Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins

Abstract

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891–31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80–87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

Antithrombin (AT),³ a plasma glycoprotein and a member of the serpin superfamily of proteins, is a major regulator of the coagulation cascade. Its primary targets are thrombin, factor Xa (fXa), and factor IXa (fIXa) (1). It has also been suggested to inhibit several other coagulation enzymes (2–6), albeit with much weaker inhibitory efficiency. Antithrombin alone is a rather poor inhibitor of factors IIa, Xa, and IXa and requires the presence of heparin to exhibit its full anticoagulant potential.

Heparin is a highly sulfated polysaccharide that greatly enhances the rate of AT inhibition of these enzymes under physiological conditions (1). This acceleration forms the basis for heparin's use as an anticoagulant for the past several decades. Yet heparin is associated with bleeding complications and suffers from a number of other limitations. In addition, the animal origin of the drug is also a cause for concern as suggested by recent incidences of oversulfated chondroitin sulfate contaminating unfractionated heparin (UFH) preparations and resulting in numerous deaths (7–9). Although low molecular weight heparins (LMWHs) are superior to UFH with respect to therapeutic complications, the iatrogenic bleeding risk is not completely eliminated. Likewise, fondaparinux, or the minimal antithrombin binding pentasaccharide sequence (H5), is also associated with bleeding (10, 11) and lacks an effective antidote to reverse excessive anticoagulation.

The major reason for the limitations of UFH and LMWH therapies is the presence of numerous negative charges on each polymeric chain. UFH and LMWH are linear co-polymers of glucosamine and uronic acid residues that are decorated with numerous sulfate groups generating a massive polyanion (12, 13). This polyanion is capable of interacting with a large number of plasma proteins and proteins present on cells lining the vasculature, which likely induce many of the UFH and LMWH complications (14, 15). Fondaparinux displays a much better pharmacological profile primarily because of its limited number of sulfate and carboxylate groups.

To design better anticoagulants that are less polyanionic and more hydrophobic than UFHs and LMWHs, we recently prepared low molecular weight variants of lignin, called sulfated dehydropolymers (DHPs) (Fig. 1), as functional mimetics of heparin. These designed molecules were prepared in a simple two-step chemo-enzymatic process involving enzymatic coupling of 4-hydroxycinnamic acids followed by the chemical sulfation of the resulting DHPs (16). In terms of structural polydispersity and heterogeneity, sulfated DHPs are similar to LMWHs. Sulfated DHPs are composed of many oligomeric chains of varying lengths and contain different inter-monomeric linkages such as β-O-4 and β-5. Yet sulfated DHPs are completely unlike LMWHs with respect to the nature of their backbone. In contrast to the highly anionic scaffold of the heparins, sulfated DHPs possess a highly aromatic scaffold with fewer anionic groups (Fig. 1). In fact, in terms of structure, sulfated DHPs are unlike any other class of anticoagulant investigated to date, including the heparins, the coumarins, the hirudins, the peptidomimetics, and the small molecule direct inhibitors. Functionally, the sulfated DHPs display plasma and blood anticoagulation similar to that of LMWHs (17, 18). Yet mechanistically, the sulfated DHPs were found to exhibit a novel mechanism of anticoagulation involving an exosite II-mediated allosteric inhibition of thrombin (17). The DHPs represent the first example of an exclusive exosite II-dependent inactivation of the catalytic function of thrombin.

FIGURE 1.

A representative structure of sulfated DHPs. CDSO3, FDSO3, and SDSO3 were chemo-enzymatically synthesized in two steps from the corresponding starting 4-hydroxycinnamic acid monomers, caffeic acid (CA), ferulic acid (FA), or sinapic acid (SA). The average molecular mass of CDs, FDs, and SDs was in the range of 3,000–4,000 Da. Two types of common linkages present in sulfated DHPs include the β-O-4 and β-5 linkages (shown as shaded ovals).

In this work we study the interaction of sulfated DHPs with AT at a molecular level. This study reveals that the indirect antithrombin-mediated pathway may contribute to the inhibition efficiency of sulfated DHPs, thus realizing molecules that can utilize both the direct and indirect inhibition pathways. These studies uncover the ability of a specific sulfated DHP to induce potent antithrombin inhibition of free fVIIa. The antithrombin-mediated effects originate from the sulfated DHPs binding to the heparin-binding site (HBS) of the serpin through non-ionic forces that contribute more than 80% of the total binding energy. Our work supports the idea that aromatic scaffolds, which exhibit hydrophobic and hydrophilic nature, can be designed to target the HBS of antithrombin for modulation of its inhibitory functions.

EXPERIMENTAL PROCEDURES

Proteins and Chemicals

Sulfated dehydropolymers CDSO3, FDSO3, and SDSO3 (Fig. 1) were prepared in two steps from 4-hydroxycinnamic acid monomers, caffeic acid, ferulic acid, and sinapic acid as described earlier (16). Human AT and human coagulation factors VIIa, IXa, Xa, and IIa (α-thrombin) were purchased from Hematologic Technologies (Essex Junction, VT). Stock solutions of proteins were prepared in 20 mm sodium phosphate buffer, pH 7.4, containing 100 mm NaCl and 2.5 mm CaCl₂ (AT and thrombin) or 5 mm MES buffer, pH 6.0 (factor Xa). Factor VIIa stock solutions of proteins were prepared in 25 mm HEPES buffer, pH 7.4, containing 100 mm NaCl and 5 mm CaCl₂, and factor IXa stock solutions were prepared in 5 mm MES buffer, pH 5.5, containing 150 mm NaCl. Chromogenic substrates Spectrozyme TH, Spectrozyme FXa, Spectrozyme FIXa, and Spectrozyme FVIIa were purchased from American Diagnostica (Greenwich, CT). Protamine sulfate was obtained from Sigma and used as received. (−)-Epicatechin sulfate was prepared in the laboratory as described previously (19, 20). All other chemicals were analytical reagent grade from either Sigma or Fisher used without further purification.

