Interaction of Thrombin with Sucrose Octasulfate

Abstract

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anti-coagulant. The polyanionic glycosaminoglycan heparin modulates thrombin’s activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a Kd of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Non-ionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin’s catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the x-ray crystal structure, two molecules of SOS are bound non-equivalently to exosites II of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.

Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants

“…Protamine is a clinically used arginine-rich polypeptide that counteracts the anticoagulant activity of UFH and LMWHs [29], whereas polybrene is a quaternary amine-containing hydrophobic polymer, which has been explored as a probe of electrostatic interactions [30]. SOS is an FDA approved antacid that is known to bind in heparin-binding sites on proteins such as thrombin [31]. FXIa was first treated with saturating concentrations of SPGG2 (5 and 250 μg/mL) and the recovery of FXIa activity by the potential antidote studied spectrophotometrically through hydrolysis of FXIa substrate (S-2366) at pH 7.4 and 37 °C (Figure 3).…”

Identification of the site of binding of sulfated, low molecular weight lignins on thrombin

“…The exosite 2-mediated allosteric regulation of thrombin offers a sound possibility of an antidote strategy with non-inhibitory sulfated carbohydrates. For example, small oligosaccharides that bind in exosite 2 without inducing much direct inhibition, such as sucrose octasulfate [21], may serve as effective antidotes of CDSO3-based anticoagulants. This would impart considerable safety to anticoagulant therapy based on sulfated LMWLs.…”

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

“…The monomeric scaffolds included chalcones (compounds 1-10), flavonoids (11)(12)(13)(14)(15)(16) [30][31][32], sucrose octasulfate (17) [40], quinazolinones (18 and 19) [37], and tetrahydro-isoquinolines (20)(21)(22)(23)(24)(25)(26)(27) [36], whereas the dimeric scaffolds comprised flavonoid-quinazolinone heterodimers (28)(29)(30)(31)(32)(33)(34) [37], bis-quinazolinones homodimers (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47) [37], and bis-flavonoid homodimers (48-55). In addition to the inherent diversity of the scaffolds in this library, NSGMs also differed in the number (1 to 8) and orientation of the sulfate groups.…”

“…Briefly, the synthesis of these NSGMs was achieved in 4 to 8 steps using traditional protection-deprotection chemistry following construction of the base scaffold with appropriate substitution pattern. The final step for the generation of each NSGM was chemical sulfation using trimethylamine-sulfur trioxide at elevated temperature, as described in our studies earlier [30][31][32][33][34][35][36][37][38][39][40][41][42]. For example, the synthesis of two promising plasmin inhibitors (Scheme 1) involved exploitation of the intramolecularly hydrogen bonded 5-OH group of quercetin 60 to selectively introduce a reactive handle (propargyl group in 63 or in situ alkyl bromide) that can be coupled with either an alkyl azide containing quinazolinone unit 32a or MOM-protected quercetin 61 to eventually give inhibitors 32 and 52, respectively.…”

“…Sulfation of polyphenolic precursors was achieved using microwave assisted chemical sulfation as described earlier [30][31][32][33][34][35][36][37][38][39][40][41][42]. Briefly, to a stirred solution of polyphenol in anhydrous CH3CN (1-5 mL) at room temperature, Et3N (10 equivalents per -OH group) and Me3N:SO3 complex (6 equivalents per -OH) was added.…”