Tag Archives: fibrinogen

Men of the molecules

In memoriam of Prof. Russell Doolittle,
Prof. Eduard Lugovskoi and their friendship that outlive both of them

V. O. Chernyshenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: bio.cherv@gmail.com

Received: 02 July 2020; Accepted: 21 July 2020

In memoriam Eduard Lugovskoi and Russell Doolittle we are referring to several episodes of their life and work.  Russell Doolittle an American biochemist and his friend and colleague Ukrainian scientist Eduard Lugovskoi, both studied fibrinogen structure and functions and finally united their efforts in the revealing of the new mechanism of intramolecular interactions of fibrin molecule through coiled-coil region. The results of their common work and discussions were included to the article “The fibrin Bβ125-135 site is involved in the lateral association of protofibrils”. Valuable part of the communication dedicated to the poetry of Eduard Lugovskoi that inspired both of scientists in work and life. We are providing some remembrance of their collaboration, their letters sent to each other, fragments of handwriting and common photo of Russell Doolittle and Eduard Lugovskoi.

Novel monoclonal antibody to fibrin(ogen) αC-region for detection of the earliest forms of soluble fibrin

N. E. Lugovska1, I. M. Kolesnikova1, Ye. M. Stohnii1, V. O. Chernyshenko1*,
A. V. Rebriev1, O. P. Kostiuchenko1, G .K. Gogolinska1, N. A. Dziubliuk2,
L. D. Varbanets2, T. M. Platonova1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Zabolotny Institute of Microbiology and Virology,National Academy of Sciences of Ukraine, Kyiv;
*e-mail: bio.cherv@gmail.com

Received: 08 May 2020; Accepted: 30 June 2020

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).

Identification of the binding site for plasminogen kringle 5 in the α-chain of fibrin(ogen) D-fragment

L. G. Kapustianenko*, T. V. Grinenko, A. V. Rebriev,
O. I. Yusova, A. A. Tykhomyrov

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: kapustyanenko@biochem.kiev.ua

Received: 17 May 2020; Accepted: 30 June 2020

The interaction of the fifth kringle of Glu-plasminogen with fibrin triggers activation and initiation of fibrinolysis, yet the site on fibrin that binds kringle 5 remains unknown.  The aim of our work was to determine an amino acid sequence in the D-fragment of fibrin(ogen) molecule, which is complementary to the lysine-binding site (LBS) in kringle 5. We studied the interaction between kringle 5 of plasminogen with polypeptide chains of the D-fragments of fibrin and cyanogen bromide fragments FCB-2 and t-NDSK and showed that kringle 5 bound specifically to α- and γ-chains of the D-fragment and the α-chain of FCB-2. Tryptic peptides of D-fragment α-chain were obtained, separated by their ability to bind with the immobilized kringle 5, and then all studied peptides were characterized by MALDI-TOF analysis. The critical amino acid residues of the α-chain of D-fragment, which provide its interaction with kringle 5, turned out to be α171Arg and/or α176Lys. The binding site of Glu-plasminogen complementary to the LBS of kringle 5 is located within Аα168Ala−183Lys, a sequence in a weakly structured loop between two supercoils in the α-chain of the D-fragment of the fibrin(ogen) molecule.

Structure and function of fibrinogen BβN-domains

L. Medved*, S. Yakovlev

Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA;
*e-mail: Lmedved@som.umaryland.edu

Received: 17 May 2020; Accepted: 30 June 2020

Fibrinogen is a polyfunctional plasma protein involved in various physiological and pathological processes through the interaction of its multiple domains with different ligands and cell receptors. Among fibrinogen domains, two BβN-domains are formed by the N-terminal portions of its two Bβ chains including­ amino acid residues Bβ1-64. Although their folding status is not well understood and the recombinant disulfide-linked (Bβ1-66)2  fragment corresponding to a pair of these domains was found to be unfolded, some data suggest that these domains may be folded in the parent molecule. In contrast, their major functional properties are well established. Removal of fibrinopeptides B (amino acid residues Bβ1-14) from these domains upon fibrinogen to fibrin conversion results in the exposure of multiple binding sites in fibrin βN-domains (residues β15-64). These sites provide interaction of the βN-domains with different proteins and cells and their participation in various processes including fibrin assembly, fibrin-dependent angiogenesis, and fibrin-dependent leukocyte transmigration and thereby inflammation. The objective of this review is to summarize the current view of the structure and function of these domains in fibrinogen and fibrin and their role in the above-mentioned processes.

