Implications of posttranslational changes of fibrinogen on its reactivity and function

Fibrinogen je vaţan protein primarne i glavni protein sekundarne hemostaze. Nakon povrede, on se dejstvom trombina pretvara u nerastvorni fibrin koji se dalje umreţava, pri čemu nastaje fibrinska mreţa koja ojačava krvni ugrušak na mestu povrede. Da bi fibrinogen obavljao svoju biohemijsku ulogu, bitni su odreĎeni faktori, u koje spadaju njegove posttranslacione modifikacije i interakcije sa drugim proteinima. Posttranslacione modifikacije fibrinogena utiču na njegovu strukturu, strukturu fibrina i interakcije sa drugim proteinima. Fibrin nije pasivna mreţa koja samo daje potporu krvnom ugrušku, već je aktivna struktura koja reguliše svoju sintezu i razgradnju interakcijom sa brojnim proteinima. Zato je vaţno otkriti nove proteine koji sa njim interaguju, a koji imaju uticaj na proces zarastanja povreda.U okviru ove diseretacije, optimizovana je procedura za izolovanje i analizu fibrinogena. Upotrebom dvostrukog taloţenja etanolom dobijen je visoko prečišćen fibrinogen, pogodan za dalju karakterizaciju.Kako je poznato da fibrinogen interaguje sa IGFBP-3 proteinom, postavilo se pitanje da li još neki protein iz grupe vezujućih proteina za IGF, pri fiziološkim uslovima, ima tu sposobnost. Upotrebom većeg broja afinitetnih metoda je pokazano da IGFBP-1 interaguje sa fibrinogenom i da je to opšta fiziološka pojava. Značaj ove interakcije treba sagledavati imajući u vidu da i IGFBP-1 podstiče zarastanje tkivnih povreda, samostalno i kao transporter IGF molekula.Brojne patologije pri kojima se javljaju i koagulopatije, kao što su dijabetes melitus tipa 2 i ciroza jetre, karakteriše izmenjena koncentracija i struktura fibrinogena, što za posledicu ima stvaranje abnormalnog, trombogenog fibrina. Detaljno izučavanje pojedinačnih proteina uključenih u koagulopatiju moţe dati bliţu sliku mehanizma odgovornog za ovu pojavu. Sa druge strane, promene na nivou posttranslacionih modifikacija i strukture fibrinogena sa starenjem mogu doprineti boljem razumevanju prisustva ili odsustva odreĎenih patologija kod starijih ljudi.Struktura fibrinogena sa starenjem se menja. Primenom lektinskog eseja uočeno je povećanje visoko-manoznih i/ili hibridnih N-glikana, tri-/tertaantenarnih kompleksnih glikana sa većim sadrţajem Gal i GlcNAc. Spektrofluorimetrijska analiza je pokazala da kod zdravih ljudi preko 60 godina starosti, fibrinogen ima kompaktniju tercijarnustrukturu...Fibrinogen is an important protein of primary and main protein of secondary hemostasis. Upon injury, fibrinogen is converted to insoluble fibrin by the action of thrombin. Fibrin further cross-links and creates fibrin network which reinforces blood clotting at the site of injury. There is a significant contribution of posttranslational modifications as well as interactions with other proteins necessary for fulfillment of fibrinogen biochemical role. Posttranslational modifications of fibrinogen influence its structure, fibrin structure and interactions with other proteins. Fibrin is not only a passive network that supports blood clot, but also an active structure that regulates its synthesis and degradation by interacting with many different proteins. For this reason, it is important to identify new proteins which interact with fibrinogen and may also have a role in wound healing.The procedure for isolation and analysis of fibrinogen was optimized in this dissertation. Application of double precipitation using ethanol resulted in highly purified fibrinogen, suitable for further characterisation.Since it is known that fibrinogen interacts with IGFBP-3 protein, the question was raised weather some other protein from the family of the IGF-binding proteins has this ability under physiological conditions. By using several affinity methods, it was shown that IGFBP-1 interacts with fibrinogen and this is a general physiological event. The significance of this interaction should be evaluated taking into consideration that IGFBP-1 itself may have beneficial effect on tissue wound healing, alone and as a transporter of the IGF molecule.Several pathologies accompanied by coagulopathies, such as diabetes mellitus type 2 and cirrhosis, are characterised by altered concentration and structure of fibrinogen, which in turn creates abnormal, thrombogenic fibrin. Detailed study of individual proteins included in coagulopathy may enable closer look at mechanisms responsible for this outcome. On the other hand, changes in posttranslational modifications and structure of fibrinogen with aging may lead to better understanding of presence or absence of certain pathologies associated with ageing.The structure of fibrinogen alteres with aging. An increase of high-mannose and/or hybrid N-glycans, tri-/tetraantennary complex glycans with greater amounts ofGal and GlcNAc was detected by lectin array..

