Nonspecific interaction between plasminogen and modified magnetic iron oxide nanoparticles.

The magnetic particles modified with silicon dioxide (SiO2) and amino groups (-NH2), as well as the magnetic particles modified with human serum albumin (HSA) were synthesized using the approaches we developed before and characterized by physico-chemical methods in this study. Plasminogen was chosen as a model protein since plasminogen plays a major role in the fibrinolytic system and plasminogen level correlates with different pathologies and conditions. For the first time it has been carried out qualitative and quantitative assessment of plasminogen nonspecific binding (noncovalent adsorption) by the particles in buffer and plasma solutions. The fibrinolytic activity of plasminogen on the surface of the particles has been measured by the aid of commercially available kits and appeared to be 28-30% of its initial value. Plasminogen desorption from the surface of particles was studied in phosphate buffer with NaCl and ε-aminocaproic acid. Despite nonspecific plasminogen binding is an undesirable process, the data obtained is valuable for further modification of particles for high-specific proteins extraction from biological fluids or transport of plasminogen by the particles. The perspectives of particles modified with SiO2 and -NH2, and particles modified with HSA for isolation of protein analytes and their quantitative assessment thereafter have been discussed.

Alcohol-Related Liver Disease Is Rarely Detected at Early Stages Compared With Liver Diseases of Other Etiologies Worldwide.

BACKGROUND & AIMS: Despite recent advances in treatment of viral hepatitis, liver-related mortality is high, possibly owing to the large burden of advanced alcohol-related liver disease (ALD). We investigated whether patients with ALD are initially seen at later stages of disease development than patients with hepatitis C virus (HCV) infection or other etiologies. METHODS: We performed a cross-sectional study of 3453 consecutive patients with either early or advanced liver disease (1699 patients with early and 1754 with advanced liver disease) seen at 17 tertiary care liver or gastrointestinal units worldwide, from August 2015 through March 2017. We collected anthropometric, etiology, and clinical information, as well as and model for end-stage liver disease scores. We used unconditional logistic regression to estimate the odds ratios for evaluation at late stages of the disease progression. RESULTS: Of the patients analyzed, 81% had 1 etiology of liver disease and 17% had 2 etiologies of liver disease. Of patients seen at early stages for a single etiology, 31% had HCV infection, 21% had hepatitis B virus infection, and 17% had nonalcoholic fatty liver disease, whereas only 3.8% had ALD. In contrast, 29% of patients seen for advanced disease had ALD. Patients with ALD were more likely to be seen at specialized centers, with advanced-stage disease, compared with patients with HCV-associated liver disease (odds ratio, 14.1; 95% CI, 10.5-18.9; P < .001). Of patients with 2 etiologies of liver disease, excess alcohol use was associated with 50% of cases. These patients had significantly more visits to health care providers, with more advanced disease, compared with patients without excess alcohol use. The mean model for end-stage liver disease score for patients with advanced ALD (score, 16) was higher than for patients with advanced liver disease not associated with excess alcohol use (score, 13) (P < .01). CONCLUSIONS: In a cross-sectional analysis of patients with liver disease worldwide, we found that patients with ALD are seen with more advanced-stage disease than patients with HCV-associated liver disease. Of patients with 2 etiologies of liver disease, excess alcohol use was associated with 50% of cases. Early detection and referral programs are needed for patients with ALD worldwide.

Oxidation-induced modifications of the catalytic subunits of plasma fibrin-stabilizing factor at the different stages of its activation identified by mass spectrometry.

Plasma fibrin-stabilizing factor (pFXIII) is a heterotetrameric proenzyme composed of two catalytic A subunits (FXIII-A2) and two inhibitory/carrier B subunits (FXIII-B2). The main function of the protein is the formation of cross-links between the polypeptide chains of the fibrin clot. The conversion of pFXIII into the enzymatic form FXIII-A2* is a multistage process. Like many other blood plasma proteins, pFXIII is an oxidant-susceptible target. The influence of distinct sites susceptible to oxidation-mediated modifications on the changes in the structural-functional characteristics of the protein remains fully unexplored. For the first time, a set of the oxidation sites within FXIII-A2 under ozone-induced oxidation of pFXIII at different stages of its activation have been identified by mass spectrometry, and the extent as well as the chemical nature of these modifications have been explored. It was shown that the set of amino acid residues susceptible to oxidative attack and the degree of oxidation of these residues in FXIII-A2 of non-activated pFXIII, pFXIII activated by Ca2+ and fully activated pFXIII treated with thrombin and Ca2+ significantly differ. The obtained data enable one to postulate that in the process of the proenzyme conversion into FXIII-A2*, new earlier-unexposed amino acid residues become available for the oxidizer while some of the initially surface-exhibited residues are buried within the protein globule.