Direct and Indirect Inhibition of Coagulation Proteinases

Both direct and indirect inhibition of coagulation factors thrombin, fVIIa, fIXa, and fXa by sulfated DHPs was determined through a chromogenic substrate hydrolysis assay (1, 17, 21, 22). For the thrombin and factor Xa assays, a 10-μl sample of the DHP at concentrations ranging from 0.035 to 10,000 μg/ml was diluted with 930 μl of 20 mm Tris-HCl buffer, pH 7.4, containing 100 mm NaCl, 2.5 mm CaCl₂, and 0.1% PEG8000 at room temperature in a 20,000 polyethylene glycol-coated polystyrene cuvette, followed by addition of 10 μl of the proteinase solution to give 4 nm thrombin or fXa. After 10 min of incubation at room temperature, 50 μl of 2 mm chromogenic substrate was added (Spectrozyme FXa for factor Xa and Spectrozyme TH for thrombin) (1, 17). For fIXa, a 10-μl sample of DHP at concentrations ranging from 0.1 to 10,000 μg/ml was diluted with 930 μl of 100 mm HEPES buffer, pH 8, containing 100 mm NaCl and 10 mm CaCl₂ (22). For fVIIa, a 10-μl sample of DHP at concentrations ranging from 1 to 10,000 μg/ml was diluted with 930 μl of 25 mm HEPES buffer, pH 7.4, containing 100 mm NaCl, 5 mm CaCl₂, and 33% ethylene glycol (21). The final concentrations of fIXa and fVIIa in these experiments were 10 and 20 nm, respectively. Following incubation of samples for 10 min, the residual activity of fVIIa or fIXa was determined by adding 50 μl of 2 mm chromogenic substrate Spectrozyme FVIIa or Spectrozyme FIXa, respectively. Thrombin, fXa, and fVIIa assays were run at 25 °C, and fIXa assay was run at 20 °C. The residual enzyme activity was determined from the initial rate of increase in absorbance at 405 nm. Relative residual proteinase activity at each concentration was calculated using the activity measured under otherwise identical conditions, except for the absence of the sulfated DHP. Indirect inhibition of thrombin by sulfated DHPs was performed at a fixed 100 nm concentration of antithrombin, and for indirect inhibition of factors VIIa, IXa, and Xa, a 200 nm concentration of the serpin was used. Except for the presence of AT, the indirect inhibition assay was run in an otherwise identical manner to the direct inhibition assay. Logistic Equation 1 was used to fit the dose dependence of residual proteinase activity to obtain the IC₅₀.

In Equation 1, Y is the ratio of residual proteinase activity in the presence of inhibitor to its absence (fractional residual activity); Y_M and Y_O are the maximum and minimum possible values of the fractional residual proteinase activity; IC₅₀ is the concentration of the inhibitor that results in 50% inhibition of enzyme activity; and HS is the Hill slope. HS does not represent cooperativity because sulfated DHPs are highly complex species that may possess multiple binding modes and geometries. Sigmaplot 8.0 (SPSS, Inc., Chicago) was used to perform nonlinear curve fitting in which Y_M, Y_o, IC₅₀, and HS were allowed to float.

Neutralization of Inhibitory Effects by Protamine Sulfate

To assess the reversibility of inhibitory effects of sulfated low molecular weight lignins, the thrombin inhibition by FDSO3 was measured in the presence of a fixed concentration of protamine sulfate. A 10-μl sample of FDSO3 at concentrations ranging from 0 to 1820 nm was diluted with 100 μl of 10 mg/ml protamine sulfate and 830 μl of 20 mm Tris-HCl buffer, pH 7.4, containing 100 mm NaCl, 2.5 mm CaCl₂, and 0.1% PEG8000 at room temperature in a 20,000 polyethylene glycol-coated polystyrene cuvette, followed by addition of 10 μl of thrombin solution to give 4 nm thrombin. After 10 min of incubation at room temperature, 50 μl of 2 mm Spectrozyme TH was added (1, 17). The residual enzyme activity was calculated from the initial rate of increase in absorbance at 405 nm, which was set to 100% in the absence of FDSO3. The fractional residual thrombin activity at different concentrations of the inhibitor was calculated from the ratio of slope of A₄₀₅ increase in the presence of FDSO3 to that in its absence.

Antithrombin Binding Studies

Antithrombin interaction studies were performed at 25 °C and in 20 mm sodium phosphate buffer, containing 0.1 mm EDTA and 0.1% (w/v) PEG8000, adjusted to either pH 6.0 or 7.4. Antithrombin solutions in pH 6.0 buffer were prepared by fresh dilution from a stock solution at pH 7.4 (>100-fold). No significant losses in inhibitor activity were noted over the time frame of the experiments performed in this study. Fluorescence experiments were performed using a QM4 fluorometer (Photon Technology International, Birmingham, NJ) in pH 7.4, I 0.15, 25 °C buffer. Equilibrium dissociation constants (K_D) for the sulfated DHP-antithrombin complex were determined by titrating the DHP into a solution of plasma antithrombin and monitoring the decrease in the fluorescence at 340 nm (λ_ex = 280 nm). The measured K_D values are apparent affinities of interaction considering that sulfated DHPs are mixtures of species, of which a selected few preferentially bind antithrombin (see below). Sulfated DHPs also absorb at 280 nm and induce small inner filter effects. Inner filter effects were calculated from A₂₈₀ values of sulfated DHPs and the geometry of fluorometer cell using the equation developed by Parker and Barnes (see Ref. 23). These effects were found to be ∼1–3% for CDSO3, 10–20% for FDSO3, and 15–25% for SDSO3. These effects were subtracted from the fluorescence of the complex to obtain the change in fluorescence due to antithrombin binding (ΔF_obs). The slit widths on the excitation and emission side were 1 and 2 mm, respectively. The decrease in fluorescence signal due to the formation of the complex was fit to the quadratic equilibrium binding Equation 2 to obtain the K_D value of interaction. In this equation, ΔF represents the change in fluorescence due to the formation of the complex following each addition of the activator ([DHP]_O) from the initial fluorescence F_O, and ΔF_max represents the maximal change in fluorescence observed on saturation of antithrombin ([AT]_O). A binding stoichiometry of 1:1 was assumed for the DHP-antithrombin interaction.

Salt Dependence of Sulfated DHP-Antithrombin Interaction

The contribution of ionic and non-ionic binding energy to antithrombin-sulfated DHP interaction was determined by measurement of K_D,obs in buffers of varying ionic strength. In the absence of any added NaCl, the ionic strength (I) of the buffer is either 0.025 (pH 6.0) or 0.035 (pH 7.4). Higher ionic strengths were achieved by adding NaCl. The non-ionic contribution to the interaction was obtained from the y intercept of the linear plot of log K_D,obs versus log [Na⁺], according to Equation 3 (24, 25).

In this equation, K_D,_NI is the dissociation constant at [Na⁺] = 1 m, and slope “m” carries information about the number of charge-charge interactions (Z) and the fraction of monovalent counterions released per negative charge following DHP binding to antithrombin (Ψ).

Competitive Binding of Sulfated DHPs to Antithrombin in the Presence of Heparin (UFH), H5, and Epicatechin Sulfate (ECS)

The apparent equilibrium dissociation constant (K_D,app) of the sulfated DHP-antithrombin complex was measured in the presence of fixed concentrations of UFH, H5, and ECS spectrofluorometrically at pH 7.4, I 0.15, 25 °C. The concentration of the competitors chosen for competitive studies corresponded to an antithrombin saturation of 50–95%. The decrease in intrinsic tryptophan fluorescence was analyzed in the same manner as above using Equation 2 to obtain the K_D,app. Assuming the validity of competitive binding, the Dixon-Webb relationship (Equation 4) was used to predict the apparent dissociation constant (K_D,calc) of the sulfated DHP-antithrombin complex.

where K_competitor refers to the equilibrium dissociation constant of the competitor (H, H5, or ECS)-antithrombin complex, which was measured independently under otherwise identical conditions.

