Mechanism of Poly(acrylic acid) Acceleration of Antithrombin Inhibition of Thrombin:  Implications for the Design of Novel Heparin Mimics

Abstract

The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive ∼2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269. To better understand this interesting phenomenon, we have studied the mechanism of PAAdependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide-and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K D of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the "bridging" mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics.

Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins

“…Small non-carbohydrate motifs (Ͻ1000 Da) have shown promise (20,32,37), but targeting the PBS has proved to be difficult (19,42). Larger non-carbohydrate motifs of the size of heparin (or LMWH) are difficult to design (33,34) or find in nature. Only a couple of natural non-polysaccharide, macromolecular structures, condensed tannins and lignins (28,29), are known that can be exploited for binding to highly electropositive-binding sites on proteins.…”

“…These free energies of binding are significantly higher than those of the monomeric or oligomeric non-carbohydrate ligands studied to date. For example, the polyacrylic acids displayed binding energies between 5.1 and 6.1 kcal/mol at pH 7.4, I 0.05, 25°C, whereas ⌬G 0 for small aromatic sulfates was in the range of 4.2-5.3 kcal/mol at pH 7.4, I 0.15, 25°C (19,(32)(33)(34). With respect to comparable polysaccharide ligands, these affinities are only 4 -32-fold weaker.…”

“…In contrast, the antithrombin affinities of non-saccharide ligands, e.g. small aromatic sulfates such as ECS and CS (19,32) and larger polymers such as polyacrylic acids (33,34), have been measured using 6-p-toluidinylnaphthalene-2-sulfonate, an external probe. In this study, we found that the interaction of our synthetic DHPs with antithrombin resulted in a decrease in intrinsic tryptophan fluorescence (Fig.…”

“…The HBS, a considerably large region of the serpin, is usually thought of as a contiguous domain made up of the pentasaccharide-binding site (PBS) and extended heparin-binding site (EHBS). It has been well established that the high affinity pentasaccharide H5 binds antithrombin in the PBS (36), whereas small aromatic activators, such as ECS, bind primarily to the EHBS (19,34,37). Thus, to determine whether sulfated DHPs bind antithrombin in the HBS, competitive binding studies with ECS and heparin pentasaccharide H5 were performed.…”

Intravenously administered nanoparticles increase survival following blast trauma

“…S3). PAA is used during nanoparticle synthesis to enable easy collection and subsequent resuspension of particles, and also has been implicated as an anticoagulant (21). In our small-scale study, we found that particles with PAA were at least as effective as particles made without PAA at reducing lethality, and thus we used PAA in all subsequent work.…”