Earlier we reported that during the human fibrinogen to fibrin transition a neoantigenic determinant was exposed in the Bβ119-133 fragment, where a hinge locus is situated. The fibrin-specific mAb FnI-3c and its Fab-fragment with epitope in this fragment inhibited the lateral association of protofibrils. We suggested that the epitope coincided with a site involved in this process. In this work we investigated the epitope location more precisely and defined a functional role for its exposure in the hinge locus of the molecule. It was found that mAb FnI-3c bound to human, horse and rabbit fibrins, all of which have Lys in the position corresponding to human BβK130, but not to bovine and rat fibrins, which have other amino acid residues in this position, strongly suggesting that BβK130 provides the integral part of the epitope. This fact, homology data, and structural biological analysis of the amino acid sequences around BβK130 indicate that the site of interest is localized within Bβ125-135. The synthetic peptides Bβ121-138 and Bβ125-135, unlike their scrambled versions, bound to mAb FnI-3c in SPR analysis. Both peptides, but not their scrambled versions, inhibited the lateral association of protofibrils. The FnI-3c epitope is exposed after fibrinopeptide A cleavage and desA fibrin monomer formation. Structural biological analysis of the fibrinogen to fibrin transition showed a distinct increase of flexibility in the hinge locus. We propose that the structural transformation in the fibrin hinge regions leads to the conformation necessary for lateral association of protofibrils. K e y w o r d s: fibrinogen to fibrin transition, coiled-coil connector, protofibril lateral association, hinge region, neoantigenic determinant. introduction Fibrinogen is a dimer, with each subunit of the molecule being formed by three polypeptide chains: Aα, Bβ and γ. The molecule consists of a central E, two peripheral D, and two extended αC regions [1]. The central E-region consisting of (Aα1-104, Bβ1-133, γ1-72) 2 is connected to the two peripheral Dregions (Aα105-219, Bβ134-461, γ73-411) by two long flexible coiled-coil connectors (Aα48-161, Bβ79-193, γ23-135). The N-terminal parts of the connectors (Aα48-104, Bβ79-133, γ23-62) belong to the E region and the C-terminal parts (Aα105-161, Bβ134-193, γ63-135) to the D regions. A hinge locus (α99-110, β130-155, γ70-100) is located in the middle part of the coiled-coil region [2, 3]. The extended αC regions (Aα220-610) consist of unfolded flexible segments (Aα220-391) and more structured αC-domains (Aα392-610). Intermolecular binding of the fibrin polymerization sites A:a as well as putative C:c sites [4], leads to protofibril formation. The protofibrils associate laterally, initially forming fibrils and, subsequently, a three-dimensional fibrin net. At the stage of protofibrils and fibril formation, inter-protofibril binding
Aims.The main purpose of the study was to obtain fibrin-specific monoclonal antibodies (mAbs), to carry out immunoenzyme methods for quantification of soluble fibrin and D-dimer in human blood plasma and to verify the methods at the pregnancy with the risk of fetal loss (RFL). Methods. Usual hybridoma technique was used for production of mAbs. MAbs were isolated from cultural media by affinity chromatography on protein G-and fibrin-sepharoses. ELISA and Western-blot analysis were used to localize the epitopes for mAbs obtained. Results. Partly denaturated human fibrin desAABB was used for mice immunization. As a result three hybridomas have been obtained, which produce mAbs reacting with human fibrinogen without cross-reaction with fibrinogen. Competitive ELISA testified that epitopes for these mAbs in fibrin molecule practically coincided. ELISA and immunoblot analysis with cyanogen bromide and plasmin degradation products of fibrin showed that epitopes for these mAbs were situated in fibrin fragment Bbeta118-134. Thus this region is the neoantigenic determinant being exposed during fibrinogen-fibrin transformation. Fibrin-specific mAb 1A-5C was used in double-sandwich ELISA as a "catch"-antibody and our mAb II-4d -as a "tag"-antibody for soluble fibrin quantification. We obtained