Competitive binding studies were also performed by titrating UFH into a solution of sulfated DHP-antithrombin complex at pH 7.4, I 0.15, 25 °C. The increase in intrinsic tryptophan fluorescence due to displacement of sulfated DHP by UFH was used to monitor the competitive process. The concentration of the sulfated DHPs chosen for these competitive studies corresponded to ∼50% saturation of antithrombin. The increase in fluorescence was analyzed using Equation 2 to obtain the K_D,app.

Affinity Capillary Electrophoresis of CDSO3-Antithrombin Interaction

The interaction of CDSO3 with AT was studied using a P/ACE MDQ^TM Beckman capillary electrophoresis system (Fullerton, CA). Electrophoresis was performed at a constant voltage of 8 kV and a constant pressure of 0.1 p.s.i. An uncoated fused silica capillary (inner diameter of 75 μm) of 31.2 cm total length with a 5-mm detection window at 21.0 cm length from the injection point was used. During electrophoresis, the capillary was held at a constant temperature of 25 °C. Sequential washes of 1 m HCl for 10 min, high purity water for 3 min, 1 m NaOH for 10 min, and high purity water for 3 min at 20 p.s.i. were used to activate a new capillary. Before each electrophoretic run, the capillary was rinsed with the run buffer for 3 min at 20 p.s.i. Electrolysis buffers containing antithrombin at the desired concentration (108 or 215 nm) were prepared from a 43 μm stock solution. A 75 μm CDSO3 solution in deionized water was injected at the anodic end using 0.5 p.s.i. pressure for 5 s to give an injection plug of less than 5% of the total capillary volume. Electrophoresis was monitored at the cathodic end by following absorbance at 214 nm.

Virtual Screening of a CDSO3 Library

SYBYL 7.2 (Tripos Associates, St. Louis, MO) was used for molecular visualization, minimization, and manipulation of protein and CDSO3 structures. Molecular modeling experiments were performed on an IRIX 6.5-based SGI Tezro graphical work station. GOLD, version 3.2 (26), was used for docking and scoring experiments. Energy minimization followed the protocol established earlier on sulfated oligosaccharides binding to antithrombin (27) and is described in more detail in the supplemental material. The coordinates for the activated form of antithrombin were extracted from the crystal structure of the antithrombin-thrombin-pentasaccharide ternary complex (Protein Data Bank entry 1TB6). A library of 11 β-5 CDSO3 oligomers, ranging from dimers to pentamers, was prepared using SYBYL 7.2. The structures of the specific 11 oligomers screened in this study are shown in supplemental Fig. S1. The oligomeric structures were docked onto the heparin-binding site of the activated form of antithrombin using GOLD version 3.2 (26) following our earlier established protocol on identifying high affinity and high specificity sequences that recognize the inhibitor (27). The binding site in antithrombin was defined as a collection of all atoms within 20 Å from the C^ζ atom of Phe-121 in the D-helix of antithrombin. This definition of the binding site covers all known important heparin-binding site residues, including Lys-11, Arg-13, Arg-46, Arg-47, Trp-49, Lys-114, Phe-121, Lys-125, Arg-129, Arg-132, Lys-133, and Lys-136. Docking was performed using a genetic algorithmic search with 300,000 iterations. In this search, GOLD starts with a population of 100 arbitrarily docked ligand orientations, evaluates them using the GOLD “fitness” function, and improves their average fitness by an iterative optimization procedure that is biased toward high scores. In this study, we used 10 genetic algorithmic runs for each oligomer to identify the best geometry possible for each structure. The best geometries were scored using the modified form of the GOLD scoring function, as described in our previous report (27). Additional details on virtual screening and scoring experiments are provided as supplemental material.

RESULTS

A Specific Sulfated DHP, SDSO3, Preferentially Inhibits Factors IXa and VIIa in the Presence of Antithrombin

Three sulfated DHPs were synthesized using a chemo-enzymatic process optimized in our laboratory (16). The chemo-enzymatic process involves horseradish peroxidase-catalyzed oxidative coupling of 4-hydroxycinnamic acid monomers followed by sulfation using triethylamine-sulfur trioxide complex to yield the sulfated DHPs. The oxidative coupling reaction involves the free radical-based coupling of monomers to a growing oligomer chain. In this process, several inter-monomeric linkages, including β-O-4, β-5, β-β, and 5–5, are formed, of which the β-O-4 and β-5 linkages are the most common (16, 28, 29). Three 4-hydroxycinnamic acid monomers, caffeic acid, ferulic acid, and sinapic acid, were selected to synthesize CDSO3, FDSO3, and SDSO3, respectively (Fig. 1). The major difference between these monomers is the presence of mono- or di-substitution ortho to the 4-hydroxy group on the aromatic ring. The presence of a single –OH or –OMe group at the 3-position of the aromatic ring allows reactions to occur at the 5-position, thereby generating β-5 inter-monomeric linkage, which is not possible for the 3,5-disubstituted ring present in sinapic acid. Therefore, CDSO3 and FDSO3 are primarily made up of β-O-4 and β-5 linkages, whereas SDSO3 primarily contains β-O-4 linkage. With respect to CDSO3 and FDSO3, the former has the potential to possess a 3-OSO₃⁻ group, whereas the latter has a 3-OMe group, which cannot be sulfated. These structural differences are likely to introduce significant physicochemical and topological differences in the sulfated DHP oligomers.

Previous studies have shown that sulfated DHPs are potent direct inhibitors of factor Xa and thrombin (17). IC₅₀ values in the range of 32–239 nm (fXa) and 20–94 nm (thrombin) were observed (Table 1). With respect to direct inhibition of fIXa, CDSO3 and FDSO3 displayed reasonable inhibitory potency (IC₅₀ = 3.4 and 1.7 μm, respectively), whereas SDSO3 was essentially ineffective (IC₅₀ > 28.5 μm). Likewise, the sulfated DHPs did not directly inhibit fVIIa (IC₅₀ > 23.6 μm, see Table 1) suggesting a high level of selectivity in the direct recognition and inhibition of coagulation enzymes.

TABLE 1.

Physical and enzyme inhibition parameters of sulfated DHPs

	Massa	Chain lengthb	IC50 (nm)c,d
			Thrombin		Factor Xa		Factor IXa		Factor VIIa
			Directe	Indirect	Directe	Indirect	Directe	Indirect	Directe	Indirect
	Da
CDSO3	∼3320	5–13	20	58	32	55	3382	1651	>29,000f	>29,000f
FDSO3	∼4120	8–15	28	73	76	132	1725	1087	>23,640	>23,640
SDSO3	∼3550	4–11	94	46	239	105	>28,500f	1732	>28,500	356

^a Data were taken from Ref. 16.

^b Data were taken from Ref. 17.

