No abstract

No abstract

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.

Aim. One of the approaches for studying structure and functions of proteins is their limited proteolysis. Proteolytic fragments of macromolecules can preserve the biological activity and can be used for the study of their structural and functional peculiarities. Thus, the characterization of new proteolytic enzymes and determination of the specificity of their action can be of interest for exploration. In the present work, we focused on the action of protease from the venom of Gloydius halys halys on fibrinogen, the crucial protein of blood coagulation system. Methods. Products of fibrinogen hydrolysis by protease from the venom of G. halys halys were studied by SDS-PAGE electrophoresis and western-blot analysis using monoclonal antibodies ІІ-5 Сand 1-5A targeted to 20‒78 and 549‒610 fragments of fibrinogen Aα-chain. Molecular weights of hydrolytic products were determined using MALDI-TOF analysis on Voyager DE PRO (USA). Sequence of hydrolytic products were predicted by «Peptide Mass Calculator» soft ware. Results. SDS-PAGE showed that protease from the venom of Gloydius halys halys initially cleaved Аα-chain of fibrinogen molecule. Western-blot analysis confirmed that this protease specifically cleaves off fragment of C-terminal parts of Аα-chain with apparent molecular weight of 22 kDa. Cleaved fragment was identified by MALDI-TOF analysis as the 21.1 kDa polypeptide. «Peptide Mass Calculator» predicted that such a fragment corresponded to Аα414-610 residue of fibrinogen molecule. Thus, we showed that studied protease cleaved peptide bond AαK413-L414 with the formation of stable partly hydrolyzed fibrinogen desAα414-610. Conclusions. The use of protease from the venom of Gloydius halys halys would allow obtaining the unique partly hydrolyzed fibrinogen des Aα 414‒610 that is suitable for the study of structure and functions of fibrinogen αС-regions.