Tag Archives: plasminogen

Plasminogen modulates formation of reactive oxygen species in human platelets

A. A. Tykhomyrov, D. D. Zhernosekov, M. M. Guzyk, V. V. Korsa, T. V. Grinenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: artem_tykhomyrov@ukr.net

Reactive oxygen species (ROS) are considered to be important signalling molecules controlling many platelet functions. ROS production has been shown to be augmented by platelet activation, however, plasminogen (Pg) has not been studied in the context of modulating intraplatelet ROS levels. The aim of this study was to investigate the ability of different Pg forms to affect platelet metabolic activity/survival and intracellular ROS production in resting and activated platelets. Platelets isolated from donor plasma were pre-treated with Glu- or Lys-Pg (1.2 µM) and activated by thrombin (1.0 NIH unit/ml) or collagen (1.25 mg/ml). MTT assay was adapted to estimate total mitochondrial dehydrogenase activity, while intracellular ROS levels were monitored with the use of H2DCF-DA probe by flow cytometry. Lys-Pg was shown to slightly, but significantly, mitigate MTT reduction (P < 0.05 vs. control platelets). Two-fold elevation in metabolic activity of platelets stimulated by thrombin as compared to untreated cells was observed. However, this activation was less exhibi­ted in the case of platelets pre-incubated with either Glu- of Lys-Pg, with a predominant effect of Lys-Pg. Unlike thrombin, collagen treatment dramatically suppressed metabolic activity of platelets by 60% compared to control (P < 0.05). Glu- or Lys-Pg pre-incubation had no effects on the activity of collagen-stimulated platelets. Two subpopulations of platelets were observed with distinct characteristics of intracellular ROS formation. Elevated ROS production was demonstrated in these populations of both thrombin- and collagen-treated platelets. Pg (Lys-form to greater extent) enhanced intracellular ROS generation in thrombin-stimulated platelets. These findings suggest that augmented ROS generation within platelets pre-treated with Pg followed by their stimulation may result in down-regulation of their survival and functional activity. This study adds to our understanding one more possible mechanism of Pg impact on the platelet function.

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.

Plasminogen and its fragments in rat brain: a plausible role for astrocytes in angiostatin generation

A. A. Tykhomyrov1, V. S. Nedzvetsky2,3, C. A. Ağca3,
V. V. Korsa1, T. V. Grinenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Dnipropetrovsk National University, Dnipro, Ukraine;
3Bingöl University, Bingöl, Turkey;
e-mail: artem_tykhomyrov@ukr.net

The purpose of the present study was to examine the plasminogen localization and to detect levels of its fragments (angiostatins) in various regions of rat brain as well as to establish whether rat brain astrocytes could be involved in angiostatin production. It was shown immunohistochemically that plasminogen is distributed broadly in the various brain regions, with predominant expression in meningeal layer and IV, V, and VI layers or cerebral cortex, dentate gyrus, meningeal and Purkinje cells, molecular and granular layers of cerebellum, as well as vessel walls. Angiostatin polypeptides were detected by Western blot analysis mostly in the cerebral cortex and were represented by 50 and 40-30 kDa polypeptides. In the whole cell lysates from primary cultures of rat astrocytes, immunoreactive polypeptides with Mm ~ 92, 84, 65-60, 50, 40, 38-30 kDa, correspon­ding to native plasminogen and a variety of its truncated products, including angiostatin polypeptides, were revealed. Incubation of astrocytes with exogenous plasminogen resulted in gradual increasing levels of some plasminogen fragments, particularly 30 kDa protein. Moreover, this polypeptide appeared to be the single angiostatin released by astrocytes in vitro. We report here for the first time that astrocytes are one of the cell types in CNS that could be responsible for angiostatin formation and releasing.

Role of plasminogen/plasmin in functional activity of blood cells

D. D. Zhernossekov, E. I. Yusova, T. V. Grinenko

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

The article deals with the data concerning structural peculiarities of plasminogen/plasmin molecule, which define the specificity of intermolecular interactions and provide the variety of its biological functions. The main principles of the modern classification of plasminogen receptors and factors, which modulate their expression, have been presented. We have considered the mechanisms regulating both plasmin formation and activity on the surface of cells, fibrin and proteins of extracellular matrix. The data of previous investigators and our own results, concerning the influence of plasminogen/plasmin on platelet aggregation induced by different agonists, have been summarized. The participation of plasminogen/plasmin in atherogenesis and angiogenesis mediated­ by endotheliocyte receptors has been discussed. Special attention was given to plasminogen/plasmin pro-inflammatory function, which is realized by regulatory processes of activation, secretion, migration and apoptosis of monocytes and macrophages.