^c The IC₅₀ values for direct and indirect inhibition of factors IIa, Xa, IXa, and VIIa were determined at pH 7.4 and at 25 °C in appropriate buffers through spectrophotometric measurement of residual proteinase activity following incubation of the enzyme and the inhibitors for a fixed time period of 10 min (under “Experimental Procedures”).

^d Errors (±1 S.E., not shown here to conserve space) were in the range of 5–15%.

^e Direct inhibition values were compiled from Ref. 17.

^f An estimated value was based on the highest concentration of the anticoagulant used in the experiment.

An important point to address at this time was whether the inhibitory effects observed by these novel molecules are reversible. To assess the reversibility of the phenomenon, we used protamine sulfate, a polycation that is expected to interact with the multiple sulfate and carboxylate groups of inhibitors. Thrombin and FDSO3 were selected as a model coagulation factor and DHP, respectively, to evaluate reversibility. The direct inhibition experiments were performed in a manner described above, except for the presence of protamine sulfate at 1 mg/ml. The fractional residual thrombin activity was measured to be 0.80, 0.88, and 0.81 at 24, 240, and 1820 nm FDSO3 in the presence of protamine sulfate, whereas in the absence of the polycation it was found to be 0.64, 0.22, and 0.21 (see supplemental Fig. S2). The results indicate that thrombin inhibition by FDSO3 is essentially completely reversed in the presence of protamine sulfate.

To assess the influence of antithrombin on the sulfated DHP-mediated direct inhibition of coagulation factors, the inhibition experiments were performed in the presence of a fixed concentration (100 or 200 nm) of the serpin. A sigmoidal decrease in enzyme activity with sulfated DHP concentrations on a semi-log plot was observed, which yielded IC₅₀ values of 55–132 and 46–73 nm against fXa and thrombin, respectively (Fig. 2 and Table 1). A subtle difference in the behavior of the three sulfated DHPs in the presence of antithrombin can be noted. Whereas the IC₅₀ value against fXa increases ∼1.7-fold for CDSO3 and FDSO3 in the presence of antithrombin as compared with the IC₅₀ in its absence, the IC₅₀ value decreases ∼2.3-fold for SDSO3 in the presence of antithrombin. Both direct and indirect inhibition pathways are expected to operate simultaneously, and possibly synchronously, in the presence of antithrombin. Yet it appears that the serpin-mediated pathway is a competing side reaction for CDSO3 and FDSO3, but not for SDSO3.

FIGURE 2.

Direct and indirect inhibition of factor IXa (A) and factor VIIa (B) by SDSO3. The inhibition of factor IXa and factor VIIa by SDSO3 in the presence (open triangles) and absence (closed triangles) of antithrombin was determined spectrophotometrically through a chromogenic substrate hydrolysis assay following incubation of the enzymes with the anticoagulants at pH 7.4 and 20 °C (fIXa) or 25 °C (fVIIa) for 10 min. Indirect inhibition of both enzymes with enoxaparin (closed circles) is shown for comparative purposes. Solid lines represent sigmoidal fits to the data to obtain values of IC₅₀ and Hill slope (Equation 1), as described under “Experimental Procedures.”

In the presence of antithrombin, all three sulfated DHPs displayed reasonably good inhibition of fIXa with IC₅₀ values in the range of 1.1–1.7 μm (Table 1). Interestingly, CDSO3 and FDSO3 inhibit fIXa nearly 1.6–2.0 times better in the presence of the serpin than in its absence, whereas the increase in similar efficiency for SDSO3 is >16-fold. Yet all three sulfated DHPs are much weaker than enoxaparin (IC₅₀ = 18 ± 4 nm; see Fig. 2) in inhibiting fIXa in the presence of antithrombin.

The difference between the three sulfated DHPs becomes more pronounced with the study of fVIIa inhibition in the presence of antithrombin. CDSO3 and FDSO3 did not induce any inhibition (IC₅₀ > 23.6 μm), whereas an IC₅₀ of 356 nm was observed for SDSO3 inhibition of fVIIa in the presence of the serpin (Fig. 2 and Table 1). In comparison, the enoxaparin-antithrombin complex was completely inactive against free fVIIa even at concentrations as high as 222 μm (data not shown). In combination, these results suggest that the presence of antithrombin appears to deplete the inhibition potency of CDSO3 and FDSO3 against factors IIa and Xa, although it significantly enhances the efficiency of SDSO3 inhibiting all four coagulation factors studied, especially factors IXa and VIIa. Alternatively, SDSO3 is a selective and potent inhibitor of free fVIIa in the presence of antithrombin.

Sulfated DHPs Bind Antithrombin with High Affinity

Previously, we have used fluorescence spectroscopy for measuring the affinity of saccharide and non-saccharide ligands for antithrombin. For saccharide ligands such as heparin and heparin pentasaccharide, a 30–40% increase in intrinsic tryptophan fluorescence affords a convenient signal for the determination of the K_D value of the interaction (30, 31). In contrast, the antithrombin affinities of non-saccharide ligands, e.g. small aromatic sulfates such as ECS and CS (19, 32) and larger polymers such as polyacrylic acids (33, 34), have been measured using 6-p-toluidinylnaphthalene-2-sulfonate, an external probe. In this study, we found that the interaction of our synthetic DHPs with antithrombin resulted in a decrease in intrinsic tryptophan fluorescence (Fig. 3). A possible explanation for this decrease could be the inner filter effect. Yet even at low sulfated DHP concentrations, where inner filter effects are minimally present or are nonexistent, a characteristic decrease is observed (see supplemental Fig. S3). Subtraction of inner filter effects due to background absorption shows that the decrease reaches a plateau at high ligand concentrations suggesting a phenomenon of molecular interaction. The reason for the quenching of intrinsic antithrombin fluorescence is not clear at present and requires detailed investigation.

FIGURE 3.

Interaction of sulfated DHPs with antithrombin at pH 7.4, I 0.15, 25 °C. The decrease in intrinsic fluorescence of antithrombin (λ_ex = 280 nm, λ_em = 340 nm) that accompanies binding of CDSO3 (○), FDSO3 (□), and SDSO3 (Δ) to antithrombin was used to determine the K_D value of sulfated DHP-antithrombin complex. Solid lines represent nonlinear fits to the data using quadratic Equation 2. See “Experimental Procedures” for details.

