Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins: OPPORTUNITIES FOR POTENT, SELECTIVE MODULATION OF ANTITHROMBIN FUNCTION*

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.Open in a separate windowFIGURE 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.