Coagulation Proteins Research Articles

Pharmacological targeting of coagulation factor XI attenuates experimental autoimmune encephalomyelitis in mice.

Multiple sclerosis (MS) is the most common causes of non-traumatic disability in young adults worldwide. MS pathophysiologies include the formation of inflammatory lesions, axonal damage and demyelination, and blood brain barrier (BBB) disruption. Coagulation proteins, including factor (F)XII, can serve as important mediators of the adaptive immune response during neuroinflammation. Indeed, plasma FXII levels are increased during relapse in relapsing-remitting MS patients, and previous studies showed that reducing FXII levels was protective in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Our objective was to determine if pharmacological targeting of FXI, a major substrate of activated FXII (FXIIa), improves neurological function and attenuates CNS damage in the setting of EAE. EAE was induced in male mice using murine myelin oligodendrocyte glycoprotein peptides combined with heat-inactivated Mycobacterium tuberculosis and pertussis toxin. Upon onset of symptoms, mice were treated every other day intravenously with anti-FXI antibody, 14E11, or saline. Disease scores were recorded daily until euthanasia for ex vivo analyses of inflammation. Compared to the vehicle control, 14E11 treatment reduced the clinical severity of EAE and total mononuclear cells, including CD11b+CD45high macrophage/microglia and CD4+ T cell numbers in brain. Following pharmacological targeting of FXI, BBB disruption was reduced, as measured by decreased axonal damage and fibrin(ogen) accumulation in the spinal cord. These data demonstrate that pharmacological inhibition of FXI reduces disease severity, immune cell migration, axonal damage, and BBB disruption in mice with EAE. Thus, therapeutic agents targeting FXI and FXII may provide a useful approach for treating autoimmune and neurologic disorders.

Quantification of proteomic profile changes in the hemolymph of crayfish during in vitro coagulation

Hemolymph is the circulatory fluid that fills the body cavity of crustaceans, analogous to blood in vertebrates. Hemolymph coagulation, similar to blood clotting in vertebrates, plays a crucial role in wound healing and innate immune responses. Despite extensive studies on the clotting process in crustaceans, no comparative quantitative analysis of the protein composition of non-clotted and clotted hemolymph in any decapod has been reported. In this study, we used label-free protein quantification with high-resolution mass spectrometry to identify the proteomic profile of hemolymph in crayfish and quantify significant changes in protein abundances between non-clotted and clotted hemolymph. Our analysis identified a total of two-hundred and nineteen proteins in both hemolymph groups. Furthermore, we discussed the potential functions of the top most high and low-abundant proteins in hemolymph proteomic profile. The quantity of most of the proteins was not significantly changed during coagulation between non-clotted and clotted hemolymph, which may indicate that clotting proteins are likely pre-synthesized, allowing for a swift coagulation response to injury. Four proteins still showed abundance differences (p < 0.05, fold change>2), including C-type lectin domain-containing proteins, Laminin A chain, Tropomyosin, and Reverse transcriptase domain-containing proteins. While the first three proteins were down-regulated, the last one was up-regulated. The down-regulation of structural and cytoskeletal proteins may affect the process of hemocyte degranulation needed for coagulation, while the up-regulation of an immune-related protein might be attributed to the phagocytosis ability of viable hemocytes during coagulation.

Comparative study on the effect of different salts on surimi gelation and gel properties

The effects of four representative salts, KCl, NaCl, CaCl2 and MgCl2, on surimi gel properties were comprehensively investigated. During gelation, monovalent salts are prone to convert α-helix to β-sheet while divalent salts tend to change α-helix to β-turn, thus altering the protein conformation differently. Monovalent salts extract and swell myofibrillar proteins more effectively, generating an ordered and smooth gel network with appreciable water-holding capacity (WHC). Ca2+ activates transglutaminase (TGase) to crosslink surimi proteins by forming ε-(γ-glutamyl) lysine bonds, while Mg2+ forms salt bridges with adjacent proteins, leading to a gel with more developed protein complexes. However, excessive divalent salts would induce serious protein coagulation, forming poor networks and lowering water retention. Ionic strength within 0.2–0.5 effectively reinforced the gel structure, while the WHC of surimi gels reached its peak with an ionic strength of around 0.6. At the optimal salt concentration of 1.5%, K+ with a larger ionic radius showed inferior effects on forming ionic bonds among proteins, whereas other metal ions induced more hydrophobic interactions within surimi gels. Mg2+, with higher oxidability than other metal ions, could enhance the formation of disulfide bonds and inhibit the browning caused by the Maillard reaction, thus improving gel whiteness. This study provides new insides into the roles of common salts during surimi gelation and gel properties and is expected to guide further gelation innovation and gel product development.