An equivalent limiting decrease of ∼100% was obtained for all three sulfated DHPs at pH 7.4 as well as at pH 6.0, which could be fitted with the standard quadratic binding Equation 2 to obtain the K_D of interaction. The apparent binding affinities of CDSO3, FDSO3, and SDSO3 were found to be 0.4, 2.6, and 3.2 μm at pH 7.4, I 0.15, and 25 °C, respectively (Table 2), corresponding to binding energies of between 7.4 and 8.6 kcal/mol. These free energies of binding are significantly higher than those of the monomeric or oligomeric non-carbohydrate ligands studied to date. For example, the polyacrylic acids displayed binding energies between 5.1 and 6.1 kcal/mol at pH 7.4, I 0.05, 25 °C, whereas ΔG⁰ for small aromatic sulfates was in the range of 4.2–5.3 kcal/mol at pH 7.4, I 0.15, 25 °C (19, 32–34). With respect to comparable polysaccharide ligands, these affinities are only 4–32-fold weaker. For example, LMWHs display plasma antithrombin affinities of ∼100 nm (35). Likewise, the affinities are ∼8–64-fold weaker than that of heparin pentasaccharide binding to plasma antithrombin under equivalent conditions (30, 31). The results indicate that all three sulfated DHPs are potent antithrombin ligands.

TABLE 2.

Interaction of sulfated DHPs with antithrombin at pH 6.0 and 7.4

	KD,obs at 100 mm NaCla	Slopeb	ΔG0Ic	Interceptb	KD,NI	ΔG0NI	ΔG0NId
	nm		kcal/mol		μm	kcal/mol	%
pH 6.0
CDSO3	208 ± 36e	1.04 ± 0.11	1.23 ± 0.13	− 5.74 ± 0.08	1.8 ± 0.1	7.8 ± 0.1	86.4
FDSO3	496 ± 46	1.39 ± 0.14	1.65 ± 0.17	− 5.00 ± 0.10	10.0 ± 0.2	6.8 ± 0.1	80.5
SDSO3	1062 ± 56	1.27 ± 0.24	1.50 ± 0.28	− 4.80 ± 0.23	15.8 ± 0.2	6.5 ± 0.1	81.4
pH 7.4
CDSO3	394 ± 43	1.16 ± 0.24	1.44 ± 0.30	− 5.27 ± 0.21	5.4 ± 0.1	7.2 ± 0.1	83.3
FDSO3	2657 ± 107	1.14 ± 0.10	1.42 ± 0.12	− 4.36 ± 0.99	43.7 ± 0.2	5.9 ± 0.1	80.7
SDSO3	3204 ± 133	0.88 ± 0.27	1.09 ± 0.34	− 4.60 ± 0.28	25.1 ± 0.2	6.3 ± 0.1	85.2

^a Data were measured through titrations using the change in intrinsic protein fluorescence at 340 nm (λ_ex = 280 nm) as signal of binding. See details under “Experimental Procedures.”

^b The slope and intercept values were calculated from linear regressional analysis of log K_D,obs versus log[Na⁺] as described by Equation 2.

^c Free energy of binding was due to ionic forces at 100 mm NaCl.

^d Non-ionic binding energy contribution to the total is expressed as percentage.

^e Error represents ±2 S.E.

At pH 6.0, plasma antithrombin is known to bind sulfated oligosaccharides with higher affinity than that at pH 7.4. For example, the affinity for heparin pentasaccharide H5 increases 25-fold because of the formation of additional ionic and non-ionic interactions (30). In contrast, the affinities of CDSO3, FDSO3, and SDSO3 were found be 0.2, 0.5, and 1.1 μm at pH 6.0, I 0.125, and 25 °C, respectively (Table 2), implying an increase of only 2–5 times from the values at pH 7.4. Comparing the affinities of the three sulfated DHPs under both pH conditions shows that CDSO3 is the most potent antithrombin ligand, whereas SDSO3 is the least.

Non-ionic Binding Energy Dominates the Interaction of Sulfated DHPs with Antithrombin

To determine the nature of interactions made by the sulfated DHPs with plasma antithrombin, the dissociation constant was measured (K_D,obs) as a function of NaCl concentration. The K_D,obs values for all three sulfated DHPs were measured in spectrofluorometric titrations at pH 7.4 and 6.0, as described above. The log K_D,obs values increased linearly with log[Na⁺] at both pH 7.4 and 6.0 (Fig. 4) in a manner similar to that observed with heparin and heparin pentasaccharide (30, 31). According to the protein-polyelectrolyte theory (24, 25), the contribution of non-ionic forces (K_D,NI) to the interaction can be obtained from the intercept of the linear plot, whereas the slope carries information on the number of ion pair interactions (Z) and the fraction of monovalent counterions released per negative charge following ligand binding.

FIGURE 4.

Dependence of the equilibrium dissociation constant of sulfated DHP-antithrombin complex on the ionic strength of the medium at pH 7.4 (A) and pH 6.0 (B). The K_D,obs of CDSO3 (○), FDSO3 (□), and SDSO3 (Δ) binding to antithrombin at varying salt concentrations at pH 7.4 and 6.0 was determined through spectrophotometric titrations. Solid lines represent linear regression fits using Equation 3. Error bars in symbols represent ±1 S.E.

The slopes of the lines for CDSO3, FDSO3, and SDSO3 were found to be 1.0, 1.4, and 1.3, respectively, at pH 6.0 (Table 2). This indicates that all three sulfated DHPs form a similar number of ion pair interactions with the serpin. These interactions result in a nearly 1.2–1.7 kcal/mol of binding energy due to ionic forces at pH 6.0. The intercepts of the linear plots gave values of −5.7, −5.0, and −4.8 at pH 6.0 for CDSO3, FDSO3, and SDSO3, respectively, corresponding to the respective K_D,NI of 1.8, 10.0, and 15.8 μm (Table 2). These results indicate that the free energy of binding due to non-ionic forces is 7.8, 6.8, and 6.5 kcal/mol for CDSO3, FDSO3, and SDSO3, respectively.

The results at pH 7.4 are conceptually similar. The free energies of binding due to ionic forces (ΔG⁰_I) at pH 7.4 were calculated to be 1.4, 1.4, and 1.1 kcal/mol for CDSO3, FDSO3, and SDSO3, respectively, which are equivalent to those at pH 6.0 (Table 2). Likewise, the intercepts at pH 7.4 increase to −5.3, −4.4, and −4.6 corresponding to K_D,NI of 5.4, 43.7, and 25.1 μm for CDSO3, FDSO3, and SDSO3, respectively. This implies that the non-ionic binding energy (ΔG⁰_NI) for the three sulfated DHPs is between 5.9 and 7.2 kcal/mol.

Overall, all three sulfated oligomers exhibit a slope of ∼1 in the double logarithmic plot of affinity versus ionic strength of the medium. This value is substantially lower than slopes of 3–5 observed with heparin and heparin pentasaccharide under nearly similar conditions (30). Interestingly, the slope for sulfated DHPs remains essentially identical at both pH 6.0 and 7.4 possibly suggesting minimal change in the level of ionic interactions between the two conditions. This observation sharply contrasts an increase in slope at the lower pH for antithrombin binding to heparin pentasaccharide (30).