Characterisation of the binding of dihydro-alpha-lipoic acid to fibrinogen and the effects on fibrinogen oxidation and fibrin formation

A reduced form of the alpha-lipoic acid, dihydro-alpha-lipoic acid (DHLA) is a potent, naturally occurring antioxidant which can be consumed as food constituent or as supplement at doses up to 600 mg/day. DHLA has inhibitory effect on coagulation as it can reduce concentrations of some coagulation factors. In this study, a direct interaction between DHLA and fibrinogen, the main protein in coagulation, is described. Binding constant for DHLA/fibrinogen complex is of moderate strength (104) and interaction probably occurs in D regions of fibrinogen, as shown by docking simulations. Fibrinogen stability remains the same with only marginal structural changes in its secondary structure favouring more ordered molecular organisation upon DHLA binding. Fibrinogen with bound DHLA forms fibrin with thicker fibers, as measured by coagulation assay and is protected from oxidation to certain extent. Obtained results support beneficial effects of DHLA on fibrinogen and consequently on coagulation process, suggesting that DHLA supplementation may be indicated for persons with an increased risk of developing thrombotic complications, particularly those whose fibrin is characterised by increased oxidative modification and formation of thinner and less porous fibers. Also, DHLA in complex with fibrinogen can be located at site of injury where it may exert antioxidant effects.This is the peer-reviewed version of the article: International Journal of Biological Macromolecules, 2020, 147, 319-325, doi: [https://dx.doi.org/10.1016/j.ijbiomac.2020.01.098]The published version: [http://cer.ihtm.bg.ac.rs/handle/123456789/3378

Ovalbumin adsorption on different types of microplastic

Microplastics (MPs) are small in size, have low densities, can exist in the atmosphere for a long time and can easily be spread by wind. Microplastics are plastic fibers, particles or films with diameters smaller than 5 mm and they have shown different effects on proteins. The objective of this study was to investigate adsorption affinity of different types of MPs (polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC)) with ovalbumin. Ovalbumin, isolated from chicken egg white, was used in this study. Plastics were mixed with ovalbumin for 1,2,4 and 19 h and then the absorbance of the remaining protein in the solution was measured at 280nm. In addition, isotherm mathematical model to calculate the adsorption affinity of ovalbumin for MPs was used. We determined affinity constants by using Langmuir isotherm models for different particle size (PS 120 m and PS 500 m), different type of plastics (PET, PS and PVC) and pH values (3 and 7,2). Adsorption experiment results showed that adsorption depends on type of plastics. Our results showed that PVC did not adsorb protein, however, PET and PS have interacted with protein. Adsorption capacities of all analysed MPs increase with pH of solution. Under different pH values, MPs and protein have different charges that may affect adsorption characteristics. With increase of pH from 3 to 7, the level of protein adsorption on MPs increased 14 times for PS (smaller in size) and 5 times for PS (bigger size) and PET

Binding affinity ovalbumin on different type of microplastics using Langmuir isotherm

Microplastics are plastic fibers, particles or films with diameters smaller than 5 mm and they have shown different effects on proteins [1]. Microplastics (MPs) are small in size, have low densities, can exist in the atmosphere for a long time and can easily be spread by wind [2]. The objective of this study was to investigate adsorption affinity of different types of MPs (polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC)) with ovalbumin. In this study ovalbumin, isolated from chicken egg white, was used. Plastics were mixed with ovalbumin for 1,2,4 and 19 h and then the absorbance of the remaining protein in the solution was measured at 280nm. In addition, Langmuir isotherm mathematical model to calculate the adsorption affinity of ovalbumin for MPs was used. We determined affinity constants by using Langmuir isotherm models for different particle size (PS 120 μm and PS 500 μm), different types of plastics (PET, PS and PVC) and pH values (3 and 7,2). Adsorption experiment results showed that adsorption depends on type of plastics. Our results showed that PVC did not adsorb ovalbumin, however, PET and PS have interacted with protein. Adsorption capacities of all analysed MPs increase with pH of solution. Under different pH values, MPs and protein have different charges that may affect adsorption characteristics. With increase of pH from 3 to 7, the affinity for protein adsorption increased 1.4 times for PS (smaller in size) while for PS (bigger size) and PET protein affinity was two times higher at pH 3

