M. L. Konstantinova

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.

Oxidized modification of fragments D and E from fibrinogen induced by ozone

Ozone-induced free-radical oxidation of fragments D and E from fibrinogen has been studied. The methods of elastic and dynamic light scattering in combination with electrophoresis of unreduced samples have shown the acceleration of enzymatic covalent crosslinking of molecules of oxidation-modified fragment D under the action of factor XIIIa. UV and IR spectroscopy shows that free-radical oxidation of amino acid residues of polypeptide chains catalyzed by ozone affects the cyclic and amino groups, giving rise to generation of mainly oxygen-containing products. Comparison of the IR spectra obtained for the oxidation-modified D and E fragments revealed more significant transformation of functional groups for the D fragment. EPR spectroscopy showed that the rotational correlation time of spin labels bound to the ozonized proteins decreased in comparison with the non-ozonized proteins. The rotation correlation time of the radicals covalently bound to the ozonized D and E fragments suggests that D fragment of fibrinogen is more sensitive to free-radical oxidation followed by local structural changes. Possible causes of different degrees of oxidation for fragments D and E are discussed.

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.

Nature of active intermediate particles formed during ozone-induced oxidation

139 Ozone as a representative of reactive oxygen spee cies (ROS) is one of the most toxic components of the atmosphere. A number of studies have shown that ozone is able to generate other ROS, including HO • , , H 2 O 2 , etc. [1, 2]. In some cases, these secondary ROS can be even more deleterious than the ozone molecules themselves. It is now generally recognized that proteins are among the main targets of ROS. Under the action of ROS, proteins undergo oxidative modifications, which disturb their structures and functions. Oxidationnmodified proteins accumulate in the course of aging, oxidative stress, and various diss eases [3]. It was shown that fibrinogen is 20 times more sensitive to oxidative modification than other major plasma proteins (albumin, immunoglobulins, transs ferrin, and ceruloplasmin) [4]. Therefore, fibrinogen, which accounts for approximately 4% of the total plasma proteins, is an easily vulnerable target for oxii dants. It was previously shown that a number of proteins (fibrinogen [5, 6], fibrinnstabilizing factor [7], bovine serum albumin [8], etc.) are involved in oxidative proo cesses under the influence of ozone. In studying the ozonization of fibrinogen, it was found that moderate oxidation leads to a decrease in the content of not only reactive groups (NH x , > SH, etc.), but also to a marked reduction in the content of СН 2 groups, whose reactivity compared to that of NH x and SHHgroups is smaller by 7 and 4 orders of magnitude, respectively [9]. This interesting feature of the reaction could not be attributed to the molecular reaction of ozone but, rather, was due to the action of an as yet unidentified secondary ROS and required further explanation. Since ozone in the liquid phase can form highly toxic secondary ROS, the discriminaa O 2 • − tion between the effects of the molecular oxidation and the free radical oxidation caused by the generation of free radicals under the influence of ozone during ozonation is one of the key problems in understanding the mechanism of oxidation of different biological tarr gets with ozone. In this study, we investigated this problem using fibrinogen as an example. The oxidation of fibrinogen under the action of ozone was performed as described previously [6]. The amount of ozone in the reactor varied from 5 × 10 –8 to 2 × 10 ⎯7 moles. The generation of hydroxyl radicals generated under the …

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.

Oxidation-induced modification of the fibrinogen polypeptide chains

By using the mass-spectrometry method, the oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains induced by its oxidation have been studied. The αC-region has been proven to be the most vulnerable target for the oxidizer (ozone) as compared with the other structural elements of the Aα chain. The Bβ chain mapping shows that the oxidative sites are localized within all the structural elements of the chain in which the β-nodule exhibits high susceptibility to oxidation. The γ chains are the least vulnerable to the oxidizer action. The results obtained demonstrate convincingly that the self-assembly centers dealing with interactions of knob “A”: hole “a” are not involved in oxidative modification. It is concluded that the numerous oxidative sites revealed are mainly responsible for inhibiting lateral aggregation of protofibrils. The part of amino acid residues subjected to oxidation is supposed to carry out the antioxidant function.

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.

Study of Human Fibrinogen Oxidative Modification using Differential Scanning Calorimetry

For the first time, with the aid of differential scanning calorimetry, the thermal denaturation of fibrinogen under induced oxidation was studied. All fibrinogen structural elements detected by DSC (D region, αC-domain, and E region) are subjected to oxidation. Structural changes in fibrinogen molecule were characterized by the denaturation temperature, denaturation enthalpy, and van’t Hoff enthalpy.

The strengthening role of D:D interactions in fibrin self-assembly under oxidation

The effect on ozone-induced oxidation on the self-assembly of fibrin in the presence of fibrin-stabilizing factor FXIIIa of soluble cross-linked fibrin oligomers was studied in a medium containing moderate urea concentrations. It is established that fibrin oligomers were formed by the protofibrils cross-linked through γ-γ dimers and the fibrils additionally cross-linked by through α-polymers. The oxidation promoted both the accumulation of greater amounts of γ-γ dimers and the formation of protofibrils, fibrils, and their dissociation products emerging with increasing urea concentrations, which have a high molecular weight. It is concluded that the oxidation enhances the axial interactions between D-regions of fibrin molecules.