Protein disulfide isomerase uses thrombin-antithrombin complex as a template to bind its target protein and alter the blood coagulation rates.

During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Naringin binds to protein disulfide isomerase to inhibit its activity and modulate the blood coagulation rates: Implications in controlling thrombosis.

Currently used antithrombotic drugs are beset with several drawbacks which necessitates the need for new and cheaper alternatives. Protein disulfide isomerase (PDI) is secreted in the blood plasma in cellular stress conditions and initiates the thrombus formation. A screening of library of natural compounds revealed that naringin had a high binding affinity for the PDI (-8.2 kcal/mol). Recombinant PDI was purified using the affinity chromatography. Incubation of purified PDI (3 µM) with naringin (0-100 µM, pH 7.4, 25 °C) partially modulated its conformation. Consequently, the fluorescence emission spectra of the PDI binding to naringin were assessed using the Stern-Volmer equation, which indicated an association constant of 2.78 × 104 M-1 suggesting an appreciable affinity for the naringin, with a unique binding site. An insulin turbidity assay showed that PDI activity is decreased in the presence of naringin indicating inhibition. Molecular dynamic simulation studies showed the changes in the PDI structure on binding to the naringin. Incubation of naringin (80 µM) in fresh human plasma along with exogenous PDI (175 nM) showed a significant delay in the intrinsic and extrinsic coagulation pathways. We show that naringin is able to modulate the PDI conformation and activity resulting in altered blood coagulation rates.

Flavonoids-induced redox cycling of copper ions leads to generation of reactive oxygen species: A potential role in cancer chemoprevention.

Flavonoids, a class of polyphenols are known to be effective inducers of apoptosis and cytotoxicity in cancer cells. It is believed that antioxidant activity of polyphenols cannot fully account for induction of apoptosis and chemotherapeutic prevention in various cancers. In this article, by employing single cell alkaline gel electrophoresis (comet assay), we established that antioxidants, flavonoids such as (myricetin=MN, fisetin=FN, quercetin=QN, kaempferol=KL and galangin=GN) can cause cellular DNA breakage, also act as pro-oxidant in presence of transition metal ion such as copper. It was observed that the extent of cellular DNA breakage was found significantly higher in presence of copper. Hydroxyl radicals are generated as a sign of flavonoids' pro-oxidant nature through redox recycling of copper ions. Further, a dose-dependent inhibition of proliferation of breast cancer cells MDA-MB-231 by MN was found leading to pro-oxidant cell death, as assessed by MTT assay. Since levels of copper are considerably elevated in tissue, cell and serum during various malignancies, suggesting that cancer cells would be more subject to copper induced oxidative DNA breakage. Such a copper dependent pro-oxidant cytotoxic mechanism better explains the anticancer activity and preferential cytotoxicity of dietary phytochemicals against cancer cells.

Chemotherapeutic Drugs and Plasma Proteins: Exploring New Dimensions.

In the last few decades, advances in the cancer chemotherapy have been a marked success. A large number of anticancer drugs currently in use include drugs based on platinum complexes such as cisplatin, base analogues such as 5-florouracil and some ruthenium drugs. This review provides a bird's eye view of interaction of a number of clinically important drugs currently in use that show covalent or non-covalent interaction with serum proteins. Platinum drug-cisplatin interacts covalently and alters the function of the key plasma protease inhibitor molecule -alpha-2-macroglobulin and induces the conformational changes in the protein molecule and inactivates it. 5-fluorouracil (5-FU) is extensively metabolized and at physiological concentrations, is found to be associated with Human Serum Albumin (HSA). Similarly ruthenium compounds bind tightly to plasma proteins- serum albumin and serum transferrin, modifying their biological activity and increasing the toxicity of drug to cancer cells. Insight into varied anticancer drug- protein interaction will go a long way in understanding in totality of the mechanism of action of any anticancer drug and its possible effects/side effects.

Conformational behavior of alpha-2-macroglobulin: Aggregation and inhibition induced by TFE.

Alpha-2-macroglobulin (α2M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body's innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α2M. TFE was sequentially added over a range of 0-20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α2M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α2M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α2M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α2M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α2M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α2M undergoes conformational and functional changes when exposed to TFE.

Identification of a new alpha-2-macroglobulin: Multi-spectroscopic and isothermal titration calorimetry study.

A α2M homologue was isolated from sheep (Ovis aries) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep α2M was found to be a large tetrameric glycoprotein of 630 kDa with monomeric subunit of 133 kDa each. Each subunit of sheep α2M was found to be made up of two fragments of 102 and 31 kDa respectively. The proteinase inhibitor from sheep was found to have Stokes radius of 79Ǻ, which makes it much more compact than its human homologue. It entraps only 1 mol of trypsin per mole of inhibitor, like its caprine counterpart. The use of isothermal titration calorimetry has become gold standard for exploring thermodynamics of binding interactions. In this study, binding interaction of trypsin with alpha-2-macroglobulin is studied using ITC. The thermodynamic signatures--enthalpy change (ΔH), entropy change (ΔS) and Gibb's free energy change (ΔG), along with number of binding sites (N) and affinity constant (K) are explored for α2M-trypsin binding for the first time for any known α2M molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin-α2M interaction is enthalpy driven event.

Reactive oxygen species and anti-proteinases.

Reactive oxygen species (ROS) cause damage to macromolecules such as proteins, lipids and DNA and alters their structure and function. When generated outside the cell, ROS can induce damage to anti-proteinases. Anti-proteinases are proteins that are involved in the control and regulation of proteolytic enzymes. The damage caused to anti-proteinase barrier disturbs the proteinase-anti-proteinases balance and uncontrolled proteolysis at the site of injury promotes tissue damage. Studies have shown that ROS damages anti-proteinase shield of the body by inactivating key members such as alpha-2-macroglobulin, alpha-1-antitrypsin. Hypochlorous acid inactivates α-1-antitrypsin by oxidizing a critical reactive methionine residue. Superoxide and hypochlorous acid are physiological inactivators of alpha-2-macroglobulin. The damage to anti-proteinase barrier induced by ROS is a hallmark of diseases such as atherosclerosis, emphysema and rheumatoid arthritis. Thus, understanding the behaviour of ROS-induced damage to anti-proteinases may helps us in development of strategies that could control these inflammatory reactions and diseases.