Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS5 containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS5 activated the inhibitor nearly 30-fold, nearly 2 to 3-fold higher than our first generation flavan-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.

BackgroundThe antithrombin–heparin/heparan sulfate (H/HS) and thrombin–H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG)–protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics.MethodologyAnalyses of solvent accessibility and exposed surface areas, gyrational mobility, symmetry, cavity shape/size, conserved water molecules and crystallographic parameters were performed for 12 X-ray structures, which include 12 thrombin and 16 antithrombin chains. Novel calculations are described for gyrational mobility and prediction of water loci and conservation.ResultsThe solvent accessibilities and gyrational mobilities of arginines and lysines in the binding sites of the two proteins reveal sharp contrasts. The distribution of positive charges shows considerable asymmetry in antithrombin, but substantial symmetry for thrombin. Cavity analyses suggest the presence of a reasonably sized bifurcated cavity in antithrombin that facilitates a firm ‘hand-shake’ with H/HS, but with thrombin, a weaker ‘high-five’. Tightly bound water molecules were predicted to be localized in the pentasaccharide binding pocket of antithrombin, but absent in thrombin. Together, these differences in the binding sites explain the major H/HS recognition characteristics of the two prototypic proteins, thus affording an explanation of the specificity of binding. This provides a foundation for understanding specificity of interaction at an atomic level, which will greatly aid the design of natural or synthetic H/HS sequences that target proteins in a specific manner.

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 pre-* This work was supported, in whole or in part, by National Institutes of Health Grants HL069975 and HL090586. This work was also supported by Grant EIA 0640053N from the American Heart Association National Center, Grant 6-46064 from the A. D. Williams Foundation, and a grant from the Mizutani Foundation for Glycoscience, Japan.

Tryptophan 49 of antithrombin, the primary inhibitor of blood clotting proteinases, has previously been implicated in binding the allosteric activator, heparin, by chemical modification and mutagenesis studies. However, the X-ray cocrystal structure of the antithrombin-pentasaccharide complex shows that Trp49 does not contact the bound saccharide. Here, we provide a detailed thermodynamic and kinetic characterization of heparin binding to a Trp49 to Lys variant of antithrombin and suggest a model for how Trp49 participates in heparin binding and activation. Mutation of Trp49 to Lys resulted in substantial losses of 16-24% in heparin-binding energy at pH 7.4, I 0.15, and 25 degrees C. These losses were due to both the loss of one ionic interaction ( approximately 30%) and the loss of nonionic interactions ( approximately 70%). Rapid kinetics analyses showed that the mutation minimally affected the initial weak binding of heparin to antithrombin or the rate constant for the subsequent conformational activation of the serpin. Rather, the principal effect of the mutation was to increase the rate constant for reversal of the conformational activation step by 70-100-fold, thereby destabilizing the activated conformation. This destabilization could be accounted for by the disruption of a network of interactions involving Trp49, Glu50, and Lys53 of helix A and Ser112 of helix P, which stabilizes the activated conformation.