Tag Archives: fibrinogen

Validation of the diagnostics algorithm to monitor coagulation parameters in pregnant women

D. S. Korolova1, A. O. Pavlenko1, A. Altorjay2, S. I. Zhuk3,
I. V. Us3, Y. Tsaryk1, A. Suranyi2, V. O. Chernyshenko1*

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Albert Szent-Györgyi Medical School, University of Szeged, Hungary;
3P. L. Shupyk National Medical Academy of Postgraduate Education, Kyiv, Ukraine;
*e-mail: bio.cherv@gmail.com

Received: 19 May 2023; Revised: 05 June 2023;
Accepted: 07 June 2023; Available on-line: 11 July 2023

Thrombotic events are among the most dangerous complications of pregnancy. Therefore, selection of appropriate tests and standardization of techniques used for accurate diagnostics of blood coagulation system state is of great importance. In this present study, we monitored several molecular markers of the dangers of intravascular thrombus formation and estimated the platelet function in pregnant women during­ gestation. We performed independent measurements using the same methodology for different cohorts of patients recruited in Kyiv (Ukraine) and in Szeged (Hungary). D-dimer and soluble fibrin were measured using ELISA. Protein C (PC) level was estimated using chromogenic substrate assay. Fibrinogen concentration was measured by spectrophotometry using thrombin-like enzyme. Platelet function was estimated by aggregometry­. Statistical data analysis was performed using the Kruskal-Wallis test. Statistically significant increases of fibrinogen concentration from first to third gestational trimester was shown for both studied cohorts of patients (5-6 mg/ml at third trimester on average). Applied methods allowed us to detect the same tendencies of decreases in PC level as well as the appearance of moderate amounts of D-dimer (up to 300 ng/ml) and SF (up to 10-15 ug/ml). Platelet function was increased on the first trimester of pregnancy and decreased during­ following trimesters slightly. Results indicated the changes in the blood coagulation system of pregnant women during gestation with the same effectiveness independently of the selected cohorts, time and place of measurements. The application of the proposed diagnostics algorithm may allow estimating the risk of thrombotic complications during pregnancy.

Overall hemostasis potential of blood plasma and its connection to molecular markers of the hemostasis system in patients with stenosis of coronary artery

N. V. Storozhuk1, L. V. Pyrogova2, Т. М. Chernyshenko2,
O. P. Kostyuchenko2, T. M. Platonova2, O. B. Storozhuk1,
B. G. Storozhuk1, R. Yu. Marunich2,
G. K. Bereznytsky2, E. M. Makogonenko2*

1MI Pirogov Vinnytsia National Medical University, Vinnytsia, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: ymakogonenko@gmail.com

Received: 01 April 2021; Accepted: 22 September 2021

The correlation relationships between hemostatic potential parameters and concentrations of molecular markers of the hemostasis system: soluble fibrin (sf), D-dimer (DD), fibrinogen (Fg) and protein C (PC) in patients with stenosis of coronary artery 6 months after coronary angioplasty have been investigated. It was found  three directions of changes in the state of the patients hemostasis system: an increasing in fibrinolytic activity (C) ~18% of patients; an increasing in coagulative activity (B) ~31% of patients; and maintaining of the balance between coagulation and fibrinolysis (A) ~51% of patients. In patients with signs of stenosis without angina pectoris, a strong Pearson correlation was shown between the half-life of the clot and the overall hemostatic potential (OHP) (r = 0.75, P << 0.05), a moderate relationship between concentrations of sf and D-dimer (r = 0.67, P <0.05), almost complete connection between coagulation potential (CP) and OHP (r = 0.975, P << 0,05) and strong connection between CP and fibrinolytic potential (FP) (r = 0.80, P << 0.05). In patients with signs of stable angina pectoris, almost complete connection was found between the concentration of sf and D-dimer (r = 0.981, P << 0.05), CP and OHP (r = 0.979, P << 0.05) and a strong connection between CP and FP (r = 0.846, P << 0.05). Possible functional mechanisms of connection between these parameters are discussed.

Thromboelastographic study of fibrin clot and molecular basis of maximum clot firmness

D. S. Korolova1*, Y. M. Stohnii1, V. I. Gryshchuk1, S. I. Zhuk2,
I. V. Us2, T. M. Chernyshenko1, O. P. Kostiuchenko1, K. P. Klymenko1,
O. M. Platonov1,3, O. I. Ivashchenko3, V. O. Chernyshenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Shupyk National Medical Academy of Postgraduate Education, Kyiv, Ukraine;
3ESC “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: d.korolova@gmail.com

Received: 29 January 2021; Accepted: 23 April 2021

Maximum clot firmness (MCF) is the main parameter of thromboelastography (TEG) reflecting the stability of a clot. In this work, we looked for markers that can influence the enhancement of MCF and detec­ted molecular markers and blood clotting parameters that can be involved in such mechanisms. Blood samples of pregnant women with placental disorders were collected in the Kyiv Perinatal Center. TEG was performed on whole blood in EXTEM and INTEM tests. APTT, INR, fibrinogen concentration and platelet aggregation were measured using traditional laboratory approaches. D-dimer was detected in sandwich ELISA using monoclonal antibodies III-3B and II-4D. The relative cross-linking activity of factor XIIIa was measured by the direct quantification of the cross-linked γ-chain of fibrin using Western-Blotting with monoclonal antibody II-4D. D-dimer and fibrinogen concentrations, clotting time in the APTT test, INR and rate of platelet aggregation did not correlate with the MCF. However, we found positive correlations of MCF with factor XIIIa activity: 0.51 and 0.87 for EXTEM and INTEM, respectively. These data indicate that for normal and slightly increased fibrinogen concentrations, fibrin clot firmness will depend mostly on the activity of factor XIIIa. Thus the direct determination of factor XIIIa activity in blood plasma of patients can be relevant for predicting the risk of intravascular coagulation. Evaluation of the content and activity of individual clotting factors or other components of the coagulation system can be useful additions to the TEG diagnostics and should not be neglected.

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.