Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins

Abstract

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.

Heparan sulfate and heparin interactions with proteins

“…Furthermore, distinct carbohydrate structures can sometimes fulfil the role of HS/heparin, as has been shown for CS/DS extracted from brittlestars, which activated FGF-2 [85]. However, as was the case for AT, FGF-1 signalling correlated strongly with protein stabilization induced by the polysaccharides, whereas FGF-2 signalling did not [79]. For the FGFs, this complex situation is underpinned by the detection of widely ranging heparin binding properties among FGF subfamily members, which possess varied numbers of HBPs, identified by Lys residues (the chemistry of Arg residues proving thus far more problematic), that ranged from 1 (FGF-9) to 3 (FGF-1) and their binding properties (K d values from 38 to 620 nM and k ass varied over 20-fold) [86].…”

“…This reinforces the idea that binding, even with low affinity, does not necessarily equate to activity. Furthermore, non-carbohydrate compounds can bind both the pentasaccharide binding site and the extended heparin binding sites of AT, leading to enhanced anticoagulant activity [79], suggesting that alternative classes of compounds may find therapeutic roles. The structure of AT in the heparin-bound, intermediate state illustrates such issues.…”

Chemical sulfation of small molecules—advances and challenges

“…The field of sulfated, small, aromatic mimetics of highly sulfated GAGs has put forward some interesting initial results 27–32,73. Yet, the difficulties associated with chemical sulfation highlighted in the current review and the absence of large chemical library of structurally diverse, sulfated aromatic molecules are the major challenges in discovering new drugs targeting the role of sulfated biomolecules.…”

“…For example, several large and small aromatic H/HS mimetics including sulfated flavonoids27,28, benzofurans29, isoquinolines30 and sulfated dehydropolymers of the lignin-type31,32 have been designed to modulate the function of coagulation proteins such as antithrombin, thrombin and factor Xa. The design of such highly sulfated, non-natural molecules suggests a strong possibility of discovering novel sulfated non-carbohydrate pharmaceutical agents.…”

Small molecule probes that perturb a protein–protein interface in antithrombin

“…Thus, the probes were incubated with α-antithrombin (time variable), and aliquots of this solution were used to inhibit thrombin-mediated hydrolysis of a peptide containing 4-nitroaniline (Spectrozyme ® TH, from American Diagnostica); this type of assay is a standard test for thrombin activity (performed in the presence of heparin). 20,21 In the event, thrombin was most active when inhibited by samples of α-antithrombin that had been incubated with ddd - 1as’f (Figure 5a). This observation is consistent with the assertion made above, that ddd - 1as’f promotes α-antithrombin deactivation by oligomerization well.…”