Effect of fibrin degradation products on fibrinolytic process

T. A. Yatsenko, V. М. Rybachuk, O. I. Yusova, S. M. Kharchenko, T. V. Grinenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: topolius@yandex.ua

Fibrin clot lysis by plasminogen/plasmin system results in fibrin degradation products formation with subsequent release into bloodstream. The fragments contain specific binding sites for fibrinolytic system components and can interact with them. In this study, we investigated the way in which fibrin fragments effect fibrinolytic process. We have shown that high molecular weight products of fibrin degradation and fibrin fragments of DDE-complex and DD, but not end product Е3, stimulate plasmin formation. Additionally, components of DDE-complex mixture of fragments Е1 and Е2 have potentiation ability. The intermediate fibrin fragments hmFDPs and DDE attenuate clot lysis by plasmin and hmFDPs protect plasmin from α2-antiplasmin inhibition but under further fragmentation to endpoint fibrin fragments loose this ability. The plasma inhibitors reduce fibrinolytic system activity generated by the degradation products. Thus, fibrin fragments formed during the clot lysis can bind and move out fibrinolytic system components from clot volume and in this way result in clot resistance to hydrolysis.

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.

Plasminogen and angiostatin levels in female benign breast lesions

A. A. Tykhomyrov1, I. L. Vovchuk2, T. V. Grinenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Odessa I. I. Mechnikov National University, Ukraine;
e-mail: artem_tykhomyrov@ukr.net

It is known that benign breast tissue exhibit relatively low angiogenic capacity. Activation of angiogenesis in mammary pre-malignant lesions could be associated with disease progression and high risk of transformation into the breast cancer. However, insight into the underlying molecular mechanisms involved in angiogenesis regulation in non-cancerous breast pathologies is still poorly defined. The purpose of the present study was to determine levels of plasminogen and its proteolytic fragments (angiostatins) in mammary dysplasia (mastopathy and breast cyst) and benign neoplasms (fibroadenomas). Plasminogen and angiostatins were analyzed using immunoblotting and quantified by densitometric scanning. The significant increase in plasminogen levels was found in fibrocystic, cysts, and non-proliferatious fibroadenoma masses (4.7-, 3.7-, and 3.5-fold, respectively) compared to healthy breast tissues (control). In the same benign lesions, 6.7-, 4-, and 3.7-fold increase in plasminogen 50 kDa fragment (angiostatin) levels as compared with control were also observed. Activation of matrix metalloproteinase-9, which was detected using gelatine zymography, could be responsible for plasminogen cleavage and abundance of angiostatin in fibrocystic and cyst masses. In contrast, dramatic decrease of both plasminogen and angiostatin levels (3.8- and 5.3-folds, respectively) was shown in tissues of proliferatious form of fibroadenoma in comparison with that of the dormant type of this neoplasm. Based on the obtained results, we concluded that angiostatin, a potent vessel growth inhibitor and anti-inflammatory molecule, can play a crucial role in pathophysiology of non-cancerous breast diseases. Further studies are needed to evaluate potential diagnostic and clinical implications of these proteins for prediction and therapy of benign breast pathologies.

Laureates of the Palladin Prize of the National Academy of Sciences of Ukraine (1985–1986)

R. P. Vynogradova, V. M. Danilova

Palladin Institute of Biochemistry, National Academe of Sciences of Ukraine, Kyiv;
e-mail: valdan@biochem.kiev.ua

The article presents the information about the life and scientific activity of the Palladin Prize laureats.  In 1985, S. O. Kudinov was awarded the Prize for his monograph ”Ca2+ transport systems in nerve cells”. In 1986,  A. Ya. Rozanov, A. I. Treshchinsky, Yu. V. Chmelevsky became the Palladin Prize laureats for the monograph “Enzymatic processes and their correction under extreme conditions” (K., Zdorov’ya, 1985. 208 p.).