The new era of anticoagulation: factor XI and XII inhibitors

The two last decades have witnessed a revolution in the field of anticoagulation, mainly due to the advent of direct anticoagulant with targeted action against single coagulation proteins. However, the residual risk of cardio- and cerebrovascular events, particularly in some critical settings, and the risk of major bleeding still represent unmet medical needs. Preclinical studies and experience from families with genetic deficiencies of factor XI or XII (FXI and FXII) allowed to identify these factors involved in the contact pathway of coagulation as potential targets for new anticoagulant approaches. To date, several pharmacological classes of FXI and FXII inhibitors have been developed, including antisense oligonucleotides, monoclonal antibodies, small molecules, natural inhibitors, and aptamers, and various molecules are currently under phase 2 or 3 clinical investigation. Particularly, promising results have been obtained in patients undergoing major orthopedic surgery, in those with end-stage kidney disease, atrial fibrillation and acute coronary syndrome. This review summarizes current knowledge on FXI and FXII inhibitors, with a particular focus on their pharmacological properties and potential clinical indications.

Inflating Pressure and Not Expiratory Pressure Initiates Lung Injury at Birth in Preterm Lambs.

Rationale: Inflation is essential for aeration at birth, but current inflating pressure settings are without an evidence base. Objectives: To determine the role of inflating pressure (ΔP), and relationship with positive end-expiratory pressure (PEEP), in initiating early lung injury pathways in the preterm lamb lung. Methods: 124-127d steroid exposed preterm lambs (n=45) were randomly allocated (8-10/gp) to 15 min of respiratory support with placental circulation and 20 or 30 cmH2O ΔP, with an initial high PEEP (maximum 20 cmH2O) recruitment manoeuvre known to facilitate aeration (dynamic PEEP), and compared to dynamic PEEP with no ΔP or 30 cmH2O ΔP and low (4 cmH2O) PEEP. Lung mechanics and aeration were measured throughout. after an additional 30 min of apnoeic placental support, lung tissue and bronchoalveolar fluid were analysed for regional lung injury, including proteomics. Results: 30 cmH2O ΔP and dynamic PEEP resulted in quicker aeration, better compliance, but higher tidal volumes (often >8 ml/kg, all p<0.0001; mixed effects) and injury. ΔP 20 cmH2O with dynamic PEEP resulted in the same lung mechanics and aeration, but less energy transmission (tidal mechanical power), as ΔP 30 cmH2O with low PEEP.. Dynamic PEEP without any tidal inflations resulted in the least lung injury. Use of any tidal inflating pressures altered metabolic, coagulation and complemented protein pathways within the lung. Conclusions: Inflating pressure is essential for the preterm lung at birth, but also the primary mediator of lung injury. Greater focus is needed on strategies that identify safest application of pressure in the delivery room.

Size Matters? A Comprehensive In Vitro Study of the Impact of Particle Size on the Toxicity of ZnO.

This study describes a comparative in vitro study of the toxicity behavior of zinc oxide (ZnO) nanoparticles and micro-sized particles. The study aimed to understand the impact of particle size on ZnO toxicity by characterizing the particles in different media, including cell culture media, human plasma, and protein solutions (bovine serum albumin and fibrinogen). The particles and their interactions with proteins were characterized in the study using a variety of methods, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Hemolytic activity, coagulation time, and cell viability assays were used to assess ZnO toxicity. The results highlight the complex interactions between ZnO NPs and biological systems, including their aggregation behavior, hemolytic activity, protein corona formation, coagulation effects, and cytotoxicity. Additionally, the study indicates that ZnO nanoparticles are not more toxic than micro-sized particles, and the 50 nm particle results were, in general, the least toxic. Furthermore, the study found that, at low concentrations, no acute toxicity was observed. Overall, this study provides important insights into the toxicity behavior of ZnO particles and highlights that no direct relationship between nanometer size and toxicity can be directly attributed.