Covalent structure of single-stranded fibrin oligomers cross-linked by FXIIIa.

FXIIIa-mediated isopeptide γ-γ bonds are produced between γ polypeptide chains of adjacent monomeric fibrin. Despite the use of the different methodological approaches there are apparently conflicting ideas regarding the orientation of γ-γ bonds. To identify the orientation of these bonds a novel approach has been applied. It was based on self-assembly of soluble cross-linked fibrin protofibrils ongoing in the urea solution of moderate concentrations followed by dissociation of protofibrils in the conditions of increasing urea concentration. The oligomers were composed of monomeric desA fibrin molecules created by cleavage of the fibrinopeptides A from fibrinogen molecules with thrombin-like enzyme, reptilase. The results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated an emergence of the double-stranded rod-like fibrin protofibrils. For the first time, the protofibrils are proved to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures. Since no accumulation of α polymers has been found the covalent structure of soluble single-stranded fibrin oligomers is entirely brought about by γ-γ bonds. The results of this study provide an extra evidence to support the model of the longitudinal γ-γ bonds that form between the γ chains end-to-end within the same strand of a protofibril.

Ozone-induced oxidative modification of plasma fibrin-stabilizing factor.

The plasma fibrin-stabilizing factor (pFXIII) function is to maintain a hemostasis by the fibrin clot stabilization. The conversion of pFXIII to the active form of the enzyme (FXIIIа) is a multistage process. Ozone-induced oxidation of pFXIII has been investigated at different stages of its enzyme activation. The biochemical results point to a decrease of an enzymatic activity of FXIIIа depending largely on the stage of the pFXIII conversion into FXIIIа at which oxidation was carried out. UV-, FTIR- and Raman spectroscopy demonstrated that chemical transformation of cyclic, NH, SH and S-S groups mainly determines the oxidation of amino acid residues of pFXIII polypeptide chains. Conversion of pFXIII to FXIIIa proved to increase protein sensitivity to oxidation in the order: pFXIII

Ozone-induced damage of fibrinogen molecules: identification of oxidation sites by high-resolution mass spectrometry.

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Ozone-induced oxidative modifications of the fibrinogen Aα, Bß, and γ polypeptide chains upon addition of various amounts of the oxidiser were studied by mass spectrometry. Amino acid residues located on all three chains and main structural parts of the protein were revealed to be involved in oxidation. The αC-connector was shown to be most vulnerable to oxidation as compared to other structural parts while the E region turned out to be the most protected area of the protein. For the first time, it was established that numerous amino acid residues responsible for the conversion of fibrinogen to fibrin remain unaffected upon fibrinogen oxidation. The data obtained in this study indicate that none of the identified residues, which are considered crucial for the binding of both hole "a" and hole "b" to knob "A" and knob "B", respectively, as well as those responsible for the thrombin binding to fibrinogen E region, have been subjected to chemical alterations under moderate oxidation. The data on fibrinogen oxidation acquired in the current study enable one to assume that some of the structural fibrinogen parts and easily oxidisable residues could be endowed with antioxidant properties. New findings presented here could be essential for the detection of adaptive molecular mechanisms capable of mitigating the detrimental action of reactive oxygen species (ROS) on the functioning of oxidatively damaged fibrinogen. Data are available via ProteomeXchange with identifier PXD012046. Highlights Various oxidative modifications were detected in fibrinogen by mass spectrometry αC-connector has been shown to be most susceptible to oxidation E region proved to be least vulnerable to the action of the oxidising agent Some of the Met residues in the fibrinogen structure could operate as ROS scavengers.

Fibrin self-assembly is adapted to oxidation.