The loss in ionic interactions is amply made up by the gain in non-ionic binding energy by the sulfated DHPs. A massive 5.9–7.8 kcal/mol binding energy arises from the contribution of non-ionic forces at pH 7.4 and 6.0, which represents 80.5–86.4% of the total binding energy (Table 2). In comparison, the heparin pentasaccharide-antithrombin interaction is characterized by ∼40–60% ΔG⁰_NI (30, 31), whereas the non-ionic binding energy contribution in our sulfated flavonoids interacting with plasma antithrombin was found to be ∼55–73% (19, 32). Sulfated DHPs appear to be the first example of antithrombin ligands for which the dominant force of recognition is non-ionic binding energy. Although the origin of non-ionic interactions is unclear at the present time, it is possible that aromatic groups as well as sulfate groups play a significant role.

Sulfated DHPs Bind in the Heparin-binding Site of Antithrombin

Sulfated DHPs resemble UFH and LMWH in the presence of several negatively charged groups, and thus it can be expected that the oligomers are likely to engage the electropositive HBS of antithrombin. The HBS, a considerably large region of the serpin, is usually thought of as a contiguous domain made up of the pentasaccharide-binding site (PBS) and extended heparin-binding site (EHBS). It has been well established that the high affinity pentasaccharide H5 binds antithrombin in the PBS (36), whereas small aromatic activators, such as ECS, bind primarily to the EHBS (19, 34, 37). Thus, to determine whether sulfated DHPs bind antithrombin in the HBS, competitive binding studies with ECS and heparin pentasaccharide H5 were performed. In addition, studies were also performed using UFH¹, which contains a proportion of high affinity chains that are known to simultaneously engage both the PBS and EHBS (38).

The affinity of each sulfated DHP in the presence of all three competitors was measured spectrofluorometrically. The addition of 64 nm H5⁴ resulted in an ∼15% increase in antithrombin fluorescence at 340 nm, as expected, which underwent gradual quenching (∼100%) with increasing concentrations of CDSO3 in a manner similar to that described above for non-competitive experiments (data not shown). The decrease in fluorescence could be fitted using the quadratic binding equation to give the apparent dissociation constant of the interaction (K_D,app) of 0.68 ± 0.07 μm at pH 7.4, I 0.15, 25 °C. Assuming ideal competitive binding, the Dixon-Webb relationship (Equation 4) predicts a decrease in the binding affinity of CDSO3 from 0.39 to 0.79 μm (Table 3), which compares favorably to the measured affinity.

TABLE 3.

Competitive binding of CDSO3 and heparin-binding site ligands, UFH, H5, and ECS, to antithrombin at pH 7.4, I 0.15, 25 °C

[Competitor]O	KD,appa	KD,calcb
μm	μm	μm
H5
0.064	0.68 ± 0.07c	0.79
0.128	0.87 ± 0.09	1.18
0.321	1.07 ± 0.07	2.37
ECS
16	0.53 ± 0.05	0.49
66	0.60 ± 0.05	0.79
166	0.82 ± 0.06	1.38
332	1.47 ± 0.16	2.38
UFH
1	1.16 ± 0.08	0.66
2	1.67 ± 0.09	0.92
8	3.16 ± 0.20	2.50
16	4.25 ± 0.29	4.60

^a The apparent binding affinity (K_D,app) was measured through titrations using the change in intrinsic protein fluorescence at 340 nm (λ_EX = 280 nm) as a signal of binding. See details under “Experimental Procedures.”

^b The calculated binding affinity (K_D,calc) was obtained from Dixon-Webb relationship assuming ideal competitive binding.

^c Error represents ±1 S.E.

To better assess the competitive effect, the interaction of CDSO3 with antithrombin was studied in the presence of multiple concentrations of H5, UFH, and ECS.⁴ Table 3 lists the measured and predicted apparent dissociation constants, K_D,app and K_D,calc, respectively, whereas Fig. 5 shows a graphical comparison of the results. In the presence of a fixed concentration of H5 (ranging from 0.064 to 0.321 μm or 1.0–5.0 × K_H5), the apparent dissociation constant of the CDSO3-antithrombin complex increased ∼1.5-fold (0.68 → 1.07 μm), whereas the K_D,calc was expected to increase ∼3.0-fold (0.79 → 2.37 μm) (Fig. 5). With respect to competition with ECS, the K_D,app increases from 0.53 to 1.47 μm (a 2.8-fold increase) as the concentration of the EHBS ligand increases from 16 to 332 μm, which was expected to ideally decrease the affinity ∼4.9-fold. Finally, competition in the presence of 0.1 to 1.6 μm UFH resulted in an increase in K_D,app from 1.2–4.3 μm, which compares favorably with the expected increase from 0.66 to 4.6 μm (Fig. 5). These results suggest that competition with UFH follows closely the expected ideal Dixon-Webb behavior, whereas that with H5 and ECS is not strictly ideal.

FIGURE 5.

Binding of CDSO3 to antithrombin in the presence of heparin-binding site ligands. The effect of heparin-binding site ligands (heparin pentasaccharide (A), epicatechin sulfate (B), and full-length heparin (C)) on the apparent equilibrium dissociation constant of CDSO3 interacting with antithrombin (K_D,app) was measured in fluorescence titrations at pH 7.4, I 0.15, 25 °C. Unfilled bars show the observed K_D,app, and shaded bars represent the calculated K_D values assuming ideal competitive effect using Dixon-Webb relationship (Equation 4). Error bars in the observed K_D,app represent ±1 S.E. See text for details.

To further confirm competition binding between sulfated DHPs and UFH, binding studies were also performed in the converse manner, wherein UFH was titrated into a solution of antithrombin-sulfate DHP complex. The addition of sulfated DHP (at ∼1 K_D concentration, i.e. 0.4 μm CDSO3, 2.15 μm FDSO3, and 2.50 μm SDSO3) to antithrombin at pH 7.4, I 0.15, 25 °C resulted in 32–59% decrease in fluorescence at 340 nm. Addition of UFH resulted in essentially complete recovery of antithrombin basal fluorescence and then a further increase to reach a limiting value of 29–36% (Fig. 6, A and B). Control experiments indicated that UFH induced a maximal fluorescence change of +36% suggesting that the antithrombin-bound sulfated DHPs are nearly completely displaced by UFH. Finally, the apparent binding affinity of UFH measured in the presence of CDSO3, FDSO3, and SDSO3 was found to correlate closely with that expected on the basis of Dixon-Webb relationship confirming a competitive binding phenomenon (Fig. 6C).

FIGURE 6.

Competitive binding of UFH to antithrombin-sulfated low molecular weight lignin complexes at pH 7. 4, I 0.15, 25 °C. The titration of UFH into a solution of antithrombin-sulfated DHP complex was monitored through the increase in fluorescence at 340 nm. A, squares represent titration of UFH in the absence of a sulfated DHP, and triangles and circles represent the titration of UFH in the presence of CDSO3 and FDSO3, respectively. Competition between UFH and SDSO3 is not shown for clarity. Regression lines represent fits to the quadratic function II to derive the K_D,app values. B, maximal change in intrinsic tryptophan fluorescence observed in the presence (and absence) of CDSO3, FDSO3, and SDSO3. Shaded bars represent the experimentally measured ΔF_max values (with ±2 S.E.), and open bars are values expected considering the initial decrease with sulfated DHP. C, apparent dissociation constant of UFH measured in the absence and presence of CDSO3, FDSO3, and SDSO3. Shaded bars are the experimentally measured K_D,app values (with ±2 S.E.), and open bars are the values expected on the basis of Dixon-Webb ideal competitive relationship IV. See text for details.