Influence of Calcium-Sequestering Salts on Heat-Induced Changes in Blends of Skimmed Buffalo and Bovine Milk.

Heat-induced interactions of calcium and protein in milk lead to undesirable changes in the milk, such as protein coagulation, which can be minimized through the addition of calcium-sequestering salts prior to heat treatment. Thus, the present study investigated the influence of 5 mM added trisodium citrate (TSC) or disodium hydrogen phosphate (DSHP) on the heat-induced (85 °C and 95 °C for 5 min) changes in physical, chemical, and structural properties of buffalo and bovine skim milk mixtures (0:100, 25:75, 50:50, 75:25, and 100:0). Significant changes in pH and calcium activity as a result of TSC or DSHP addition subsequently resulted in higher particle size and viscosity as well as non-sedimentable protein level. These changes are mostly observed during heat treatment at 95 °C and increased proportionally to the concentration of buffalo skim milk in the milk mixture. Significant changes were affected by TSC addition in the 75:25 buffalo:bovine milk blend and buffalo skim milk, but for other milk samples, TSC addition effected comparable changes with DSHP addition. Overall, the addition of TSC or DSHP before heat treatment of buffalo:bovine milk blends caused changes in milk properties that could reduce susceptibility of milk to coagulation.

Immobilization of blood coagulant factor VII on polycaprolactone membrane through polydopamine grafting

Postoperative bleeding following cardiac surgeries is still an issue that deranges the medical resources and cost. The oral and injection administrations of blood coagulation protein, Factor VII (FVII), is effective to stop the bleeding. However, its short half-life has limited the effectiveness of this treatment and frequent FVII intake may distress the patients. Instead, incorporating FVII into synthetic biodegradable polymers such as polycaprolactone (PCL) that is commonly used in drug delivery applications should provide a solution. Therefore, this study aimed to immobilize FVII on PCL membranes through a cross-linkage polydopamine (PDA) grafting as an intermediate layer. These membranes are intended to provide a solution for cardiac bleeding in coagulating blood and sealing the sutured region. The membranes were evaluated in terms of its physio-chemical properties, thermal behavior, FVII release profile and biocompatibility properties. The ATR-FTIR was used to analyze the chemical functionalities of the membranes. Further validation was done with XPS where the appearances of 0.45 ± 0.06% sulfur composition and C–S peak have confirmed the immobilization of FVII on the PCL membranes. The cross-linked FVIIs were viewed in spherical immobilization on the PCL membranes with a size range between 30 and 210 nm. The surface roughness and hydrophilicity of the membranes were enhanced with a slight shift of melting temperature. The PCL-PDA-FVII0.03 and PCL-PDA-FVII0.05 membranes, with wide area of FVII immobilization released approximately only 22% of FVII into the solution within 60 days period and, it is found that the PCL-PDA-FVIIx membranes projected the Higuchi release model with non-Fickian anomalous transport. While the cytotoxic and hemocompatibility analyses showed advance cell viability, identical coagulation time and low hemolysis ratio on the PCL-PDA-FVIIx membranes. The erythrocytes were viewed in polyhedrocyte coagulated structure under SEM visualization. These results validate the biocompatibility of the membranes and its ability to prolong blood coagulation, thus highlighting its potential application as cardiac bleeding sealant.

TMEM16E regulates endothelial cell procoagulant activity and thrombosis.

