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EXPERIMENTAL ARTICLES
UDK 577.112:612.115
http://dx.doi.org/10.15407/biotech9.03.037
CALIX[4]ARENE C-145 EFFECTS ON СELLULAR HAEMOSTASIS
V. O. Chernyshenko1 D. S. Korolova1 T. V. Nikolaienko2 V. E. Dosenko3
D. O. Pashevin3 V. I. Kalchenko4 S. O. Cherenok4
N. N. Khranovska5 L. V. Garmanchuk4
E. V. Lugovskoy1 S. V. Komisarenko1
1Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv 2ESC "Institute of Biology" Taras Shevchenko National University, Kyiv 3Bohomolets Institute of Physiology of the National Academy of Sciences of Ukraine, Kyiv 4 Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine, Kyiv 5National Cancer Institute of Ministry of Health of Ukraine, Kyiv
E-mail: bio.cherv@gmail.com
Received 29.05.2016
The aim of the research was to study a potential antithrombotic sodium salt of calix[4]arene-methy-lene-bis-phosphonic acid (C-145) — on activation and aggregation of platelets in vivo, as well as on proliferation and apoptosis of endothelial cells in the cell culture.
Effects of calix[4]arene C-145 estimated in vitro after addition to the platelet rich plasma, and in vivo after intravenous injection into rabbit bloodstream in equivalent amounts (46 pM). Aggregation of platelets was induced by adenosine diphosphate and detected using aggregometer Solar AP2110. Platelet shape and cytoplasmic granularity were monitored on COULTER EPICS XL Flow Cytometer. The level of tissue-type plasminogen activator — tPA — was estimated using enzyme-linked immunosorbent assay ELISA. Effects of calix[4]arene C-145 on culture of endotelial cells cells was studied using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide — MTT-test. The population of proliferative pool of cells (G2/ M+S) was determined using flow cytometry.
Aggregometry and flow cytometry showed that calix[4]arene C-145 did not activate platelets nor affect their aggregation in vitro. However intravenous injection of calix[4]arene C-145 into the bloodstream of healthy rabbits leads to strong inhibition of platelet aggregation and changes of shape and granularity of most of the platelets after 2 hours of administration. Any additional appearance of endo-thelial cells activation marker tPA in vivo and any inhibition of calix[4]arene C-145 on proliferation of endothelial cells in cell culture did not observe.
So calix[4]arene C-145 had strong anti-platelet effect in vivo that was not a result of their direct action on platelets or endothelial cells in vitro. This allowed to assume the possibility of calix[4]arene C-145 use as an effective antithrombotic agent.
Key words: calix[4]arene, haemostasis, antithrombotic drugs, fibrin polymerization.
Calix[4]arene-methylene-bis-phosphonic acids are synthetic macrocyclic compounds obtained by cyclocondensation of para-substituted phenols and formaldehyde. Aromatic rings of calix[4]arenes form a lipophilic "cup", an interface tailored to handle macromolecules through hydrogen bonds, hydrophobicity or electrostatic interactions. Calix[4]arene C-192 and it's
sodium salt C-145 are inhibitors of blood coagulation. These calix[4]arenes selectively and with high affinity inhibit the first stage of fibrin polymerization — formation of protofibrils (IC50 = 0,510-6 and IC50 = 2,510-6 respectively). It was reported that this inhibition occurred as a consequence of direct binding of calix[4]arene with "A"-knob of fibrin molecule [1].
In vivo studies demonstrated that C-145, being injected intravenously into rabbit's bloodstream, acts as effective anticoagulant agent and its effects correspond to those shown in vitro. We did not find any significant effects of C-145 on total level of prothrombin, fibrinogen, activity of protein C and other main protein compounds of coagulation and fibrinolysis [2]. The effects of С-145 on cellular haemostasis that include platelets and endothelium remained unstudied.
Platelets and endothelium are the most sensitive compounds of haemostasis that being activated immediately after stimulation of coagulation cascade and take part in the formation of thrombus [3]. Study of platelet or endothelium responce on the action of any potential antithrombotic drug is one of the main conditions of the estimation of its effectiveness [4, 5].
