M. I. Biryukova

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<pFXIII activated by thrombin<pFXIII in the presence of calcium ions<FXIIIa. The dynamic light scattering data indicate that the three-dimensional structure of pFXIII becomes loosened due to oxidative modification. ESR spectroscopy data also point to conformational changes of the fibrin-stabilizing factor under oxidation. Taking into account these new findings it seems reasonable to assume that the inhibitory/carrier FXIII-B subunits can serve as scavengers of ROS. Hypothetically, this mechanism could help to protect the key amino acid residues of the FXIII-A subunits responsible for the enzymatic function of FXIIIa.

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.

Free-radical oxidation of plasma fibrin-stabilizing factor.

213 Fibrinnstabilizing factor (FXIII) belongs to the family of transglutaminases (endooγglutamine:ε lysine transferase, EC. 2.3.2.13) and is one of the key proteins of the blood coagulation system. Similarly to other blood clotting factors, FXIII circulates in plasma as an inactive precursor. This is a heterotett ramer (FXIIIIA 2 B 2) with a molecular weight of 320 kDa, consisting of two identical singleechain catt alytic subunits А (FXIIIIA 2 ; molecular weight, ~83 kDa) and two identical singleechain regulatory subunits B (FXIIIIB 2 ; molecular weight, ~78 kDa), which are held together by weak noncovalent bonds [1]. Factor XIII is activated in several stages. The first stage, catalyzed by thrombin, is the hydrolytic cleavv age of the peptide bond between Arg37 and Gly38 in the NH 2 terminal region of subunit А, causing the removal of the activation peptide АP from each sub unit A. As a result, the FXIIIIA 2 B 2 fibrinnstabilizing factor becomes the FXIIII B 2 factor, which still has no enzymatic activity [2]. The second activation stage requires the presence of calcium ions, which can cause dissociation of heterosubunits to form FXIIII and FXIIIIB 2. At the last stage, which also proceeds in the presence of Ca 2+ , FXIIII undergoes conformaa tional transformations, resulting in the exposure of the active site Cys314 with the formation of enzyme FXIIII (FXIIIa) [3]. The main function of factor XIII is to maintain hemostasis by covalent stabilization of the fibrin clot, which is accompanied by an increase in its mechanical strength and resistance to plasmin degradation. In the presence of the active form of fibrinnstabilizing factor, fibrin polymers undergo covalent crossslinking due to formation of ε/γglutamyl–lysine isopeptide bonds [4]. A 2 * To date, the catalytic function of FXIIII has been studied sufficiently well, whereas the role of the noncatalytic FXIIIIВ 2 subunits is less obvious. It is believed that, in the bloodstream, these subunits funcc tion as carriers of the catalytic subunits FXIIIIA 2 , proo tecting them from possible proteolytic degradation and thereby maintaining the required level of zymogen in the bloodstream [5, 6]. In addition, they fulfill a regg ulatory function by controlling the activation of FXIIIIA 2 B 2 by thrombin [7]. It is known that fibrinnstabilizing factor, similarly to many other proteins circulating in the blood plasma, may be a target for reactive oxygen species (ROS), which disturb its functional properties [8]. Previously, by …

Ozone-induced oxidative modification of fibrinogen molecules

Ozone-induced oxidation of fibrinogen has been investigated. The conversion of oxidized fibrinogen to fibrin catalyzed either by thrombin or by a reptilase-like enzyme, ancistron, in both cases is accompanied by production of gels characterized by a higher weight/length ratio of fibrils in comparison with the native fibrin gels. IR spectra of the D and E fragments isolated from unoxidized and oxidized fibrinogen suggest a noticeable transformation of functional groups by oxidation. A decrease in content of N-H groups in the peptide backbone and in the number of C-H bonds in aromatic structures, as well as a decrease in the intensity of the C-H valence vibrations in aliphatic fragments CH2 and CH3 were found. The appearance in the differential spectra of the D fragments of rather intense peaks in the interval of 1200–800 cm−1 clearly indicates the interaction of ozone with amino acid residues of methionine, tryptophan, histidine, and phenylalanine. Comparison of the differential spectra for the D and E fragments suggests that fibrinogen fragment D is more sensitive to the oxidant action than fragment E. Using EPR spectroscopy, differences are found in the spectra of spin labels bound with degradation products of oxidized and unoxidized fibrinogen, the D and E fragments, caused by structural and dynamical modifications of the protein molecules in the areas of localization of the spin labels. The relationship between the molecular mechanism of oxidation of fibrinogen and its three-dimensional structure is discussed.

