparin (H), have a potent ability to catalyze the inhibition of factor Xa (or thrombin) by added AT. Although approximately 30% of ATH molecules contain two AT-binding sites on their heparin chains, the secondary site does not solely account for the increased activity of ATH. We studied the possibility that all pentasaccharide AT-binding sequences in ATH may catalyze factor Xa inhibition. Chromatography of ATH on Sepharose-AT resulted in >80% binding of the load. Similar chromatographies of non-covalent AT + H mixtures lead to a lack of binding for AT and fractionation of H into unbound (separate from AT) or bound material. Gradient elution of ATH from Sepharose-AT gave 2 peaks, a peak containing higher affinity material that had greater anti-factor Xa catalytic activity (708 units/mg heparin) compared with the peak containing lower affinity material (112 units/mg). Sepharose-AT chromatography of the ATH component with short heparin chains (相似文献   

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.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin

Heparin (H) was previously shown to accelerate the inactivation of alpha-thrombin (T) by antithrombin III (AT) primarily by promoting the initial binding of thrombin and AT in a ternary T.AT.H complex intermediate without significantly influencing the subsequent product formation step (Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-14895). In the present study, the protein-heparin interactions which contribute to the assembly of the ternary complex intermediate and their linkage were quantitated by equilibrium binding and stopped-flow kinetic studies at pH 7.4, I 0.3, 25 degrees C, using p-aminobenzamidine (P) as a fluorescence probe. Equilibrium binding studies of the AT.H and T.H binary complex interactions monitored by the 40% enhancement in AT fluorescence or the 16-18% quenching of thrombin-bound p-aminobenzamidine fluorescence, respectively, indicated a 100-fold greater affinity of AT for heparin (KAT,H 0.23 microM) as compared to thrombin for heparin (KT,H 35-42 microM). Consistent with this large difference in affinities, rapid kinetic studies indicated that assembly of the ternary complex occurred predominantly as a bimolecular association between the AT.H binary complex and free thrombin. Thus, under pseudo-first order conditions ([AT]o, [H]o much greater than [T]o much less than [P]o), the observed thrombin inactivation rate constant (kobs) exhibited a saturable dependence on [AT]o or [H]o when [H]o much less than KT,H, reflecting a KAT,H (0.25 microM) similar to that directly determined by equilibrium binding. Moreover, competitive inhibition of the reaction by T.H binary complexes was indicated from the hyperbolic decrease in kobs produced by heparin with either high or low affinity for AT or active-site blocked thrombin at concentrations comparable to KT,H. This behavior was consistent with values of KT,H (27-51 microM) similar to those determined directly from equilibrium binding measurements. Comparison of the affinities of the binary protein-heparin interactions with the affinity of thrombin for AT.H complex in the ternary complex measured previously, indicated that the affinity of either protein for heparin was enhanced about 10-fold by the prior binding of the other protein to heparin. This linkage of the protein-heparin interactions implies that the ternary complex will be assembled at thrombin, AT, and heparin concentrations considerably lower than those predicted from previous reaction models which fail to account for this linkage.     

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.