Hydropathic interaction analyses of small organic activators binding to antithrombin

Recently we designed the first small organic ligands, sulfated flavanoids and flavonoids, that act as activators of antithrombin for accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade [Gunnarsson and Desai, Bioorg. Med. Chem. Lett. (2003) 13:579]. To better understand the binding properties of these activators at a molecular level, we have utilized computerized hydropathic interaction (HINT) analyses of the sulfated molecules interacting in two plausible electropositive regions, the pentasaccharide- and extended heparin-binding sites, of antithrombin in its native and activated forms. HINT analyses indicate favorable multi-point interactions of the activators in both binding sites of the two forms of antithrombin. Yet, HINT predicts better interaction of most activators, except for (-)-catechin sulfate, with the activated form of antithrombin than with the native form supporting the observation in solution that these molecules function as activators of the inhibitor. Further, whereas (+)-catechin sulfate recognized the activated form of antithrombin better in both the pentasaccharide- and extended heparin- binding sites, the native form was better recognized by (-)-catechin sulfate, thus explaining its weaker binding and activation potential in solution. A reasonable linear correlation between the overall HINT score and the solution free energy of binding of the sulfated activators was evident. This investigation indicates that HINT is a useful tool in understanding interactions of antithrombin with small sulfated organic ligands at a molecular level, has some good predictive properties, and is likely to be useful for rational design purposes.     

Synthesis of per-sulfated flavonoids using 2,2,2-trichloro ethyl protecting group and their factor Xa inhibition potential

The synthesis of per-sulfated flavonoids, organic compounds with multiple sulfate groups, is challenging. We present here a two-step synthesis of fully sulfated flavonoids in high overall yields using the 2,2,2-trichloroethyl moiety as a protecting group. The two-step synthesis results in exclusive formation of the per-sulfated product in contrast to common sulfating agents that yield differentially sulfated mixture of compounds. Most per-sulfated flavonoids studied are activators of antithrombin for accelerated inhibition of factor Xa, a key enzyme of the blood coagulation cascade. As a group the per-sulfated flavonoids possess a range of factor Xa inhibition potential.     

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.     

Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?

We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.     

Conformational equilibrium of the reactive center loop of antithrombin examined by steady state and time-resolved fluorescence measurements: consequences for the mechanism of factor Xa inhibition by antithrombin-heparin complexes

Activation of antithrombin by high-affinity heparin as an inhibitor of factor Xa has been ascribed to an allosteric switch between two conformations of the reactive center loop. However, we have previously shown that other, weaker binding, charged polysaccharides can give intermediate degrees of activation [Gettins, P. G. W., et al. (1993) Biochemistry 32, 8385-8389]. To examine whether such intermediate activation results from different reactive center loop conformations or, more simply, from a different equilibrium constant between the same two extreme conformations, we have used NBD covalently bound at the P1 position of an engineered R393C variant of antithrombin as a fluorescent reporter group and measured fluorescence lifetimes of the label in free antithrombin as well as in antithrombin saturated with long-chain high-affinity heparin, high-affinity heparin pentasaccharide, long-chain low-affinity heparin, and dextran sulfate. Steady state emission spectra, anisotropies, and dynamic quenching measurements were also recorded. We found that the large steady state fluorescence enhancements produced by binding of activators resulted from relief of a static quench of fluorescence of NBD in approximately 50% of the labeled antithrombin molecules rather than from any large change in lifetimes, and that similar lifetimes were found for NBD in all activated antithrombin-oligosaccharide complexes. Similar anisotropies and positions of the NBD emission maxima were also found in the absence and presence of activators. In addition, NBD was accessible to quenching agents in both the absence and presence of activators, with an at most 2-fold increase in quenching constants between these two extremes. The simplest interpretation of the partial static quench in the absence of activators, the different degrees of enhancement by different antithrombin activators, and the similar fluorescence properties and quenching behavior of the different states is that there are two distinct types of conformational equilibrium involving three distinct states of antithrombin, which we designate A, A', and B. A and A' represent low-affinity or inactive states of approximately equal energy, both having the hinge residues inserted into beta-sheet A. A is fluorescent, while A' is statically quenched. State B represents the activated loop-expelled conformation in which none of the NBD fluorophores are statically quenched, as a result of the loop, including the P1-NBD, moving away from the body of the antithrombin. Different activators are able to shift the equilibrium to the high-activity (B) state to different extents and hence give different degrees of measured activity, and different degrees of relief of static quench. The similar properties and accessibility of the NBD in the A and B conformations also indicate that the P1 side chain is not buried in the low-activity A conformation, suggesting that an earlier proposal that activation involves exposure of the P1 side chain cannot be the explanation for activation. As an alternative explanation, heparin activation may give access to an exosite on antithrombin for binding to factor Xa and hence be the principal basis for enhancement of the rate of inhibition.     