Several Distinct DHP Sequences Are Involved in the High Affinity Interaction with Antithrombin

The sulfated DHPs are a heterogeneous mixture of a large number of structures with varying chain lengths. To test whether specific sequence(s) in the mixture possess(es) high affinity for antithrombin, affinity capillary electrophoresis (ACE) was utilized. ACE has been previously utilized to assess the affinity of polyanionic polysaccharides for antithrombin (39–41), although recently we utilized this technique to profile the interaction of aromatic small molecules with the inhibitor (42). Briefly, if a polyanionic molecule interacts with a protein, its electrophoretic mobility through an open bore microcapillary will be altered as a function of the concentration of the protein. Thus, sequences in sulfated DHPs that bind to antithrombin with higher affinity will migrate differently from sequences that possess weaker or no affinity.

Fig. 7 shows the ACE profile of CDSO3 in the presence of a fixed concentration of plasma antithrombin at pH 7.4 and 8.0. In the absence of antithrombin, CDSO3 displays a broad, featureless peak in the region 9.5–19.5 at pH 7.4. In contrast, the presence of 108 nm antithrombin results in several small peaks between 5 and 9 min (Fig. 7A). These peaks increase in intensity when the concentration of the serpin was increased to 215 nm suggesting the formation of CDSO3-antithrombin complex. The small number of peaks corresponding to the complex indicates that several sequences in CDSO3 are able to bind antithrombin with higher affinity than the majority of oligomeric sequences. To assess whether reducing the electropositive character of the HBS affects the number of sequences interacting with antithrombin, ACE was performed at pH 8.0. Under this condition, the number and signal intensity of the peaks corresponding to the CDSO3-antithrombin complex decrease significantly (Fig. 7B). This supports the conclusion that a small number of distinct sequences are present in the heterogeneous CDSO3s that recognize antithrombin with higher affinity than the majority of sequences.

FIGURE 7.

Affinity capillary electrophoresis of CDSO3 in the presence of plasma antithrombin at pH 7. 4 (A) and 8.0 (B). The resolution of CDSO3 at 8 kV in 20 mm sodium phosphate buffer, pH 7.4, containing 108 and 215 nm antithrombin was monitored at 214 nm. The appearance of distinct peaks in the region 4.5–9.5 min at pH 7.4 (marked by arrow in A) or in the region 3–5.5 min at pH 8.0 (arrow in B) suggests selective recognition of certain structures in CDSO3 by antithrombin. Peak marked x is a ghost peak because of sudden, but transient, electrical disturbance.

Virtual Screening Identifies a β-5 Tetramer Sequence as a Potent Antithrombin Ligand

To determine the important structural features, especially the optimal length, of sulfated DHPs that may induce preferential recognition of antithrombin, we resorted to computerized virtual docking experiments. A combinatorial virtual library screening approach for deducing “high affinity, high specificity” sequences that recognize antithrombin was developed earlier in our laboratory (27). In this approach, oligomeric sequences were prepared in silico using an automated protocol and docked onto a pre-defined binding site, e.g. the HBS on the serpin. The docking approach utilizes a genetic algorithm to iteratively derive the best binding geometry for each oligomeric structure. The best binding geometry is then scored using GOLD, which evaluates the number and strength of hydrogen bonding, van der Waals, and Coulombic interactions present in the simulated complex (27). The GOLD scores for each oligomer present in the virtual library can then be used to sort sequences that preferentially recognize antithrombin.

A small library of 11 β-5 CDSO3 structures that are dimer to pentamer in length was generated in an automated manner using the standard molecule building module in Sybyl (see supplemental Fig. S1 for detailed structures). Although CDSO3 can consist in all possible combinations of β-5, β-O-4, β-β, and 5-5 inter-monomeric linkages, the virtual library consisted of only β-5-linked sequences to rapidly assess selectivity of interaction. Varying levels of sulfation and carboxylation of oligomeric sequences were chosen for this study. For example, the sulfation level increases from three OSO₃⁻ groups in the dimers to six in the pentamers. Likewise, the carboxylation level varies from one in several sequences to five COO⁻ groups in a pentamer (see supplemental Fig. S1). Genetic algorithm-based virtual screening of this 11-membered library shows that the majority sequences studied exhibited a GOLD score in the range of 73–88 (Table 4). Comparison with our results on a heparin/heparan sulfate oligosaccharide library shows that this score represents average to poor affinity for antithrombin (27). Importantly, the virtual screening identified a β-5 tetramer, consisting of five sulfates and four carboxylates, to possess a GOLD score of 110. This score compares favorably with a GOLD score of 140 for the high affinity heparin pentasaccharide H5 binding to antithrombin in the PBS (27). This suggests that one β-5 CDSO3 tetramer sequence out of the 11 studied binds to the HBS with reasonably high affinity. Atomic level details shows that the high GOLD score arises from the recognition of Arg-46, Arg-47, Lys-114, Lys-125, Arg-129, Arg-132, Lys-133, and Lys-136 (Fig. 8). These residues belong to the PBS and EHBS of antithrombin and are known to be important for interacting with heparin. Thus, this limited genetic algorithm-based virtual screening study supports the conclusion that distinct sequences in sulfated DHPs may exist that preferentially recognize the HBS of antithrombin with relatively high affinity.

TABLE 4.

Computerized virtual screening of selected β-5 CDSO3 oligomers interacting with antithrombin

^a Virtual screening of CDSO3 oligomers binding to the HBS of antithrombin was performed in accordance with the protocol established earlier (27). The complete experimental protocol and details regarding the oligomeric structures are provided in the supplemental material.

^b Heparin pentasaccharide GOLD score was taken from Ref. 27.

FIGURE 8.

Virtual docking of CDSO3 oligomers onto heparin-binding site in antithrombin. CDSO3 oligomers (dimers through pentamers) containing all β-5 linkages were docked onto the HBS of antithrombin (shown in ribbon diagram with critical side chain in stick format) using GOLD following a protocol established earlier (27). Shown here is a tetramer sequence (ball and stick representation) that was found to bind with a GOLD score of 110. See Table 4 and text for details.

DISCUSSION

Antithrombin is an interesting protein. It features a massive ∼20-Å-long electropositive domain, the heparin-binding site, which intimately communicates with its business end, the reactive center loop, nearly 25 Å away. This allosteric communication is important not only for regulation of coagulation (1) but also for anti-angiogenesis (43, 44). Modulators of this communication, either positive or negative, are likely to be useful in understanding mechanistic and structural aspects of the communication, and may have pharmaceutical value.

