A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

Abstract

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), akey serpin present in human plasma, remain unknown. Using ac omputational strategy on alibrary of 46 656 heparan sulfate hexasaccharides we identified ar are sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, ac losely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly,our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAGsequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.Heparin cofactor II (HCII) is as erine protease inhibitor (serpin) that circulates in human plasma at high levels. Although it has been known to selectively inhibit thrombin for several decades, [1][2][3] its true physiologic function remains to be understood. [4] HCII is known to bind to glycosaminoglycans (GAGs) such as dermatan sulfate (DS) and heparan sulfate (HS), which help mediate its inhibition of thrombin. One of the key reasons for the inability to identify HCIIs biologic role is the lack of knowledge on the specificity of HCII-GAGi nteraction.HCII is an interesting serpin. It bears considerable similarity to antithrombin (AT), another plasma serpin that mediates the anticoagulant action of heparin and fondaparinux, [5,6] two clinically used drugs.A Tand HCII are homologous in primary,secondary and tertiary structure ( Figure S1 in the Supporting Information). Yet, whereas AT binds specifically to fondaparinux sequence in heparin, [7] HCII is considered to bind non-specifically to heparin and HS. Further,a lthough both serpins display at wo-step,i nducedfit, allosteric activation mechanism in inhibiting their target enzymes, [8][9][10] no HS oligosaccharide has been discovered to induce robust activation of HCII.Identifying GAGs equences that selectively target proteins is extremely challenging.Akey reason for this is their structural complexity.H S, ah ighly anionic polymer containing variably sulfated, acetylated and epimerized residues, presents enormous structural diversity that makes comprehensive analysis of all possible sequences difficult. Thus, identifying key "needles" in this haystack, especially with synthesis [11,12] or isolation of oligosaccharides from nature nearly impossible. [13] We had earlier developed ad ual-filter computational algorithm, called combinatorial virtual library screening (CVLS) strategy,torapidly sort HS sequences into "specific" and "non-specific" bins. [14,15] We wondered whether this tool could pinpoint HS sequences that preferentially activate HCII for inhibition of thrombin. Further, we posited that such asequence,ifany,would be different from the pentasaccharide seq...

Heparin mimetics with anticoagulant activity

“…It is noteworthy that tirofiban on its own has a very short half-life and cannot be used for outpatients whereas 13 has a much longer half-life due to the presence of the idraparinux moiety. (9) molecular spacers 65,66 Recently, Oscarson and Desai generated an in silico library of 46,656 heparan sulfate hexasaccharides and found a rare sequence consisting of consecutive GlcA2S residues which could selectively target heparin cofactor II (HCII), 71 another serpin involved in the regulation of blood coagulation via inhibition of FIIa. They synthesized five unique sequences including three containing at least one GlcA2S residue (a residue rarely found in heparin).…”

“…Recently, Oscarson and Desai generated an in silico library of 46,656 heparan sulfate hexasaccharides and found a rare sequence consisting of consecutive GlcA2S residues which could selectively target heparin cofactor II (HCII), another serpin involved in the regulation of blood coagulation via inhibition of FIIa. They synthesized five unique sequences including three containing at least one GlcA2S residue (a residue rarely found in heparin).…”

So you think computational approaches to understanding glycosaminoglycan–protein interactions are too dry and too rigid? Think again!

“…1B) [18,21,22] the most optimal GAG sequence for a target protein (Fig. 1C) [11,23,24], and the structure of ternary protein 1 -GAG-protein 2 system (Fig. 1D) [21,25,26].…”

“…A case highlighting this is the interaction of GAGs with heparin cofactor II (HCII). Although generally believed to be non-specific, recent studies indicate highly selective binding of HS hexasaccharide to HCII [11].…”

Replacement of the L-iduronic acid unit of the anticoagulant pentasaccharide idraparinux by a 6-deoxy-L-talopyranose – Synthesis and conformational analysis

“…In the field of heparinoid anticoagulants, current research focuses on unmet issues such as low oral bioavailability of heparin 10 , lack of specific antidote for low molecular weight heparins 11 , and chemoenzymatic production of heparin oligosaccharides 12 – 15 . Furthermore, many research efforts have been devoted to the development of highly efficient synthetic routes to the outstanding anticoagulant pentasaccharides fondaparinux 16 – 18 and idraparinux 19 , 20 as well as the preparation of various analogues of the antithrombin-binding pentasaccharide unit of heparin as novel anticoagulant candidates 21 – 25 .…”