The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure.

Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC extraction, poly(ethylene glycol) precipitation, and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000-fold relative to the starting plasma. The apparent molecular weight of the purified factor V was 439,000 +/- 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 239,000; alpha-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.     

Mechanism of catalysis of inhibition of factor IXa by antithrombin in the presence of heparin or pentasaccharide

Because of the homology between factor IXa and factor Xa (f.IXa and f.Xa, respectively), and the critical upstream position of f.IXa in the coagulation cascade, the contribution of the heparin-derived pentasaccharide to antithrombin-mediated inhibition of f.IXa was investigated. Pentasaccharide promotes inhibition of both f.IXa and f.Xa generated in recalcified plasma. This result demonstrates that antithrombin is the predominant inhibitor of f.IXa in plasma, and that the activity of antithrombin is promoted by pentasaccharide. Kinetic experiments reveal that pentasaccharide increases the rates of antithrombin-mediated inhibition of both f.IXa and f.Xa by 2 orders of magnitude. These findings indicate that pentasaccharide-induced conformational changes in antithrombin enhance its capacity to inhibit both f.IXa and f.Xa. In the presence of Ca2+, full-length heparin produces an additional approximately 10-fold increase in the rates of inhibition of both enzymes, consistent with a template role of heparin. Heparin binding to f.Xa was previously shown to be promoted in the presence of Ca2+. Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca2+ compared with its absence. Thus, Ca2+ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa. Furthermore, our data suggest that the efficacy of pentasaccharide for prevention and treatment of thrombotic disorders may reflect its action at two sites in the coagulation system.     

A novel allosteric pathway of thrombin inhibition: Exosite II mediated potent inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988-7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2-3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17-300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.     

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.     

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)     

Heparinoid-active two sulfated polysaccharides isolated from marine green algae Monostroma nitidum

Two sulfated polysaccharides WF1 and WF3 were isolated from marine green algae Monostroma nitidum, and their structural characteristics were determined. Anticoagulant activities of WF1 and WF3 were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), antithrombin and anticoagulation factor Xa activities. The results showed that WF1 and WF3 had similar high contents of rhamnose, whereas their sulfate contents, sulfation positions, molecular sizes and linkage patterns of rhamnose residues were different. The bioassay results demonstrated that WF1 and WF3 had high anticoagulant activities, and were potent thrombin inhibitors mediated by heparin cofactor II, especially WF3. They also hastened thrombin and coagulation factor Xa inhibition by potentiating antithrombin III, but at a lower effectiveness. The differences of anticoagulant activities between WF1 and WF3 were directly due to their structural features discrepancy.     

Importance of the P2 glycine of antithrombin in target proteinase specificity, heparin activation, and the efficiency of proteinase trapping as revealed by a P2 Gly --> Pro mutation.