That is why the aim of present work was to study a potential antithrombotic sodium salt of calix[4]arene-methylene-bis-phosphonic acid (С-145) — on activation and aggregation of platelets in vivo, as well as on proliferation and apoptosis of endothelial cells in the cell culture.
Materials and Methods
Materials. ADP (Merck, Germany); anti-tPA (tissue-type plasminogen activator), clone GMA-043 mouse monoclonal IgG; goat anti-mouse IgG (H+L) alkaline phosphatase conjugate, PNPP (para-Nitrophenylphosphate), DMEM (Dulbecco's Modified Eagle's medium), MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, dimethyl sulfoxide were purchased from Sigma (USA. Cell line РАЕ (Porcine Aortic Endothelial Cells)) was a kind gift of Prof. I. T. Goot (University of London).
Methods. Sample of Calix[4]arene С-145 was dissolved in 0.9% NaCl solution to the final concentration 15 mg/ml and was injected in the marginal ear vein of rabbit in the dose of 7.5 mg/kg using Wenflon catheter (Becton Dickinson, USA), G22 (0.8 mm). This study was approved by the institution's Ethics Committee.
Blood samples were collected using Wenflon catheter (Becton Dickinson, USA), G22 (0.8 mm) before the injection and after 2, 4 and 24 h after injection. 3.8% Sodium Citrate added immediately after collection to whole blood in 1:9 ratio was used as an anticoagulant.
Platelet rich blood plasma (PRP) was prepared from human citrated blood by centrifugation at 1 000 rpm during 30 min. Platelet poor blood plasma as obtained by spinning-down PRP at 1 500 rpm during 30 min [6].
Platelet shape and cytoplasmic granularity were monitored on COULTER EPICS XL Flow Cytometer [7]. Samples of PRP (1 ml) before and after the administration of C-145 were analyzed. Scattered and transmitted light were monitored to detect any changes of platelet granularity and shape respectively.
Platelet aggregation measurements were based on changes in the turbidity of platelet-rich plasma. Aggregation was registered for 10 min using Aggregometer Solar AP2110 (Belorussia). We estimated the initial rate and final level of aggregation at 37 °C. In typical experiment 250 pl of PRP was activated by ADP (12.5 pM) [8].
The level of tPA was estimated using ELISA according to [9]. Mixture was contain 2 pl of blood plasma in 0.2 ml of TBS. Monoclonal mouse anti-tPA (clone GMA-043) IgG antibody and Goat anti-mouse IgG conjugated to alkaline phosphatase were used for the detection. PNP formed after PNPP cleavage by alkaline phosphatase was determined at 405 nm using multiplate reader Multiscan EX.
Cell line of PAE (Porcine Aortic Endothelial Cells) was incubated in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine and 40 mg/ml gentamicin at standard conditions (at 37 °C in 5% CO2) during two days. C-145 in the final concentration 300 pM in TBS was added to the culture medium. Equivalent volume of TBS was added to control samples.
Cell viability was measured by MTT-colorymetric test. The biochemical essence of this method is based on the fact that mitochondrial dehydrogenases of living cells are capable to cleave MTT (3-(4, 5-diethylthiazoly-2-yl)-2,5-diphenyltetrazolium bromide) rings with formation of insoluble purple crystals (formazan). Cells were plated at a density of PAE cells per well in 96-well plates. The cells were incubated with 20 pl complete medium containing 1 mg/ml MTT at 37 °C for 4 h followed by solubilization with 100 pl dimethyl sulfoxide. The absorbance at 540 nm was measured with a microplate reader. The cell proliferation was expressed as percentage of the viable cell number of the control (non-treated cells) and C-145-treated cells. Cell proliferation rate was calculated as [(1-ODexperimental group)/ ODcontrol group] X 100% [10].
Cell cycle determination was measured by flow cytometry [11]. For this purpose the samples were stained with propidium iodide (PI), which selectively joins with intercalating places in DNA. Cytometry was carried out on the FACS Calibur (Becton Dickinson, United
States). Special mathematical program Mod Fit LT 2.0 (BDIS, United States) for Macintosh computers was used for acquisition and data analysis. Narrowband filter 585/42 nm was used in order to measure the fluorescence of PI.