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

High fibrinogen levels are associated with poor survival in patients with liposarcoma

The aim of this study was to evaluate whether (preoperative) plasma levels of fibrinogen, an essential clotting and acute phase protein, are associated with the prognosis of patients with a liposarcoma, a subtype of sarcoma derived from adipose tissue. We performed a retrospective cohort study of 158 patients with liposarcoma treated at the Department of Orthopaedics of the Medical University of Vienna in Austria from May 1994 to October 2021. Kaplan–Meier curves as well as uni- and multivariable Cox proportional hazard models were performed to evaluate the association between fibrinogen levels and overall survival. Elevated fibrinogen was associated with adverse overall survival in cause specific hazards analysis of mortality (hazard ratio [HR] per 10 mg/dL increase: 1.04; 95% CI 1.02–1.06; p < 0.001). This association prevailed in multivariable analysis after adjustment for AJCC tumor stage (HR 1.03; 95% CI 1.01–1.05; p = 0.013). Increasing levels of fibrinogen, a routinely available and inexpensive parameter, predicts the risk of mortality in patients with liposarcoma.

The impact of extract of mistletoe on tumour cells and hemostasis of human plasma

Background: The plant Viscum album (Mistletoe) is a known source of biologically active substances, used in traditional medicine in Europe and Asia.Aim: The goal was to study cytotoxic/cytostatic effect of mistletoe extract on tumour and normal cells and its influences on certain proteins of the blood coagulation system.Setting: Mistletoe was collected in Ukraine in November 2020.Methods: Water extract of V. album, both leaves and stems, was obtained and fractionated; it was characterised using high-performance liquid chromatography (HPLC) and mass spectrometry and tested on cancer cells and blood plasma.Results: Extract demonstrated the presence of viscotoxins and carbohydrates and thermolabile compounds that enhance the activity of thrombin and factor X in plasma in the presence of calcium ions and increased the activated partial thromboplastin time (APTT) by 2.7 times. The cytotoxic/cytostatic action of the mistletoe fractions, the total extract and the fraction 10 000 Da were higher in relation to Lewis lung carcinoma (LLC) cells than with Vero cells. The IC50 of total extract for LLC cells was 35% (p 0.05) lower than that for Vero cells (1.12 ± 0.2 and 1.71 ± 0.15 mg/ml). Thus, the IC50 of the fraction 10 000 Da for LLC cells was more than by 44% (p 0.05) lower than that for Vero cells (2.07 ± 0.26 and 3.67 ± 0.41 mg/ml).Conclusion: It was shown that both extracts exhibited a cytotoxic/cytostatic effect, more pronounced against tumour cells than normal cells and enhanced the clotting of blood plasma by thermolabile compounds by facilitating plasma coagulation.Contribution: This research makes it possible to study mistletoe as a light and cheap anticancer therapy plant and cure blood coagulopathy or construct antibleeding bandages.

In vitro gastric digestion of an experimental infant formula containing both intact and hydrolyzed milk proteins

Milk protein hydrolysates may have several benefits for digestion and digestion-related complications in infants, whereas intact milk proteins have been demonstrated to provide functionality beyond their nutritional value. In this study, in vitro digestion of an experimental infant formula containing both intact milk proteins and a milk protein hydrolysate was determined. Relative to an intact milk protein control formula, the experimental formula displayed a higher initial protein digestion during simulated gastric digestion as illustrated by a larger proportion of smaller peptides and higher level of available amino groups during digestion. Gastric protein coagulation was not affected by the hydrolysate addition. Further in vivo studies should demonstrate whether partial replacement of the protein source by a hydrolysate and observed differences in in vitro protein digestion result in overall altered protein digestion and absorption kinetics or affect functional gastrointestinal disorders as has been demonstrated for full hydrolysate formula.

Proteomics of the haemolymph of the terrestrial crustacean Porcellio scaber reveals components of its innate immunity under baseline conditions

The terrestrial crustacean Porcellio scaber is an established test organism in environmental research. We analysed the haemolymph proteome of P. scaber using a classical proteomic approach based on one-dimensional gel electrophoresis and tandem mass spectrometry. Using a publicly available protein database and our P. scaber transcriptome data, we have identified 76 proteins involved in cytoskeleton formation, protein degradation, vesicular transport, genetic information processing, detoxification, carbohydrate and lipid metabolism reflecting haemocyte metabolic activity, active intracellular transport, and intercellular communication. Compared with the data reported for other crustaceans, 28 of these P. scaber proteins have been linked to its immunity, among them hemocyanin, α-2-macroglobulin, phenoloxidase 3, superoxide dismutase, glutathione S-transferase, haemolymph clottable protein, and histones H4 and H2B. Our results thus provide a firm base for studying the innate immune response of P. scaber at the level of the haemolymph proteome. This knowledge is of particular importance in ecotoxicity studies with various environmental stressors where understanding physiological changes is important to reveal possible modes of action.