Physicochemical characterisation of dihydro-alpha-lipoic acid interaction with human serum albumin by multi-spectroscopic and molecular modelling approaches

The binding of a popular food supplement and well-known antioxidant, dihydro-alpha-lipoic acid (DHLA) to human serum albumin (HSA) was characterised. The binding was monitored by several spectroscopic methods together with the molecular docking approach. HSA was able to bind DHLA with moderate affinity, 1.00±0.05×104 M-1. Spectroscopic data demonstrated that the preferential binding site for DHLA on HSA is IIA (Sudlow I). Both experimental and molecular docking analysis identified electrostatic (salt bridges) and hydrogen bonds as the key interactions involved in DHLA binding to HSA. Molecular docking confirmed that the Sudlow I site could accommodate DHLA and that the ligand is bound to the protein in a specific conformation. The molecular dynamic simulation showed that the formed complex is stable. Binding of DHLA does not affect the structure of the protein, but it thermally stabilises HSA. Bound DHLA had no effect on the susceptibility of HSA to trypsin digestion. Since DHLA is a commonly used food supplement, knowledge of its pharmacokinetics and pharmacodynamic properties in an organism is very important. This study further expands it by providing a detailed analysis of its interaction with HSA, the primary drug transporter in the circulation

Atypical antipsychotic clozapine binds fibrinogen and affects fibrin formation

Clozapine is an atypical antipsychotic used for the treatment of schizophrenia. The prescribed target daily doses may reach 900 mg. Literature studies report a connection between clozapine usage and thrombosis development. Our in vitro study aimed to provide insight into molecular bases of this observation, investigating clozapine binding to fibrinogen, the main plasma protein involved in hemostasis. Fibrinogen/clozapine interaction was confirmed by protein fluorescence quenching, with an affinity constant of 1.7 × 105 M−1. Direct interactions did not affect the structure of fibrinogen, nor fibrinogen melting temperature. Clozapine binding affected fibrin formation by reducing coagulation speed and thickness of fibrin fibers suggesting that in the presence of clozapine, fibrinogen may acquire thrombogenic characteristics. Although no difference in fibrin gel porosity was detected, other factors present in the blood may act synergistically with altered fibrin formation to modify fibrin clot, thus increasing the risk for development of thrombosis in patients on clozapine treatment. ORAC and HORAC assays showed that clozapine reduced free radical-induced oxidation of fibrinogen. All observed effects of clozapine on fibrinogen are dose-dependent, with the effect on fibrin formation being more pronounced.This is the peer-reviewed version of the article: Gligorijević, N.; Vasović, T.; Lević, S. M.; Miljević, Č.; Nedić, O.; Nikolić, M. Atypical Antipsychotic Clozapine Binds Fibrinogen and Affects Fibrin Formation. International Journal of Biological Macromolecules 2020, 154, 142–149. [https://doi.org/10.1016/j.ijbiomac.2020.03.119

A thin layer chromatographic comparison of raw and soluble starch hydrolysis patterns of some alpha-amylases from Bacillus sp isolated in Serbia

Several natural isolates of Bacillus strains namely 5B, 12B, 16B, 18 and 24B were grown at two different temperatures in submerged fermentation for the production of raw-starch-digesting alpha-amylases. All strains except Bacillus sp. 18 produced more alpha-amylase at 37 degrees C. The hydrolysis of raw cornstarch followed the same pattern. Efficient hydrolysis was obtained with alpha-amylases from Bacillus sp. 5B, 12B, 16B and 24B grown at 37 degrees C and Bacillus sp. 18 grown at 50 degrees C. Zymography after isoelectric focusing showed that alpha-amylases were produced in multiple forms, from 2 to 6, depending on the strain when they were growing at 37 degrees C, while growth at 50 degrees C induced only 1 or 2 isoforms. Thin layer chromatography (TLC) analysis of the hydrolysis products of raw corn and soluble starch by alpha-amylases revealed the production of various mixtures of oligosaccharides. In most cases, G3 was the most dominant product from soluble starch while G2, G3 and G5 were the main products of raw starch hydrolysis. This indicates that the obtained alpha-amylases could be used for starch liquidification or short-chain-oligosaccharide formation, depending on the type of starch (raw or soluble) used for the hydrolysis

