Tag Archives: fibrinogen

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.

Inventive activity of the Departments of Protein Structure and Function, and Molecular Immunology of the Palladin Institute of Biochemistry of NAS of Ukraine. Part II. National breakthrough in the study and diagnostics of human hemostasis system

N. E. Lugovska

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: nlugovsk@mail.ru

The scientists of Protein Structure and Function, and Molecular Immunology Departments of the Palladin Institute of Biochemistry (NAS of Ukraine) under the supervision of member of NASU and NAMSU, prof. S. V. Komisarenko  and corresponding member of NASU prof. E. V. Lugovskoy have made the real breakthrough in the field of research of the mechanisms of fibrin polymerization and formation of fibrin framework of thrombi.  The immunodiagnostic test-systems for the evaluation of the risk of thrombus formation  were developed for the first time. Researches have obtained the monoclonal antibodies to fibrinogen, fibrin, D-dimer and their fragments. These monoclonal antibodies were used as molecular probes for the localization of newly detected fibrin polymerization sites. Obtained antibodies with high affinity interact with fibrinogen, D-dimer and soluble fibrin – main markers of the risk of thrombus formation. They were used for the development of the immunodiagnostic test-systems to quantify these markers in human blood plasma for the evaluation of the state  of haemostasis system, detection of prethrombotic states, disseminated intravascular coagulation, detection of thrombosis and monitoring of antithrombotic and fibrinolytic therapy. The successful trial of developed test-systems was carried out in clinics of Ukraine, and the State registration was obtained for the implementation of them into the clinical practice. Presented works were awarded State prize of Ukraine in Science and technology.

A novel mechanism controlling the growth of hemostatic thrombi

V. K. Lishko, I. S. Yermolenko, N. P. Podolnikova, T. P. Ugarova

School of Life Sciences, Arizona State University, Tempe, AZ USA

Current knowledge of the mechanisms of blood coagulation does not provide an answer to one pivotal question: why is, in contrast to a pathological thrombus, the growth of normal hemostatic clot after blood vessel injury suddenly terminated? In the present paper, we summarize the results of our investigations that give an answer to this question. We show that the surface of fibrin clot in the circulation is coated with a thin metastable layer of fibrinogen which is not able to support adhesion of blood cells. Consequently, platelets and leukocytes, the cells expressing adhesive integrins, are incapable of consolidating­ their grip on the surface and washed away by blood flow, thereby preventing the thrombus propagation. The cells that escaped this fibrinogen shield and reached a solid fibrin matrix use an additional mechanism – the ability to activate plasminogen bound either to the surface of cells or to fibrin. Plasmin formed at the interface between the cells and the clot locally degrades fibrin resulting in the fragmentation of the surface rendering it unstable, non-adhesive and therefore non-thrombogenic. Thus, the growth of hemostatic thrombus is halted by two mechanisms, fibrinogen- and plasminogen-dependent, both of which are based on the same principle – the generation of the mechanically unstable, non-adhesive surface.