Tag Archives: platelets

Blood coagulation parameters in rats with acute radiation syndrome receiving activated carbon as a preventive remedy

V. Chernyshenko1, E. Snezhkova2, M. Mazur2, T. Chernyshenko1,
T. Platonova1, O. Sydorenko2, E. Lugovskoy1, V. Nikolaev2

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: bio.cherv@gmail.com;
2RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv

Received: 13 December 2018; Accepted: 20 March 2019

Radiation-induced coagulopathy (RIC) is one of the major causes of death during acute radiation syndrome (ARS). The aim of this study was to characterize the responses of the hemostasis system to ARS of a moderate level on the 1st and 9th days after irradiation. We aimed to identify molecular markers of the blood coagulation system that are most affected by ARS and to estimate the enterosorption effect on the development of irradiation-induced changes. Platelet aggregation rate, activated partial thromboplastin time (APTT) and fibrinogen concentration were determined by standard methods. Level of protein C (PC) was measured using­ chromogenic substrate S2366 (p-Glu-Pro-Arg-pNa) and Agkistrodon halys halys snake venom activa­ting enzyme. Functionally inactive forms of prothrombin (FIFPs) were determined using two activators in parallel – thromboplastin or prothrombin activator from Echis multisqumatis venom. Rats of both irradia­ted groups had a higher risk of intravascular clotting in comparison to both control groups. Statistically significant shortening of clotting time in the APTT test (24 ± 4 s vs. 33 ± 5 s) and increased fibrinogen concentration (4.2 ± 0.6 mg/ml vs. 3.2 ± 0.3 mg/ml) were detected. Both parameters were normalized on the 9th day after irradiation. However the platelet count was decreased (0.3∙106 ± 0.05∙106 1/μl vs. 0.145∙106 ± 0.04∙106 1/μl) due to the impaired megakaryocytic function. The level of PC was decreased after X-ray irradiation (70 ± 10%) and partly restored on the 9th day after irradiation (87 ± 10%). Administration of activated carbon (AC) inhibited the drop in the PC concentration after X-ray irradiation (86 ± 15%) and accelerated its restoration on the 9th day (103 ± 14%). The statistically significant accumulation of FIFPs was detected in blood plasma of irradia­ted rats at the 1st and 9th days after irradiation. No FIFPs were found in any irradiated rat treated with AC. Characterization of the hemostasis system of rats that were exposed to a semilethal dose of X-rays allowed us to select parameters that can be used for monitoring of ARS development. Apart of from basic coagulation tests (APTT) and the measurement of platelet aggregation, fibrinogen and protein C level we can recommend the determination of FIFPs as a useful tool for estimation of the hemostasis response after irradiation with X-rays. This test indicates the intravascular thrombin generation and can help predict thrombotic complication or disseminated intravascular coagulation. Determination of FIFPs in blood plasma of irradia­ted rats allowed us to study the enterosorption effect on the development of irradiation-induced changes. It was shown that enterosorption with AC prevented accumulation of FIFPs which appears to be a newly discovered anti-thrombotic effect of therapy with AC. ARS influenced hemostasis by inducing thrombin generation (indicated by FIFPs generation), low-grade inflammation (indicated by PC concentration decrease) and thrombocytopenia. Enterosorption with AC minimizes inflammation and pro-coagulant processes caused by a moderate dose of X-ray irradiation. Accumulation of FIFPs can be assumed to be one of the most sensitive markers of the blood coagulation response to X-ray irradiation.

Preparation of highly-concentrated autologous platelet-rich plasma for biomedical use

V. Chernyshenko1, K. Shteinberg2, N. Lugovska1, M. Ryzhykova1,
T. Platonova1, D. Korolova1, E. Lugovskoy1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: bio.cherv@gmail.com;
2‘Dr. Zapolska Clinic’, Kyiv, Ukraine

Received: 21 December 2018; Accepted: 20 March 2019

Cell therapy with platelets is a widely accepted approach for wound healing and tissue regeneration in medicine. However, with most available methods poorly concentrated platelet suspensions (up to 0.3∙106 1/µl) or suspensions of mostly inactivated or lost platelets are obtained. In this study, we aimed to develop a simple and effective method for preparing a suspension of native and resting platelets with over 1∙106 1/µl. Platelet-rich plasma (PRP) was obtained from fresh blood of healthy donors (n = 5) collected using different amounts of heparin as the anticoagulant. Samples of PRP were spun down and re-suspended in auto­logous blood plasma. Count and vitality of platelets in each sample were determined by aggregation study on the Solar AP2110 aggregometer. Platelet shape and cytoplasmic granularity that indicate the nativity of platelets were monitored on the COULTER EPICS XL Flow Cytometer. This study of aggregation of platelets in PRP obtained using various amounts of heparin allowed us to reduce final concentrations to the amount that effectively prevented clotting and did not affect platelet reactivi­ty (5 U/ml). PRP concentrated 5 times with a total concentration of cells of 1∙106 1/µl was able to be activated by adenosine diphosphate (ADP) (aggregation rate 54 ± 7%). The amount of cells with altered shape and granularity in concentrated suspension was not higher than 20%. This finding means that the platelets would still be able to release a number of growth factors and other biologically active compounds after stimulation or injection into tissue during cell therapy. The decrease in heparin concentrations also minimizes haemorrhage in the injection site supporting biomedical use of the suspension. A simple and effective method for preparation of highly-concentrated PRP (1.2∙106 1/µl) for biomedical use was developed. Aggregometry and flow cytometry proved that obtained platelets were resting and able to be activated. Being autologous, the preparation can be widely used for cell therapy without additional precautions.

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.

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.