Antipsychotic clozapine binds catalase and preserves its activity in oxidative environment

Oxidative stress undoubtedly accompanies mental disorders, and the pleiotropic effects of atypical antipsychotics, recommended drugs in the treatment of psychosis, are not clarified at the molecular level. Catalase is one of the key enzymes of the primary antioxidant protection system. This work studied the binding of second-generation antipsychotic drug Clozapine to commercial bovine liver catalase. Using various spectroscopic methods under simulated physiological conditions, we found moderate binding affinity of clozapine for catalase (Ka ~ 2x105 M-1), the binding influenced the secondary and tertiary structure of protein (according to UV-VIS and CD spectroscopy) and it managed to slightly increase its thermal stability. In AAPH induced oxidation experiments, we found that clozapine efficiently protects catalase from free-radicals oxidation and preserves its activity. Clozapine affects catalase activity in dose dependant manner, having no significant effect at lower concentrations but significantly inhibiting enzyme at saturating concentrations. In conclusion, our results indicate that the effect of direct binding of clozapine to catalase can be both beneficial and harmful and that this effect is dose dependent.The Biochemistry Global Summit, 25th IUBMB Congress, 46th FEBS Congress, 15th PABMB Congress, July 9-14, 2022, Lisbon, Portuga

Stabilization of C-phycocyanin by immobilization in alginate beads

C-Phycocyanin (C-PC), the major protein of cyanobacteria Arthrospira platensis, is a phycobiliprotein with potent biological activity. It has several beneficial effects, including anti-oxidant, anti-inflammatory, immunomodulatory, and anti-cancer. A significant challenge for the broader application of C-PC in the food industry is its stability in food processing conditions, such as increased light exposure, temperature, and high pressure and drying. This work aimed to investigate if the immobilization of C-PC onto alginate beads could improve its stability. C-PC was immobilized by dropping the solution of C-PC and 1% alginate (final concentration) in the solution of 2% CaCl2. Both protein/alginate mixture and CaCl2 were kept at pH 4. Immobilized CPC was treated for 30 min at 65°C, by high pressure up to 4500 bar, and incubated under light exposure for a month. Alginate beads with immobilized C-PC were also left to dry in the fridge and kept for a month. C-PC was extracted from alginate beads by immersing them in 20 mM phosphate buffer, pH 7. The stability of C-PC was assessed by a color change and UV-VIS spectroscopy. Immobilized C-PC was stable under all tested conditions, with only small aggregation and color change appearing after high-pressure treatment. Immobilization of C-PC by alginate thus shows promise for its efficient stabilization under food processing conditions

Dihydro-alpha-lipoic acid binds and protects fibrinogen from oxidation and affects fibrin formation

A reduced form of the alpha-lipoic acid, dihydro-alpha-lipoic acid (DHLA) is a potent, naturally occurring antioxidant that is found in higher amounts in plants like spinach, broccoli, potatoes, tomatoes, carrots, beets and rice. DHLA can be consumed as a food supplement as well, at doses up to 600 mg/day. DHLA has an inhibitory effect on coagulation as it can reduce concentrations of some coagulation factors. This study investigated a direct interaction between DHLA and fibrinogen, the main protein in coagulation and hemostasis. DHLA binds fibrinogen with a moderate straight. Calculated constant from spectrofluorimetric titration for DHLA/fibrinogen complex was 104 M -1. Fibrinogen stability remains the same with only marginal structural changes in its secondary structure favouring more ordered molecular organisation upon DHLA binding, as determined by Fourier transform infrared spectroscopy. Coagulation assay showed that fibrinogen with bound DHLA forms fibrin with thicker fibres, as measured by coagulation assay and is protected from oxidation to a certain extent. Docking analysis showed that DHLA may bind fibrinogen in its D regions, which are directly involved in the fibrin formation. Obtained results support beneficial effects of DHLA on fibrinogen and consequently on coagulation process, suggesting that DHLA supplementation may be indicated for persons with an increased risk of developing thrombotic complications, particularly those whose fibrin is characterised by increased oxidative modification and formation of thinner and less porous fibres. Although further investigation is needed, our results suggest that DHLA in complex with fibrinogen can be located at the site of injury where it may exert antioxidant effects.45th FEBS Congress, Molecules of Life: Towards New Horizons, Ljubljana, Slovenia, July 3-8, 202