The primary modulator of antithrombin function is the heparin pentasaccharide sequence, a highly specific motif honed by nature through years of evolution. Several other polysaccharide-based motifs have been studied as HBS ligands (45, 46), yet achieving selectivity of recognition has been a challenge. Small non-carbohydrate motifs (<1000 Da) have shown promise (20, 32, 37), but targeting the PBS has proved to be difficult (19, 42). Larger non-carbohydrate motifs of the size of heparin (or LMWH) are difficult to design (33, 34) or find in nature. Only a couple of natural non-polysaccharide, macromolecular structures, condensed tannins and lignins (28, 29), are known that can be exploited for binding to highly electropositive-binding sites on proteins. The sulfated DHPs studied here are chemo-enzymatically prepared low molecular weight variants of natural lignins (16).

Sulfated DHPs represent a collection of diverse structures. The multiple inter-monomeric linkages present in sulfated DHPs coupled with the variability in chain length generate numerous structural topologies. The presence of multiple sulfate and carboxylate groups induces high aqueous solubility and an ability to interact with multiple lysines and arginines. At the same time, the highly aromatic backbone introduces significant hydrophobicity in the motifs. This dual hydrophilic and hydrophobic property, which is not possible for polysaccharides, makes sulfated DHPs a library of unique and diverse structures possessing significant variability in their ability to interact with proteins.

An example of functional variability in the sulfated DHP sequences is displayed by the inhibition of selected coagulation factors in the presence of antithrombin. Although the inhibitory potency of CDSO3 and FDSO3 against factors IIa, IXa, and Xa reduces 1.5–3-fold in the presence of antithrombin from that in the absence of the serpin, the potency of SDSO3 increases 2–16-fold. Particularly striking is the observation that SDSO3 activates antithrombin to better inhibit free factor VIIa (Fig. 2 and Table 1). In contrast, heparin activates antithrombin to inhibit fVIIa only in the presence of tissue factor (4, 5). The mechanistic basis for SDSO3 activation of antithrombin inhibition of free fVIIa is not clear at the present time, but it probably arises from the modulation of the reactive center loop conformation due to binding in the HBS. This presents an interesting opportunity for regulating the cellular initiation phase of hemostasis (47, 48).

All three sulfated DHPs studied bind to antithrombin. Under physiological conditions, CDSO3 binds to the serpin with sub-micromolar affinity (Fig. 3 and Table 2), which is comparable with that of heterogeneous LMWHs (35). To the best of our knowledge, CDSO3 appears to be the most potent, non-carbohydrate antithrombin recognition scaffold reported to date. The antithrombin affinities of the other two sulfated DHPs are modest in comparison with that of LMWHs (and UFH) but much better than other non-carbohydrate, antithrombin-binding polymers (33, 34).

A major point of difference between sulfated DHPs and LMWHs is the nature of interaction with antithrombin. Sulfated DHPs display a much lower ionic component and a massive 80–87% non-ionic contribution to binding energy (Table 2). The essentially non-ionic interaction of the sulfated DHPs with the highly electropositive HBS of antithrombin appears to suggest an energetic contribution by the hydrophobic scaffold. For example, the lysines and arginines of the HBS may interact with the aromatic rings of sulfated DHPs through cation-π interactions. An internal cation-π interaction between Trp-49 and Lys-53 has been hypothesized to play a role in the antithrombin activation mechanism (49). Inter-molecular cation-π interactions, the probability of which is higher because of the presence of multiple aromatic rings in DHPs, may explain the high non-ionic contribution. Yet a more plausible explanation is that the non-ionic binding energy arises from the hydrogen bonding component resident in each ion pair (50). For example, the arginines bind to sulfate and/or carboxylate groups such that the interaction involves a significant component of hydrogen bond energy, which may not involve the phenomenon of the release of counter ions. It is possible that specific sequences of sulfated DHPs generate a majority of binding energy because of the formation of specific sulfate-arginine ion pairs.

Competitive binding studies suggest that the presence of H5 and ECS, PBS, and EHBS ligands, respectively, weakens the affinity of CDSO3 for antithrombin, albeit less than that predicted on the basis of ideal competition (Fig. 5 and Table 3). The presence of full-length heparin, which simultaneously engages the PBS and EHBS, decreases the affinity of sulfated DHPs in a manner predicted on the basis of an ideal competitive effect. One explanation for less than ideal competition with exclusive PBS and EHBS ligands is the possibility of simultaneous binding of smaller DHP sequences in the EHBS and H5 in the PBS. Such simultaneous and additive binding of a heparin tetrasaccharide in the PBS and a non-saccharide EHBS ligand has been noted earlier (19, 42). Likewise, a DHP sequence may avoid competition with ECS by engaging the PBS resulting in weakened EHBS competition. This suggests that optimal DHP sequence(s), especially the higher affinity sequence(s), is(are) likely to bind to antithrombin in both the PBS and the EHBS.

The presence of multiple negative charges on a DHP oligomer ensures binding to the HBS, just as heparin chains devoid of the H5 sequence also interact with the serpin (38). Yet, distinct DHP sequences may be present that bind to antithrombin with higher affinity and selectivity. Affinity capillary electrophoresis (ACE) is a particularly robust technique to elucidate such interactions (39–41). ACE displayed a small number of peaks assignable to antithrombin-DHP complexes under physiological conditions (Fig. 7) suggesting preferential binding of selected DHP oligomers to antithrombin. Likewise, genetic algorithm-based virtual screening suggested that only one β-5 oligomer out of the 11 present in a library bound in the HBS with relatively higher affinity (Fig. 8 and Table 4). This sequence contains several sulfate and carboxylate groups suggesting that recognition “in silico,” and possibly in solution, is driven by an anionic scaffold. This does not contradict the primarily non-ionic interaction (80–87% ΔG⁰_NI, see Table 2) observed in solution because each sulfate-arginine interaction carries significant non-ionic energy (hydrogen bond), as discussed above (50). The identification of one “high affinity” sequence in a small computational screen suggests that such additional sequences are possible. Thus, it is to be expected that the number of high affinity sequences is likely to increase with the size of the library (27). Yet at the present time, these results suggest that distinct DHP sequences, rather than one dominant sequence, are involved in the recognition of antithrombin. It is possible that these sequences possess a common motif with peripheral differences that are not critical for antithrombin recognition.

Overall, this work suggests that selected sulfated DHP motifs potently recognize antithrombin in the heparin-binding site. The nature of the interaction of these motifs with the serpin is significantly different from that of the heparins. This may afford a heparin-like function without the heparin-like adverse effects. The chemo-enzymatic origin of sulfated DHPs lends itself particularly amenable to identifying or engineering specific sulfated DHP sequence(s) that modulate antithrombin function, especially for inhibiting the initiation phase of hemostasis.