Fibrinogen is extremely susceptible to attack by reactive oxygen species (ROS). Having been suffered an oxidative modification, the fibrinogen molecules, now with altered spatial structure and function of fibrin network, affect hemostasis differently. However, the potential effects of the oxidative stress on the early stages of the fibrin self-assembly process remain unexplored. To clarify the damaging influence of ROS on the knob 'A': hole 'a' and the D:D interactions, the both are operating on the early stages of the fibrin polymerization, we have used a novel approach based on exploration of FXIIIa-mediated self-assembly of the cross-linked fibrin oligomers dissolved in the moderately concentrated urea solutions. The oligomers were composed of monomeric desA fibrin molecules created by cleaving the fibrinopeptides A off the fibrinogen molecules with a thrombin-like enzyme, reptilase. According to the UV-absorbance and fluorescence measurements data, the employed low ozone/fibrinogen ratios have induced only a slight fibrinogen oxidative modification that was accompanied by modest chemical transformations of the aromatic amino acid residues of the protein. Else, a slight consumption of the accessible tyrosine residues has been observed due to intermolecular dityrosine cross-links formation. The set of experimental data gathered with the aid of electrophoresis, elastic light scattering and analytical centrifugation has clearly witnessed that the oxidation can serve as an effective promoter for the observed enhanced self-assembly of the covalently cross-linked oligomers. At urea concentration of 1.20M, the pristine and oxidized fibrin oligomers were found to comprise a heterogeneous set of the double-stranded protofibrils that are cross-linked only by γ-γ dimers and the fibers consisting on average of four strands that are additionally linked by α polymers. The amounts of the oxidized protofibrils and the fibers accumulated in the system were higher than those of the non-oxidized counterparts. Moreover, the γ and α polypeptide chains of the oxidized molecules were more readily crosslinked by the FXIIIa. Upon increasing the urea solution concentration to 4.20M, the cross-linked double-stranded desA fibrin protofibrils have dissociated into the single-stranded fibrin oligomers, whereas the fibers dissociated into both the double-stranded desA fibrin oligomers, the structural integrity of the latter being maintained by means of the intermolecular α polymers, and the single-stranded fibrin oligomers cross-linked only by γ-γ dimers. The data we have obtained in this study indicate that the FXIIIa-mediated process of assembling the cross-linked protofibrils and the fibers constructed from the oxidized monomeric fibrin molecules was facilitated due to the strengthening of D:D interactions. The findings infer that the enhanced longitudinal D:D interactions become more essential in the assembly of soluble protofibrils when the interactions knobs 'A': holes 'a' are injured by oxidation. The new experimental findings presented here could be of help for elucidating the essential adaptive molecular mechanisms capable of mitigating the detrimental action of ROS in the oxidatively damaged fibrin self-assemblage processes.

Ozone-induced oxidative modification of fibrinogen: role of the D regions.

Native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing cross-linked fibrin clots under the influence of thrombin and fibrin-stabilizing factor (FXIIIa). The aim of this study was to investigate mechanisms of impairment of both the molecular structure and the spatial organization of fibrinogen under ozone-induced oxidation. FTIR analysis showed that ozone treatment of the whole fibrinogen molecule results in the growth of hydroxyl, carbonyl, and carboxyl group content. A similar analysis of fibrinogen D and E fragments isolated from the oxidized protein also revealed transformation of distinct important functional groups. In particular, a remarkable decay of N-H groups within the peptide backbone was observed along with a lowering of the content of C-H groups belonging to either the aromatic moieties or the aliphatic chain CH2 and CH3 units. The model experiments performed showed that the rather unexpected decay of the aliphatic CH units might be caused by the action of hydroxyl radicals, these being produced in the water solution from ozone. The observed dissimilarities in the shapes of amide I bands of the fibrinogen D and E fragments before and after ozone treatment are interpreted in terms of feasible local conformational changes affecting the secondary structure of the protein. Taken as a whole, the FTIR data suggests that the terminal D fragments of fibrinogen are markedly more susceptible to the ozone-induced oxidation than the central E fragment. The data on elastic and dynamic light scattering provide evidence that, in the presence of FXIIIa, both the unoxidized and the oxidized fibrinogen molecules bind to one another in an "end-to-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. The γ and α polypeptide chains of the oxidized fibrinogen proved to be involved in the enzymatic cross-linking more readily than those of unaffected fibrinogen. The experimental data on fibrinogen oxidation acquired in the present study, combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some reactive oxygen species actions detrimental to the protein function.