A sequence-specific heparin pentasaccharide activates the serpin, antithrombin, to inhibit factor Xa through an allosteric mechanism, whereas full-length heparin chains containing this sequence further activate the serpin to inhibit thrombin by an alternative bridging mechanism. To test whether the factor Xa specificity of allosterically activated antithrombin is encoded in the serpin reactive center loop, we mutated the factor Xa-preferred P2 Gly to the thrombin-preferred P2 Pro. Kinetic studies revealed that the mutation maximally enhanced the reactivity of antithrombin with thrombin 15-fold and decreased its reactivity toward factor Xa 2-fold when the serpin was activated by heparin pentasaccharide, thereby transforming antithrombin into an allosterically activated inhibitor of both factor Xa and thrombin. Surprisingly, the enhanced thrombin specificity of the mutant antithrombin was attenuated when a full-length bridging heparin was the activator, due both to a reduced rate of covalent reaction of the mutant serpin and thrombin and preferred reaction of the mutant serpin as a substrate. These results demonstrate that the reactive center loop sequence determines the specificity of allosterically activated antithrombin for factor Xa and that the conformational flexibility of the P2 Gly may be critical for optimal bridging of antithrombin and thrombin by physiologic heparin and for preventing antithrombin from reacting as a substrate in the bridging complex.     

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.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Deletion of P1 arginine in a novel antithrombin variant (antithrombin London) abolishes inhibitory activity but enhances heparin affinity and is associated with early onset thrombosis

A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.     

Serine 380 (P14) --> glutamate mutation activates antithrombin as an inhibitor of factor Xa

Heparin regulates the inhibitory activity of antithrombin. It has been proposed that residues P15 and P14 are expelled from beta-sheet A of antithrombin by heparin binding, permitting better interaction of the reactive center loop with factor Xa. We have made a P14 antithrombin variant (S380E) to create an activated inhibitory form of antithrombin in which P14 is already expelled from beta-sheet A. S380E antithrombin fluorescence is enhanced 35 +/- 5% compared with control antithrombin. There is minimal further increase in antithrombin fluorescence upon heparin binding. The variant has a 5 degrees C lower T(m) than control antithrombin. The variant is an inhibitor of proteinases and has a nearly 200-fold increased basal rate of inhibition of factor Xa, after correction for an increased stoichiometry of inhibition. This is comparable to that of antithrombin activated by high affinity heparin pentasaccharide. Full-length high affinity heparin causes only a 7-fold additional increase in rate and a large increase in stoichiometry of inhibition. In contrast, the basal rate of inhibition of thrombin is similar to that of control antithrombin but is increased 300-fold by heparin. These findings suggest that the native state of the S380E variant exists in a loop-expelled conformation that is consequently highly reactive toward factor Xa.     

Regulation of glycosaminoglycan function by osmotic potentials. Measurement of water transfer during antithrombin activation by heparin.

The sulfated glycosaminoglycan heparin is an important anticoagulant, widely used to treat and to prevent arterial thrombosis. Heparin triggers conformational changes in, and the functional activation of, the serine proteinase inhibitor antithrombin. We investigated water-transfer reactions during the activation process to explore the possibility that functional interaction between antithrombin and sulfated glycosaminoglycans can be regulated by osmotic potentials. Volume of water transferred upon heparin binding was measured from differences in free energy change, Delta(Delta G), with osmotic stress, pi. Osmotic stress was induced with chemically inert probes that are geometrically excluded from the water-permeable spaces of antithrombin and from intermolecular spaces formed during the association reaction. The free energy change, Delta G, for the antithrombin/heparin interaction was calculated from the dissociation constant, determined by functional titrations of heparin with antithrombin at fixed concentrations of the coagulation protease factor Xa. The effect of osmotic stress was independent of the chemical nature of osmotic probes but correlated with their radius up to radius >17 A. In mixtures including a large and a small probe, the effect of the large probe was not modified by the small probe added at a large molar excess. With an osmotic probe of 4-A radius, the Delta(Delta G)/pi slope corresponds to a transfer of 119 +/- 25 water molecules to bulk solution on formation of the complex. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures revealed complex surfaces with smaller hydration volumes in the bound relative to the free conformation. The residue distribution in, and atomic composition of, the pockets containing atoms from residues implicated in heparin binding were distinct in the bound versus free conformer. The results demonstrate that the heparin/antithrombin interaction is linked to net water transfer and, therefore, can be regulated in biological gels by osmotic potentials.     

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.     

Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.     

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.