earlier D-dimer-specific mAb III-3b and showed that epitope for this mAb in human D-dimer was localized in Bbeta155-160 fragment. It has been shown that mAb III-3b reacts also with oligomeric plasmin degradation products of fibrin. Thus Bbeta155-160 region is the neoantigenic determinant, being exposed during fibrin degradation by plasmin. MAb III-3b was used in double-sandwich ELISA as a "catch"-antibody and our mAb II-4d -as a "tag"-antibody for D-dimer quantification. There are a lot of data on the direct relation between the RFL and thrombophilia. So this complication of pregnancy may be accompanied by the increase of soluble fibrin and D-dimer quantity. We performed quantifications of soluble fibrin and D-dimer in blood plasma of healthy pregnant women (n = 33) and pregnant women with RFL (n = 44). D-dimer concentrations varied in the range of 1 -224 ng/ml at the pregnancy period from 4 till 37 weeks. There was positive correlation between D-dimer concentration and pregnancy term both at normal pregnancy and pregnancy with RFL. The mean values of D-dimer concentration at various terms of normal pregnancy and pregnancy with RFL did not vary considerably. The increased mean values of soluble fibrin concentrations were observed at pregnancy with RFL as compared to the normal pregnancy at the terms from 4 till 24 weeks (17,87 mkg/ml and 9,03 mkg/ml correspondingly, P less 0,05). Conclusions. Three fibrin-specific mAbs have been obtained with epitopes being localized in fibrin fragment Bbeta118-134. One of these mAbs was used in double-sandwich ELISA as a "catch"-antibody for soluble fibrin quantification in human blood plasma. Soluble fibrin quantification proved to give useful diagnostic information at the pregnancy with the risk of ...
Obtaining new monoclonal antibodies (mAbs) towards fibrin(ogen) and its fragments is an important task for studying mechanisms of blood clot formation, searching for novel antithrombotic agents and developing immunodiagnostics. The aim of the present work was to create and characterize a new mAb towards the fibrin(ogen) αС-region. We surmise that having a specific mAb towards this flexible part of the molecule will allow us to study the role of the αС-region in fibrin polymerization and also to develop an approach for detecting the earliest forms of soluble fibrin by sandwich ELISA. Using hybridoma technology we оbtained mAb 1-5A to the αC-region of fibrinogen.. It was characterized using several variations of ELISA and Western blot. Application of specific proteases together with MALDI-TOF analysis allowed us to localize its epitope that is located in fragment 537-595 of the Aα-chain of fibrin(ogen). МAb 1-5A can be used as a detecting tag-antibody in sandwich ELISA for the quantification of the earliest forms of soluble fibrin which are uncleaved by plasmin and preserved C-terminal portions of αC-regions. These earliest forms of soluble fibrin are direct evidence of blood coagulation system activation, thrombin generation and the danger of intravascular thrombus formation. Their determination will provide additional, more accurate information about the state of the blood coagulation system and the risk of blood clotting, which is very important for the timely and correct selection of adequate antithrombotic therapy. MAb 1-5A effectively binds the αC-containing molecules of fibrinogen and fibrin in blood plasma. It also can be used for studying protein-protein and protein-cellular interactions of the αC-regions of fibrin(ogen). K e y w o r d s: monoclonal antibody, fibrinogen, fibrin, αC-region of the fibrin(ogen) molecule, hemostasis, immunodiagnostics.
7. Aleksandrov, A. A., Bagnenko, E. S. (2012). Psychological characteristics of women with cosmetic defects of the facial skin. Herald of psychotherapy, 41 (46), 52-66. 8. Bodnar, L. A. (2011). Clinical-psychopathological and pathopsychological characteristics of patients who turned to plastic surgeons for rhinoplasty. Mental Health, 1-2 (30-31), 4-7. 9. Sats, E. A., Slobodchikov, I. M. (2015). Features self-awareness for women-clients beauty salon. Modern Problems of Science and Education, 1.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.