Tag Archives: fibrinolysis

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.

Haemostasis modulation by calix[4]arene methylenebisphosphonic acid C-145 and its sulfur-containing analogue

V. O. Chernyshenko1, O. V. Savchuk1, S. O. Cherenok2,
O. M. Silenko2, A. O. Negelia3, L. O. Kasatkina1, L. V. Pirogova1,
V. A. Didkivskyi1, O. I. Yusova1, V. I. Kalchenko2, L. V. Garmanchuk3,
T. V. Grinenko1, E. V. Lugovskoy1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
3ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: bio.cherv@gmail.com

C-145 (octasodium salt of calix[4]arene-tetra-methylenebisphosphonic acid) was previously considered as specific anti-сoagulant agent that affects fibrin polymerization and does not notably influence other parameters of coagulation system. C-145S (octasodium salt of thiacalix[4]arene-tetra-methylenebisphosphonic acid) possessing wider hydrophobic hole was expected to be more effective antithrombotic agent than C-145. The aim of present work was to compare the action of both organic compounds on fibrin polymerization, fibrinolysis, platelets and endothelial cells. The change of turbidity during fibrin clot formation induced by APTT-reagent and digestion induced by tPA was estimated. Turbidity study was used for the estimation of polymeric fibrin hydrolysis by plasmin in the presence of thiacalix[4]arene C-145S and calix[4]arene C-145. Effects of thiacalix[4]arene C-145S and calix[4]arene C-145 on the activation of Glu-plasminogen by streptokinase were studied using chromogenic substrate S2251. Platelet aggregation study was performed using aggregometry. Stimulated Ca2+ efflux from endoplasmic reticulum and cytoplasm were determined using specific Ca2+-sensitive probes targeted to endoplasmic reticulum (Mag-Fluo-4) and cytoplasm (FURA-2) by spectrofluorimetry. Both C-145 and C-145S decreased the final turbidity of clot and prolonged clot lysis time in blood plasma in comparison to control value. C-145 was shown to be the more effective fibrinolysis inhibitor when studied in model system of polymerized fibrin desAB. C-145S but not C-145 induced concentration changes of Ca2+ in cytoplasm of resting platelets and significantly inhibited (up to 30%) Ca2+ efflux from endoplasmic reticulum of platelets activated by ADP. Both C-145 and C-145S stimulated the proliferation of endothelial cells of PAE cell line. The effect of C-145S was more prominent. In conclusion, calix[4]arene C 145S proved to be the more potent inhibitor of fibrin polymerization in comparison to C-145, which suggested earlier as anticoagulant agent. C-145S proved to have much more outlined inhibitory action on Ca2+-signaling in platelets and stimulatory effect on endothelial cells proliferation. Thus C-145 remained the most prospective molecular platform for the development of antithrombotic agent.

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.

The experimental investigation of fibrinolytic system under the influence of flocalin in conditions of acute hypoxic kidney injury

A. I. Gozhenko1, Yu. I. Gubsky2, N. D. Filipets3,
О. О. Filipets3, O. A. Gozhenko1

1State Enterprise Ukrainian Research Institute for Medicine of Transport, Odessa;
2State Institution Institute of Pharmacology and Toxicology, National Academy of Medical Sciences of Ukraine, Kyiv;
3Higher State Educational Establishment of Ukraine Bukovinian State Medical University, Chernivtsi;
е-mail: filipec.natalja@bsmu.edu.ua

In the experiments on rats subjected to acute hypoxic histochemical nephropathy, caused by sodium nitrite and 2,4-dinitrophenol, fibrinolytic activities of blood plasma, urine, renal cortex, medulla, and papilla after treatment with flocalin – the activator of KATP channels, were studied­. It was shown that in the conditions of acute kidney hypoxic injury flocalin administration resulted in the increase and essential restoration of fibrinolysis in blood plasma diminished under hypoxia, which was due to the growth of non-enzymatic fibrinolysis, whereas in urine and renal medulla the appreciable increase of enzymatic fibrinolytic activity took place. Moreover, the treatment of hypoxic nephropathy animals by flocalin resulted in the marked restoration of kidney ion regulatory and protein excretory functions that proves the positive influence of KATP channels activation on the one of the biochemical mechanisms of acute kidney injury as well as the protective effect of flocalin in relation to tubular cells of nephron. The obtained results testify to the beneficial effects of KATP channels activation in the conditions of acute hypoxic kidneys injury.