Blood Coagulation and Beyond: Position Paper from the Fourth Maastricht Consensus Conference on Thrombosis.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited.

Rapid detection of the adulterants in milk using gamma radiation

Milk and dairy adulteration practice nowadays is becoming a global concern. The present study discloses the role of gamma irradiation as a cost-effective and rapid-detection approach to detect adulterants in milk instead of using expensive and time-consuming chemical-based methods. In the present study, synthetic milk samples were prepared using different common adulterants (vegetable oil, urea, water) and a comparative analysis has been done with market milk samples along with fresh milk based upon the attenuation property. The obtained linear attenuation coefficient decreases significantly (p < 0.05) with the increasing concentration of adulterants in sample. The recorded attenuation coefficient for fresh milk was 0.379 ± 0.04 and that of adulterated samples found to be in the range of 0.296–0.188. Moreover, different protein coagulation patterns of the heat-processed milk samples were detected. The coagulated protein of adulterated milk shows a granular texture. The attenuation coefficient of milk samples decreases with the gradual increase of adulterants. The correlation between the attenuation coefficient and adulterants in milk indicated the possible use of gamma radiation as a significant adulterant-detection method.

Sensory indicators of suluguni cheese when using enzyme preparations of different origins in the technology of soft cheeses

Cheeses are food products obtained by concentration and biotransformation of the main components of milk under the influence of physicochemical factors of rennet enzyme preparations and microorganisms. Suluguni occupies an important place among cheeses in Ukraine. Suluguni cheese has been known for a long time, it was first mentioned in the book of the Pope's personal chef Bartolomeo Scipri. Industrial production of the product was established at the end of the 18th century. The main operation in the production of rennet cheese is the enzymatic coagulation of milk under the action of chymosin, resulting in the formation of a milk clot with a large part of casein and whey. In the body of animals, chymosin, similar to cheese-making technology, coagulates milk at the beginning of its digestion. Thus, the processing of milk in the process of cheese production corresponds to natural physiological processes. III groups of milk samples (n = 5) were formed to conduct the experiment. In the control group of samples, rennet enzyme of microbial origin was used for curdling milk. In the 1st experimental group of samples, an enzyme preparation from the rennet of dairy calves, extracted according to the method of Yu. Ya. Svyridenko, was used. In the II experimental group, an enzyme preparation was used, which was extracted from the rennet of dairy calves according to the method of S. V. Merzlova. The duration of protein coagulation during the production of “Suluguni” cheese using a microbial enzyme preparation is 23 min., when using enzymes of the first research group – 27 min. and when using rennet enzymes of the II research group – 33 min. In view of the output of ready-made Suluguni cheese with a commercial purpose to obtain more profit, it is advisable to use an enzyme of microbial origin. According to sensory analysis, samples using calf rennet enzyme extracted according to the method of S. V. Merzlova (I research group of samples) were characterized by pronounced cheesy, sour-milk, without extraneous tastes and smells, taste and smell characteristic of soft fresh cheese; the surface is clean without mechanical damage, elastic; the consistency of the smear is gentle, moderately dense; the color is white, uniform throughout the mass. Samples of the II experimental group were characterized by the worst organoleptic indicators.

Pendampingan Pemanfaatan Limbah Tahu Sebagai Pupuk Organik Cair (Poc) Bagi Masyarakat Kelurahan Kayu Manis

Tofu is a food product that comes from processed products (protein coagulation process) of soybeans. This home-based tofu industry, in its processing, can produce waste, namely solid waste (soybean dregs) and liquid waste. Handling of tofu liquid waste is not only a temporary treatment but needs to be done on an ongoing basis, one of which is by making valuable agricultural products having economic value. The methods for implementing this Community Self-Reliance Activity (KKM) include debriefing, team coordination, training, Liquid Organic Fertilizer (POC) application, mentoring and monitoring. The people involved were UKM Harjasari and KWT Ceria Mandiri Kayu Manis. The results of this activity indicate that community service in the form of assistance in using liquid tofu waste as a POC product can reduce environmental pollution and increase public knowledge about the proper use of natural resources. This can be seen from the increase in general understanding and its application, with an average increase of 62.28%.