Clot formation and lysis in platelet rich plasma of healthy donors and patients with resistant hypertension

I. I. Patalakh1, O. V. Revka1, O. B. Kuchmenko2, O. O. Matova2, T. F. Drobotko2, T. V. Grinenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: sedrickedel@gmail.com
2National Scientific Center “Strazhesko Institute of Cardiology” NAMS of Ukraine, Kyiv

Hemostatic balance in blood is affected by numerous factors, including coagulation and fibrinolytic proteins, the wide spectrum of their inhibitors, and blood cells. Since platelets can participate in contradictory processes, they significantly complicate the whole picture. Therefore, nowadays the development of global assays of hemostasis, which can reflect the physiological process of hemostasis and can be used for point-of-care diagnosis of thrombosis, is crucial. This paper outlines a new approach we used to analyze the capabilities of clot waveform analysis tools to distinguish the response of platelet-rich plasma from healthy donors and patients with arterial hypertension caused by stimulation of coagulation and lysis (with exogenous thrombin and recombinant tissue-type plasminogen activator, respectively). In donor plasma, when the clot degradation was accompanied by 40 IU/ml of recombinant tissue-type plasminogen activator, platelets potentiated fibrinolysis more than coagulation, which ultimately shifts the overall balance to a profibrinolytic state. At the same time, for patients with hypertension, platelets, embedded in clot obtained from platelet-rich plasma, showed a weaker ability to stimulate fibrinolysis. The obtained data gives the evidence that platelets can act not only as procoagulants but also as profibrinolytics. By simultaneously amplifying coagulation and fibrinolysis, making their rates comparable, platelets would control plasma procoagulant activity, thereby regulating local hemostatic balance, the size and lifetime of the clot. Moreover, clot waveform analysis may be used to distinguish the effects of platelet-rich plasma on clotting or lysis of fibrin clots in healthy donors and patients with essential hypertension.

Glu- and Lys-forms of plasminogen differentially affect phosphatidylserine exposure on the platelet surface

D. D. Zhernossekov, Y. M. Roka-Moiia, A. O. Tykhomyrov,
M. M. Guzyk, T. V. Grinenko

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

Plasminogen/plasmin system is known for its ability to support hemostatic balance of blood. However, plasminogen may be considered as an adhesive ligand and in this way could affect the functioning of blood cells. We showed that exogenous Lys-plasminogen, but not its Glu-form, inhibited platelet aggregation and suppressed platelet α-granule secretion. The aim of this work was to investigate the influence of Glu- and Lys-form of plasminogen on the formation of platelet procoagulant surface using phosphatidylserine exposure as a marker. Human platelets were obtained from human platelet-rich plasma (donors were healthy volunteers, men aged 30-40 years) by gel-filtration on Sepharose 2B. Phosphatidylserine exposure on the platelet surface was evaluated by flow cytometry with FITC-conjugated annexin A5. Glu- and Lys-plasminogen have different impact on the platelet functioning. Exogenous Lys-plasminogen has no significant effect on phosphatidylserine exposure, while Glu-plasminogen increases phosphatidylserine exposure on the surface of thrombin- and collagen-activated human platelets. Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation. Meanwhile effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes. The observed different effects of Glu- and Lys-plasminogen on phosphatidylserine exposure can be explained by their structural peculiarities.

The search of compounds with antiaggregation activity among S-esters of thiosulfonic acids

T. I. Halenova1, I. V. Nikolaeva1, A. V. Nakonechna2,
K. B. Bolibrukh2, N. Y. Monka2, V. I. Lubenets2,
O. M. Savchuk1, V. P. Novikov2, L. I. Ostapchenko1

1Educational and Scientific Centre “Institute of Biology”,
Taras Shevchenko National University of Kyiv, Ukraine;
2Lviv Polytechnic National University, Ukraine;
е-mail: galenovatanya@rambler.ru

According to the current understanding, the hyperactivation of platelets may lead to increased intravascular coagulation and thrombosis. Today a relevant issue is the search for new anti-thrombotic agents that are able to modulate the activity of platelet receptors, thus, influence the processes of activation and aggregation of platelets. The aim of this study was to investigate the effects of newly synthesized thiosulfonate derivatives on platelet aggregation. The activity of the compounds was tested in vitro using platelet-rich plasma. As a result of the screening test, structural formulas of four agents with high antiaggregative activity were established. These compounds inhibited ADP- and collagen-induced platelet aggregation in a dose-dependent manner. Two of these compounds were shown to be more effective inhibitors of aggregation induced by ADP (IC50 ~ 8-10 µM), as well as collagen (IC50 ~ 1.5-2.0 µM).

Dynamics of thrombin-induced exposition of actin on the platelet surface

A. A. Tykhomyrov

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

Platelets play the key role in thrombosis and are also involved in angiogenesis as well as immune and reparative responses. In the function cascade, platelets undergo a complex cell processing, and subcellular fragments, not detectable in the resting state, are exposed on platelet surface after stimulation with agonists. This study has been performed to evaluate dynamic characteristics of actin exposition on the surface of plasma membrane of thrombin-activated platelets. Using flow-cytometric assay, it has been observed that the level of actin presented on activated platelets directly depends on agonist concentration. In the case of platelet stimulation with thrombin in the highest concentration (1.0 U/ml) taken for this study, the level of actin exposed on activated platelets was up to 4.4 times higher as compared with resting cells. Confirmation of the flow cytometry data for cell-surface actin on thrombin-activated platelets was achieved by direct visualization using a confocal laser scanning microscopy. Period of actin exposition appeared to be longer than the time phase corresponding to platelet secretion stage. Functional role of platelet surface actin has required further detailed studying, however, it is thought that superficial actin could interact with various blood plasma proteins, including plasminogen and its activators, serving as a binding site and/or center for their pericellular processing.