Deciphering the role of trehalose in hindering antithrombin polymerization

Abstract

Serine protease inhibitors (serpins) family have a complex mechanism of inhibition that requires a large scale conformational change. Antithrombin (AT), a member of serpin superfamily serves as a key regulator of the blood coagulation cascade, deficiency of which leads to thrombosis. In recent years, a handful of studies have identified small compounds that retard serpin polymerization but abrogated the normal activity. Here, we screened small molecules to find potential leads that can reduce AT polymer formation. We identified simple sugar molecules that successfully blocked polymer formation without a significant loss of normal activity of AT under specific buffer and temperature conditions. Of these, trehalose proved to be most promising as it showed a marked decrease in the bead like polymeric structures of AT shown by electron microscopic analysis. A circular dichroism (CD) analysis indicated alteration in the secondary structure profile and an increased thermal stability of AT in the presence of trehalose. Guanidine hydrochloride (GdnHCl)-based unfolding studies of AT show the formation of a different intermediate in the presence of trehalose. A time-dependent fluorescence study using 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid (Bis-ANS) shows that trehalose affects the initial conformational change step in transition from native to polymer state through its binding to exposed hydrophobic residues on AT thus making AT less polymerogenic. In conclusion, trehalose holds promise by acting as an initial scaffold that can be modified to design similar compounds with polymer retarding propensity.

Trehalose: is it a potential inhibitor of antithrombin polymerization?

“…The study of molecules capable of reducing or preventing the polymerization of serpins has been carried out with different serpins [18–24], in order to then extrapolate the results to other serpins. In the study by Naseem et al [25], the effect of trehalose on the polymerization of serpins has been carried out with antithrombin. Although the authors present an in-silico study on the potential interaction of trehalose with other serpins and reference their effect in the prevention of polymerization with neuroserpin [22], the effect on the polymerization of other serpins should be explored.…”

“…This fact combined with the very (∼1 M) high concentrations strongly support a non-specific effect for their action. This is acknowledged in the final paragraph of the Naseem et al discussion [25], but they also propose that the interaction must occur in a hydrophobic area of the antithrombin. This must be demonstrated through site-directed mutagenesis, and thus define the residues involved and the type of interaction.…”

“…This must be demonstrated through site-directed mutagenesis, and thus define the residues involved and the type of interaction. Naseem et al’s [25] paper showed using intrinsic fluorescence that trehalose stabilizes against the formation of the intermediate in the presence of GdnHCl. But they then choose a concentration of GdnHCl where they have shown that the intermediate is not populated (2 M) in an experiment using BisANS and infer that the low BisANS signal is due to shielding of a hydrophobic patch (rather than simply not being populated).…”

“…Antithrombin has a dynamically regulated reactive center loop, whose conformation can affect kinetics of interaction/inhibition with different proteases. Therefore the changes induced by a heavily modified solvent could affect reactive center loop conformation rather than the process of RCL insertion as contended by Naseem et al [25]. But the most important thing is to define the effect of trehalose or its derivatives in vivo .…”

“…In summary, the paper of Naseem et al [25] describes the potential of trehalose or molecules based on this scaffold to prevent the polymerization of antithrombin. Although more efforts should be made to obtain an efficient drug for use in patients, this is a step forward toward the ultimate goal.…”