Effect of CSN3 Gene Polymorphism on the Formation of Milk Gels Induced by Physical, Chemical, and Biotechnological Factors.

During the last decade, research into genetic markers in the casein gene cluster has been actively introduced in cattle breeding programs. A special interest has been paid to the polymorphism of the CSN3 gene, responsible for the expression of the k-casein, playing a key role in protein coagulation, interaction with whey proteins, stabilization, and aggregation of casein micelles. This paper aimed to determine the effect of CSN3 genetic polymorphism on acid; rennet; acid-rennet; heat- and acid-induced as well as heat- and calcium-induced coagulation in skimmed milk; and protein-standardized milk systems (UF, NF, RO, VE). The influence of polymorphic variants of the CSN3 gene on the coagulation ability of milk proteins was assessed by the particle size of casein micelles, protein retention factor in the clot, and coagulation ability (duration of induction period, mass coagulation period, dynamic viscosity in gel point). The correlation between CSN3 gene polymorphism and protein coagulation was revealed. Milk systems obtained from CSN3 BB milk were found to have the shortest duration of coagulation, formation of better gel strength values, and increased yield compared to CSN3 AA. This study will improve the efficiency of milk processing and optimize the technology of dairy product production.

Bacterial Production of Recombinant Coagulation Factor VIII Domains.

Factor VIII (F8) is a blood coagulation protein prearranged in six domains, and its deficiency causes hemophilia A. To fashion functional F8 therapeutics, development of a recombinant F8 (rF8) domain is essential not only for F8 substitution, but also to decipher the F8-related mechanisms. In this study, we generated Glutathione S-transferase (GST)-conjugated recombinant A2 and A3 domains of F8 using Escherichia coli. The high growth rate and economically advantageous protein production system in terms of inexpensive reagents and materials in E. coli cells facilitated the completion of entire process from protein expression to purification in 3-4 days with low production cost. Subsequent assessment of these purified proteins using enzyme-linked immunosorbent assay (ELISA) and antibodies against F8 revealed enhanced detection of rF8-A2 or rF8-A3 in a concentration dependent manner, indicating the presence of the antibody-binding epitopes in these proteins. Furthermore, these proteins are suitable for generating novel antibodies against the F8 domain and F8 domain-capturing affinity columns by enabling their conjugation to GST-capturing beads. Additionally, the recombinant F8 domains produced herein can be used for various studies, which include investigating the explicit roles of the F8 domain in the coagulation process, with domain-specific binding partners, and antibodies.

Exploring effects of Simvastatin on coagulation mediators to alleviate the advancement of high cholesterol diet triggered neurodegeneration.

The objectives of our study were to investigate the possible effect of Simvastatin in ameliorating high cholesterol diet (HCD)-induced neurodegeneration and to also investigate its possible action on coagulation mediators.In silico and in vitro studies were performed to evaluate the impact of Simvastatin on prime coagulation mediators. HCD was used to induce neuropathology in wistar rats and histopathological and immunohistochemical studies were performed to evaluate the efficacy of Simvastatin in preventing the advancement of neurodegeneration in obese rats. Biochemical analyses were used to estimate changes in lipid profile, oxidative stress, inflammatory and coagulation markers. Simvastatin showed good theoretical affinity to coagulation proteins, significantly reversed changes in inflammatory and coagulation biomarkers which were induced by HCD. Enhanced fibrinolytic activity of Simvastatin was revealed through in vitro analysis. Immunohistoanalysis showed raised level of Nrf2. Histopathological studies also supported neuroprotective potential of Simvastatin in HCD fed rats. Simvastatin demonstrated reduced hypercoagulation, enhanced fibrinolysis and reversed neurodegeneration in HCD exposed rats suggesting its potential role in preventing the progression of neurodegeneration in obesity.