Self-assembly of soluble unlinked and cross-linked fibrin oligomers

Self-assembly of soluble unlinked and cross-linked fibrin oligomers formed from desA-fibrin monomer under the influence of factor XIIIa was studied in the presence of non-denaturing urea concentrations. By methods of elastic and dynamic light scattering combined with analytical ultracentrifugation, desA-fibrin oligomers formed in both the presence and absence of the factor XIIIa were shown to be ensembles consisting of soluble rod-like double-stranded protofibrils with diverse weight and size. Unlinked and cross-linked soluble double-stranded protofibrils can reach the length of 350–450 nm. The structure of soluble covalently-linked protofibrils is stabilized by isopeptide γ-dimers. Electrophoretic data indicate a complete absence of isopeptide bonds between α-chains of desA-fibrin molecules. The molecular mechanism of formation of soluble rod-like fibrin structures and specific features of its covalent stabilization under the influence of factor XIIIa are discussed.

Modification of the catalytic subunit of plasma fibrin-stabilizing factor under induced oxidation

For the first time, by using mass-spectrometry method, the oxidation-mediated modification of the catalytic FXIII-A subunit of plasma fibrin-stabilizing factor, pFXIII, has been studied. The oxidative sites were identified to belong to all structural elements of the catalytic subunit: the β-sandwich (Tyr104, Tyr117, and Cys153), the catalytic core domain (Met160, Trp165, Met266, Cys328, Asp352, Pro387, Arg409, Cys410, Tyr442, Met475, Met476, Tyr482, and Met500), the β-barrel 1 (Met596), and the β-barrel 2 (Met647, Pro676, Trp692, Cys696, and Met710), which correspond to 3.9%, 1.11%, 0.7%, and 3.2%, respectively, of oxidative modifications as compared to the detectable amounts of amino acid residues in each of the structural domains. Lack of information on some parts of the molecule may be associated with the spatial unavailability of residues, complicating analysis of the molecule. The absence of oxidative sites localized within crucial areas of the structural domains may be brought about by both the spatial inaccessibility of the oxidant to amino acid residues in the zymogen and the screening effect of the regulatory FXIII-B subunit.

The oxidative modification of cellular fibrin-stabilizing factor

For the first time, the induced oxidative modification of cellular fibrin-stabilizing factor (cFXIII) has been studied. According to the electrophoresis analysis, the conversion of oxidized cFXIII into FXIIIa resulted in producing the enzyme that significantly lost the initial enzymatic activity. At the same time, FXIIIa subjected to induced oxidation was completely devoid of enzymatic activity. The results of FTIR spectroscopy showed that the oxidation of cFXIII or FXIIIa was accompanied by profound changes both in chemical and spatial structures of the protein. The results of this study are in good agreement with our earlier assumption regarding the antioxidant role of the regulatory subunits B of plasma fibrin-stabilizing factor.

Synthesis and physicochemical properties of composites for electromagnetic shielding applications: a polymeric matrix impregnated with iron- or cobalt-containing nanoparticles

Abstract. Magnetic, magnetic resonance, and structural properties of iron and cobalt nanoparticles embedded in a polyethylene matrix were studied. The materials were prepared by thermal decomposition of cobalt or iron formate in a polyethylene melt in mineral oil and contained from 2 to 40% wt. of metal. Transmission electron microscopy data indicate that the average diameter of particles is up to 8.0 nm. According to extended x-ray absorption fine structure and Mössbauer spectroscopy studies, the particles comprise a metallic core and nonmetallic shell which is chemically bound to the surrounding matrix. Electrophysical and magnetic properties of the materials prepared were studied along with their reflection and attenuation factors in the super high frequency band. The materials were found to be suitable for use in electromagnetic shielding.

The Influence of the End Products of Plasmin-Mediated Hydrolysis of Fibrinogen and Fibrin (EF and Ef Fragments) on Fibrinogen Cross-linking

We studied the influence of the end products of plasmin-mediated hydrolysis of fibrinogen and nonstabilized fibrin (EF and Ef fragments) on covalent cross-linking of fibrinogen molecules catalyzed by a fibrin-stabilizing factor (factor XIIIa). The data on elastic and dynamic light scattering reveal no difference in the spatial structure of covalently linked fibrinogen molecules in the presence of the hydrolysis end products EF and Ef. In contrast to the inactive fragment EF, fragment Ef significantly accelerates the enzymatic reaction. This is also confirmed by electrophoresis of the reduced samples indicating a relatively fast accumulation of γ-dimers and Aα-polymers as compared to the control samples. Possible molecular mechanisms of this effect are discussed.

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.