Deciphering the role of trehalose in hindering antithrombin polymerization.

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.

Oxidized antithrombin is a dual inhibitor of coagulation and angiogenesis: Importance of low heparin affinity.

Endogenous proteins that promote vascular endothelial cell based inhibition of angiogenesis are an attractive option for antitumor therapy. Inactive cleaved and latent conformations of antithrombin (AT) are antiangiogenic, but not its native form which is an inhibitor of proteases involved in blood coagulation. Unlike native, the cleaved and latent conformations are reactive center loop inserted conformations which binds heparin with very low affinity. We use a sulfoxy modified AT to assess the role of reactive center loop insertion and heparin affinity in antiangiogenic function. Chorioallantoic membrane assay (CAM) shows that antiangiogenic activity of latent and oxidized AT are better than thalidomide, a potent antiangiogenic drug. Wound healing experiments suggest that latent and oxidized conformations can influence endothelial cell migration. Latent and cleaved conformations of AT shows an increase in α-helical content in the presence of unfractionated heparin, but not the oxidized AT. Unlike the loop inserted polymer, cleaved and latent conformations, oxidized AT has factor Xa inhibitory activity indicating that loop insertion is not necessary for antiangiogenic role. The results of our study establish that active conformation of AT can become antiangiogenic while maintaining its anticoagulant activity possibly through chelation of low affinity heparin in the vicinity of endothelial cell.

Antiangiogenic function of antithrombin is dependent on its conformational variation: implication for other serpins.

Endogenous angiogenesis inhibitor that specifically decreases tumor cell proliferation can be used to treat cancer since angiogenesis is required at every step of tumor progression and metastasis. Endothelial cells are the main target for the antiangiogenic therapy because they are non-transformed and easily accessible to angiogenic inhibitors. Antithrombin functions as a principal plasma protein inhibitor of blood coagulation proteinases and belongs to the family of serine protease inhibitors (serpins) which have common mechanism of inhibition. Antithrombin acquires a potent antiangiogenic activity upon conversion of the native molecule to cleaved or latent conformation. Cleaved and latent preparations of bovine and human plasma derived antithrombin inhibits capillary endothelial cell proliferation and the growth of human SK-NAS neuroblastoma and Lewis lung carcinoma tumors in mice but not the native antithrombin's. The native form of antithrombin binds with high affinity to vascular heparan sulfate proteoglycans containing a specific pentasaccharide sequence and it is this cofactor interaction that activates antithrombin to maximal rate of thrombin inhibition. Upon inhibitory complex formation with target proteinases the antithrombin undergoes stressed to relaxed transformation and lose their high affinity for pentasacchride. Low affinity relaxed conformation with reduced heparin binding like cleaved and latent are antiangiogenic but native high affinity heparin binding stressed conformation is not, indicating the critical importance of heparin affinity in antithrombin antiangiogenic function. Based on evidence of interactions of the endothelial cell growth factors bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial cell growth factor) with heparin like molecule in matrix, the possibility of antiangiogenic antithrombin to interfere with endothelial cell growth and angiogenesis through heparin mediated mechanism deserves serious consideration and investigation. It is also possible that cleaved and latent conformations with reduced affinity for heparins can also induce conformational change in the antithrombin which can open an epitope on the antithrombin surface for appropriate interactions on the endothelial surface for better antiangiogenic activity. This review illustrates the potential of antithrombin and other serpin family members as endogenous antiangiogenic proteins.

Analysis of surface cavity in serpin family reveals potential binding sites for chemical chaperone to reduce polymerization.

Serpin constitute about 10% of blood protein and are associated with mutations that results in aberrant intermolecular linkages which leads to polymer formation. Studies with short peptides have shown promise in depolymerization of serpins however a reactive center loop based peptide also makes the serpin inactive. A chemical chaperone based approach is a better option in terms of maintaining activity and retarding polymerization but not much is known about its binding and mechanism. Specific target for chemical chaperones and its effectiveness across many serpin is not known. We did an analysis of serpin cavity using CASTp and show that cavities are distributed throughout the molecule where the largest cavities are generally present in areas of major conformational change like shutter region, helix D and helix F. An analysis of different conformational states of serpins showed that this large cavity undergoes increase in size in latent and cleaved states as compared to native state. We targeted serpins with a variety of carbohydrate, methylamine and amino acid based chemical chaperones and selected those that have highest binding energy across different serpins to assess their ability to bind large cavities. The results show that carbohydrate based chemical chaperone like sorbitol, sucrose, arabitol and trehalose and amino acid based chaperones like dopamine, phenylalanine, arginine and glutamic acid are the most effective in binding serpins. Most of these chemical chaperone interacted with residues in the shutter region and the helix D arm at the C-terminal which are part of the largest cavities. We selected the carbohydrate based chemical chaperone with best binding energies and did experimental study under the condition that induce polymerization and show that indeed they were able to retard polymer formation with moderate effect on inhibition rates. However a fluorometric study with native antithrombin showed that chemical chaperone may effect the conformation of the proteins. Our study shows that chemical chaperones have the best binding affinities for the cavities around shutter region and helix D and that a cavity targeting based approach seems to be a better option for retarding polymerization in serpins, but a thorough analysis of its effect on folding, inhibition and cofactor binding is required.

Serpin Inhibition Mechanism: A Delicate Balance between Native Metastable State and Polymerization.

The serpins (serine proteinase inhibitors) are structurally similar but functionally diverse proteins that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. Serpins play absolutely critical role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways and are associated with many conformational diseases. Serpin's native state is a metastable state which transforms to a more stable state during its inhibitory mechanism. Serpin in the native form is in the stressed (S) conformation that undergoes a transition to a relaxed (R) conformation for the protease inhibition. During this transition the region called as reactive center loop which interacts with target proteases, inserts itself into the center of ß-sheet A to form an extra strand. Serpin is delicately balanced to perform its function with many critical residues involved in maintaining metastability. However due to its typical mechanism of inhibition, naturally occurring serpin variants produces conformational instability that allows insertion of RCL of one molecule into the ß-sheet A of another to form a loop-sheet linkage leading to its polymerization and aggregation. Thus understanding the molecular basis and amino acid involved in serpin polymerization mechanism is critical to devising strategies for its cure.