Aggregation of platelets, proliferation of endothelial cells and motility of cancer cells are mediated by the Bβ1(15)-42 residue of fibrin(ogen)

Y. M. Stohnii1, M. V. Ryzhykova1, A. V. Rebriev1,
M. D. Kuchma2, R. Y. Marunych1, V. O. Chernyshenko1*,
V. A. Shablii2, N. M. Lypova3, O. Yu. Slominskyi1,
L. V. Garmanchuk4, T. M. Platonova1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
2Institute of Cell Therapy, Kyiv, Ukraine;
3University of Louisville, USA;
4ESC “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;
*e-mail: bio.cherv@gmail.com

Received: 23 December 2019; Accepted: 27 March 2020

The fibrinogen molecule contains multiple binding motifs for different types of cellular receptors, acting as a molecular link between coagulation and cell adhesion. In this study we generated a truncated form of the fibrinogen molecule lacking the Bβ1-42 sequence by site-specific proteolysis and evaluated the role of the fragment in adhesive capabilities of platelets, endothelial and cancer cells. Fibrinogen with the removed Bβ1-42 sequence and fibrin without the Bβ15-42 fragment (desβ1-42 fibrinogen and desABβ15-42 fibrin) were obtained by proteolysis using the specific protease from the venom of Echis multisquamatis. The cleaved fragment was purified by HPLC and was identified using MALDI-TOF. ADP- and collagen-induced aggregation of washed platelets in the presence of fibrinogen desBβ1-42 was studied using an aggregometer. Proliferation of mice aortic endothelial cells (MAEC) and human umbilical vein endothelial cells (HUVEC) was studied using the fibrin desABβ15-42 as the scaffold. Cell viability was quantified by the MTT test (MAEC). Generation time was calculated for the estimation of proliferative activity of HUVEC. Lung cancer cell line Н1299 was used to evaluate cancer cell motility in vitro using the scratch assay. Direct comparison of cellular behavior in the presence of truncated vs native forms demonstrated attenuated cell adhesion in the presence of fibrinogen desBβ1-42 and fibrin desBβ15-42. The platelet aggregation rate was only slightly decreased in the presence of fibrinogen desBβ1-42 but resulted in 15-20% disaggregation of adhered platelets. We also observed the substantial decrease of generation time of HUVEC and inhibition of viability of MAEC cells grown on scaffolds of a desABβ15-42 matrix. Finally, desBβ1-42 modulated the motility of H1299 cells in vitro and suppressed the wound healing by 20% compared to the full-length fibrinogen. We postulate that fragment 1-42 of the BβN-domain of fibrinogen is not sufficient for platelet aggregation, however it may contribute to platelet clot formation in later stages. At the same time, this fragment may be important for establishing proper cell-to-cell contacts and cell viability of endothelial cells. Also, 1-42 amino acid fragment of the BβN-domain supported the migration of cancer cells suggesting that interactions of fibrinogen with cancer cells could be a target for anticancer therapy. The Bβ1-42 fragment of fibrinogen contributes to efficient intracellular interactions of different types of cells, including platelets, endothelial cells and cancer cells.

Comparative analysis of the influence of chlorine and fluorine anions on the fibrin polymerization

L. V. Pyrogova1, G. K. Bereznitsky1, G. K. Gogolinskaya1, T. M. Platonova1,
I. M. Kolesnikova1, O. O. Masenko1, R. Yu. Marunich1, P. Yu. Tsap1,
Yu. V. Ushenin2, Y. M. Makogonenko1, E. V. Lugovskoi1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2V.E. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine, Kyiv;
e-mail: ymakogonenko@gmail.com

