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Antithrombin (AT) is a serpin that inhibits mainly thrombin and fXa after being activated by binding to glycosaminoglycans as heparin and heparan sulfate. Upon binding, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. Recently, a new compound, named TMI, was discovered in our group with nanomolar affinity to antithrombin, and shown to be able to induce a partial activation of antithrombin. As TMI represents an original scaffold for structural optimizations aiming the development of new antithrombotic drugs, the present work demonstrated, through a series of molecular dynamics simulations, that TMI is able to modulate AT reactive center loop flexibility similarly to what is observed to heparin, as well as exposing AT P1 residue, Arg393. These results represent the first atomic level indication of AT conformational activation by TMI, and may offer a predictive basis for future studies aiming TMI structural optimization.

fIIa and fXa are two of the main targets of antithrombin, a serine proteases inhibitor that plays a major role in the regulation of blood clotting. The formation of ternary complexes between such molecules and glycosaminoglycans, as heparin, is the main path for inhibiting those enzymes, which may occur through two distinct mechanisms of action. While these serine proteases present distinct susceptibilities to these paths, in which fIIa demands an interaction with heparin, neither the molecular basis of this differential inhibition nor the role of fIIa glycosylation on this process is fully understood. Thus, the present work evaluated through molecular dynamics simulations the effects of glycosylation on fIIa and the consequences of heparin binding to both proteases function and dynamics. Based on the obtained data, fIIa N-linked glycan promoted an increase in the active site pocket size by stabilizing regions that encircle it, while heparin binding was observed to reverse such an effect. Additionally, heparin orientation observed on the surface of fIIa, but not fXa, allows a linear long-chain heparin binding to antithrombin in ternary complexes. Finally, the enzymes catalytic triad organization was disrupted due to a strong glycosaminoglycan binding to the proteases exosite 2. Such data support an atomic-level explanation for the higher inhibition constant of the antithrombin-heparin complex over fIIa than fXa, as well as for the different susceptibilities of those enzymes for antithrombin mechanisms of action.

Antithrombin (AT), a serine protease inhibitor, circulates in blood in two major isoforms, α and β, which differ in their amount of glycosylation and affinity for heparin. After binding to this glycosaminoglycan, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. In this process, β-AT presents the higher affinity for heparin, being suggested as the major AT glycoform inhibitor in vivo. However, either the molecular basis demonstrating the differences in heparin binding to both AT isoforms or the mechanism of its conformational activation are not fully understood. Thus, the present work evaluated the effects of glycosylation and heparin binding on AT structure, function, and dynamics. Based on the obtained data, besides the native and activated forms of AT, an intermediate state, previously proposed to exist between such conformations, was also spontaneously observed in solution. Additionally, Asn135-linked oligosaccharide caused a bending in AT-bounded heparin, moving such polysaccharide away from helix D, which supports its reduced affinity for α-AT. The obtained data supported the proposal of an atomic-level, solvent and amino acid residues accounting, putative model for the transmission of the conformational signal from heparin binding exosite to β-sheet A and the reactive center loop, also supporting the identification of differences in such transmission between the serpin glycoforms involving helix D, where the Asn135-linked oligosaccharide stands. Such intramolecular rearrangements, together with heparin dynamics over AT surface, may support an atomic-level explanation for the Asn135-linked glycan influence over heparin binding and AT activation., (Copyright © 2011 Wiley-Liss, Inc.)

2-O-Sulfo-alpha-l-iduronic acid (IdoA2S) is one of the main components of heparin, an anticoagulant and antithrombotic polysaccharide able to potentiate the inhibitory effect of antithrombin over plasma serine proteases. This monosaccharide unit adopts an equilibrium between chair (1C4) and skew-boat (2SO) forms as a function of heparin sequence size and composition. Although the prevalence of the 1C4 chair conformation in monosaccharides is understood, the reasons for the increase in 2SO contribution in the whole polysaccharide chain are still uncertain. In this context, 0.2 mus molecular dynamics simulations of IdoA2S-containing oligosaccharides indicated that stabilization due to intramolecular hydrogen bonds around IdoA2S is highly correlated (p0.001) with the expected conformational equilibrium for this residue in solution. This behavior explains the known effect of different heparin compositions, at the monosaccharide level, on IdoA2S conformation in biological solutions.

Heparin was isolated in the beginning of the 20th century and until today remains as one of the most important drugs able to interfere with the haemostatic process. Due to the side effects produced by heparin therapy, new promising drugs have been developed, as the synthetic pentasaccharide (synthetically derived from the sequence GlcN-GlcA-GlcN-IdoA-GlcN). The anticoagulant activity of this compound is based on potentiation of antithrombin (AT) inhibitory activity upon serine proteinases of clotting cascade, a mechanism based on the conformational modification of AT. In this context, we present here a molecular dynamics (MD) study of the interaction between the synthetic pentasaccharide and AT. The obtained data correctly predicted an induced fit mechanism in AT-pentasaccharide interaction, showing a solvent-exposed P1 residue instead of a hided conformation. Also, the specific contribution of important amino acid residues to the overall process was also characterized, both in (2)S(0) and (1)C(4) conformations of IdoA residue, suggesting that there is no conformational requirement to the interaction of this residue with AT. Altogether, the results show that MD simulations could be used to characterize and quantify the interaction of synthetic compounds with AT, predicting its specific capacity to induce conformational changes in AT structure. Thus, MD simulations of heparin (and heparin-derived)-AT interactions are proposed here as a powerful tool to assist and support drug design of new antithrombotic agents.

HF/6-31G** and molecular dynamics (MD) simulations were used to evaluate the performance of different atomic charge basis sets (i.e., Mulliken, Lowdin, and Electrostatic Potential Derived Charges--ESP) in heparin simulations. HF/3-21 G calculations were also used to study the NMR conformation of the IdoA residue. The results thus obtained indicated that ESP and Lowdin charges gave the better results in heparin simulations, followed by Mulliken charges, and that the minimum-energy conformation of IdoA can be different from that observed by NMR spectroscopy by less than 1 Angstrom. However, it was found that this small conformational modification is capable of inducing a change of almost 200 kJ/mol in the interactions of heparin with the surrounding environment, which is a meaningful amount of energy in the context of ligand-receptor interactions. This information can be potentially of great relevance in the design of heparin-derived antithrombotic compounds.

Molecular dynamics (MD) simulations on heparin-water-sodium systems were carried out in order to establish a simulation protocol able to represent heparin solution conformation under physiological conditions. Atomic charges suitable for heparin oligosaccharides were obtained from ab initio quantum-mechanical computations, at the 6-31G(**) level. The GROMACS forcefield, the SPC, and SPC/E water models were employed. Also heparin was simulated with IdoA residues in 1C(4) or 2S(0) conformational states. The results of the performed MD simulations are in agreement with the available experimental data, suggesting that this approach can be applied for the study of heparin interactions with its target proteins and thus play a role in the development of new antithrombotic agents.