Received: 25 April 2019; Accepted: 18 October 2019

The effect of NaCl and NaF salts in the range of 0.1-0.225 M concentrations on individual stages of fibrin polymerization was investigated, namely: the rate of fibrinogen activation by thrombin, the rate of protofibrils formation, the rate of lateral association of protofibrils, and the maximum clot absorbance value at 350 nm. It was found that the chlorine and fluorine anions equally inhibit the rate of formation of fibrin from fibrinogen and the formation of protofibrils activated with thrombin. Chlorine anions were shown to be significantly more effective than fluorine anions, inhibiting the rate of lateral association and maximal clot absorban­ce level from fibrin desA and desAB. A component of the inhibitory action of chlorine anions, not related to the ionic strength of the solution, was identified and its effect on the individual polymerization steps was shown. Chlorine anions were found to bind to a fibrin clot. Using the surface plasmon resonance method and fibrin-specific mAb FnI-3c, it was established that the rate of exposure of neoantigenic determinants of mAb in the hinge regions of the fibrinogen molecule during its transformation into fibrin under the action of thrombin is inhibited by chlorine anions in correlation with inhibition of the protofibril lateral association rate. It has been suggested that the inhibitory effect of chlorine anions consists of an ionic component and a component that blocks the conformational mobility of the molecule by the chlorine anions binding to its hinge regions and polymerization sites.

Overall hemostasis potential of the blood plasma and its relation to some molecular markers of the hemostasis system in patients with chronic renal disease of stage VD

B. G. Storozhuk1, L. V. Pyrogova2, T. M. Chernyshenko2, O. P. Kostiuchenko2,
I. M. Kolesnikova2, T. M. Platonova2, O. B. Storozhuk1, L. O. Storozhuk1,
G. K. Bereznitsky2, P. Yu. Tsap2, O. O. Masenko2,
E. M. Makogonenko2, E. V. Lugovskoy2

1Pyrogov National Medical University of Vinnytsa, Ukraine;
2Palladin Instiute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: makogonenko@biochem.kiev.ua

The values of the coagulation, overall and fibrinolysis potentials were estimated by the method of the global potential of Blomback M., as well as the values of concentrations of molecular markers of the hemostasis system: soluble fibrin (sf), D-dimer, fibrinogen (Fg) and protein C (88 patients, 52 of them men, 36 women). It was shown that hemostasis system activity in women plasma is higher than that in men plasma. The division of patients into 3 groups, depending on the concentration of sf: less than normal – sf ≤ 3, about norm – 3 < sf < 4 and more than norm – sf > 4 μg/ml, allowed establishing the growth of the parameters of both the hemostatic potential and concentrations of molecular markers in accordance with concentration of sf in the groups of patients. Paerson’s correlation analysis of the relationship between the parameters of the hemostasis potential and concentrations of molecular markers revealed an increase in the correlation relationship to the strong and very strong between the parameters of coagulation, fibrinolysis and protein C systems with an increase in the concentration of soluble fibrin in plasma of patients.

Blood coagulation and aortic wall integrity in rats with obesity-induced insulin resistance

O. S. Dziuba1, V. O. Chernyshenko1, Ie. A. Hudz1, L. O. Kasatkina1, T. M. Chernyshenko1,
P. P. Klymenko2, H. V. Kosiakova1, T. M. Platonova1, N. M. Hula1, E. V. Lugovskoy1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: oksana.dziuba86@gmail.com;
2State Institute of Gerontology of AMS of Ukraine, Kyiv

Obesity is an important factor in pathogenesis of disorders caused by chronic inflammation. Diet-induced obesity leads to dyslipidemia and insulin resistance (IR) that in turn provoke the development of type 2 diabetes and cardiovascular diseases. Thus, the aim of this work was to investigate the possible pro-atherogenic effects in the blood coagulation system and aortic wall of rats with obesity-induced IR. The experimental model was induced by a 6-month high-fat diet (HFD) in white rats. Blood samples were collected from 7 control and 14 obese IR rats. Prothrombin time (PT) and partial activated thromboplastin time (APTT) were performed by standard methods using Coagulometer Solar СТ 2410. Fibrinogen concentration in the blood plasma was determined by the modified spectrophotometric method. Levels of protein C (PC), prothrombin and factor X were measured using specific chromogenic substrates and activa­ting enzymes from snake venoms. Platelet aggregation was measured and their count determined using Aggregometer Solar AP2110. The aorta samples were stained by hematoxylin and eosin according to Ehrlich. Aortic wall thickness was measured using morphometric program Image J. Statistical analysis was performed using Mann-Whitney U Test. The haemostasis system was characterized by estimation of the levels of individual coagulation factors, anticoagulant system involvement and platelet reactivity. PT and APTT demonstrated that blood coagulation time strongly tended to decrease in obese IR rats in comparison to the control group. It was also detec­ted that 30% of studied obese IR rats had decreased factor X level, 40% had decreased level of prothrombin whereas fibrinogen concentration was slightly increased up to 3 mg/ml in 37% of obese IR rats. A prominent decrease of anticoagulant PC in blood plasma of obese rats was detected. Obese IR rats also had increased platelet count and higher rate of platelet aggregation in comparison to control animals. Histological analysis identified the disruption of aorta endothelium and tendency for the thickening of the aorta wall in the group with obesity-induced IR compared to the group of control rats. Changes of individual coagulation factors were assumed as the evidence of imbalance in the blood coagulation system. Increase of fibrinogen level, drop in PC concentration and pathological platelet reactivity were taken to corroborate the development of low-grade inflammation in obese IR rats. Instant generation of small amounts of thrombin in their blood plasma is expected. Since the aorta morphology assay detected the trend of its wall to thicken and the emergence of disruptions, we assumed there were initial stages of atherosclerosis and the danger of developing atherothrombosis. We detected an increase of blood coagulability and changes in aorta morphology in rats with obesity-induced IR which we assume indicate early development of atherosclerosis.

Ca(2+)-dependent regulation of fibrinolytic system activation on fibrin(ogen) D-domains

T. A. Yatsenko, V. M. Rybachuk, S. M. Kharchenko, T. V. Grinenko

Palladin Instiute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: tetyanaa.yatsenko@gmail.com

In the present study, we investigated whether calcium content modulation in D-domains of fibrin(ogen) was involved in fibrinolytic process activation. To investigate the effect of Ca2+-dependent changes in D-domains two types of fibrinogen fragments D and cross-linked fibrin fragments DD were obtained from plasmin hydrolysate of human fibrin(ogen): chelator-treated and without chelating agents. The study of plasminogen activation by tissue-type plasminogen activator on D- and DD-fragments had shown the intensification of plasmin formation in case of EDTA pretreatment of fragments. The proenzyme activation rate on DD also increased in the presence of EGTA in concentration-dependent manner. Potentiating effect of EGTA-pretreated DD-fragment on plasminogen activation by tPA was decreased in the presence of Ca2+. Activation rate reduction was observed according to the increase of CaCl2 concentration in the reaction medium. The intensification of plasminogen activation potentiation by chelator-treated fibrin(ogen) D-domain containing fragments and subsequent potentiation decrease in the presence of Ca2+ indicated the requirement of Ca2+-dependent changes in D-domains for plasminogen activation sites exposure and initiation of fibrinolysis.

Mapping of residues of fibrinogen cleaved by protease II of Bacillus thuringiensis var. israelensis IMV B-7465

E. M. Stohniy1, V. O. Chernyshenko1*, N. A. Nidialkova2, A. V. Rebriev1,
L. D. Varbanets2, V. E. Hadzhynova1, T. M. Chernyshenko1,
I. M. Kolesnikova1, E. V. Lugovskoy1

1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
2Zabolotny Institute of Microbiology and Virology, National Academy
of Sciences of Ukraine, Kyiv;
*e-mail: bio.cherv@gmail.com

The limited proteolysis of macromolecules allows obtaining the fragments that preserve the structure and functional properties of the whole molecule and could be used in the study of proteins structure and function. Proteases targeted to fibrinogen and fibrin are of interest as the tool for obtaining of functionally active fragments of fibrin(ogen) and for the direct defibrination in vivo. That is why the aim of the present work was to study the proteolytic action of Protease II (PII) purified from Bacillus thuringiensis var. israelensis IMV B-7465 on fibrinogen.
Hydrolysis products of fibrinogen by PII were analysed by SDS-PAGE under reducing conditions with further immunoprobing using the mouse monoclonal 1-5A (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibodies. It was shown that PII cleaved preferentially the Aα-chain of fibrinogen splitting off the peptide with apparent molecular weight of 10 kDa that corresponded the C-terminal part of Aα-chain of fibrinogen molecule.
MALDI-TOF analysis of hydrolysis of fibrinogen was performed using a Voyager-DE. Results analyzed by Data Explorer 4.0.0.0 allowed to detect the main peak occurring at mass/charge (M/Z) ratio of 11 441 Da. According to “Peptide Mass Calculator” this peptide corresponded to fragment Аα505-610 of fibrinogen molecule. The result showed that PII cleaves the peptide bond AαAsp-Thr-Ala504-Ser505.
Thus, PII can be used for the obtaining of unique fragments of fibrinogen molecule. As far as αC-domain contains numerous sites of fibrin intermolecular interactions we can consider PII as a prospective agent for their study and for the defibrination.