Results and Discussion
Effects of C-145 on platelets. Action of C-145 on platelet branch of haemostatic system was estimated by the study of ADP-induced platelet aggregation in PRp. Number of platelets before the C-145 administration and after 2, 4 and 24.0 h after its injection into the rabbit's bloodstream was also calculated. PRP of rabbit taken before the injection of C-145 was used as the control probe. It was shown that in 2 hours after the administration of 46 pM of C-145 platelets lost their ability for aggregation. After 24 h the rate of platelet aggregation was not higher then 9 ± 5% against 35 ± 8% in PRP of control group (Fig. 1, A).
Number of platelets after the administration of C-145 was dramatically decreased from 180±30 thousands per 1 pl. The restoration of platelets count was observed in 24 h after injection of C-145 but nor number of platelets, nor their ability to aggregate did not restored completely till 48 h (Fig. 1, B).
Flow cytometry study showed that in 2 h after C-145 injection the pool of intact platelets in PRP decreased more then in 2 times (Fig. 1, C). Number of intact platelets in PRP did not reach control value (80 ± 4%) in 24 and 48 h.
In vitro studies demonstrated that C-145, being added to the PRP of healthy rabbit at the final concentration of 46 pM, did not change shape or granularity during incubation up to 3 h (Fig. 2, B) and also did not decrease the platelet aggregation (Fig. 2, E). The effects of C-145 injected intravenously in the equivalent amount are shown on the Fig. 2 (panels C and F).
Presented data allowed us to assume that C-145 injected into the rabbit's bloodstream
A
40 n 35 30 -25 -20 -
0 4
Time after C-145 injection, h
24
B
0 4
Time after Q-145 înjecth
24
C
Time after C-145 injection, h
¿Jj
24
Fig. 1. Platelet aggregation rate (A), number of platelets (B) and value of intact platelets (C) in platelet rich plasma of rabbits after intravenous administration of 46 pM of C-145:
0 — parameters before the injection (control); 4 — after 4 h; 24 — after 24 h after injection. Data represent
the means ± SE, *P < 0.05 as compared to the control
0
4
Contra)
In Vitro C-145
In vivo C-145
10 10 FS LOG 10 10
D
E
F
OS 18 -
CD <
Time, s
Time, s
Time, s
Fig. 2. Flow cytometry and aggregation study of platelets during C-145 administration in vitro and in vivo:
Flow cytometry of resting platelets of rabbit in platelet rich blood plasma (A), after addition of 46 pM of C-145 to platelet rich blood plasma (PRP) in vitro (B) and after intravenous administration of equivalent C-145 in vivo (C). The distribution of platelets according to the correlation between shape and granulation. LgSS — parameter of platelets granulation: LgFS — parameter of platelets shape. Traces are typical for 3 independent experiments. Aggregation of rabbit platelets in PRP (D), after addition of 46 pM of C-145 to platelet rich blood plasma (PRP) in vitro (E) and after intravenous administration of equivalent C-145 in vivo
(F). Traces are typical for 3 independent experiments
at the final concentration of 46 pM has strong anti-platelet action. Using flow cytometry and aggregometry we did not observed any similar effects of equivalent concentrations of C-145 in vitro.
Effects of C-145 on endothelial cells. As far as C-145 demonstrated prominent effects on platelets, it was very important to investigate its effects on another compound of cellular haemostasis — endothelium [12].
For the evaluation of C-145 effects on endothelial cells in vivo we determined the level of tPA in blood plasma of rabbits after intravenous administration of 46 pM of C-145. Tissue-type plasminogen activator is secreted into the bloodstream and is common marker of endothelial cells activation [13].
As it is shown on Fig. 3, confident difference between level of tPA before and after the C-145 administration was not observed. Thus we can assume that endothelial cells remained intact and were not activated after C-145 administration.
Direct study of C-145 action on endothelial cells was carried-out using PAE cell culture
that was synchronized in G0/G1 phase of cell cycle. For this cells were planted 500 000 ml1 in 100 ml and cultivated during 2 days. Model of used cells culture leaded to the formation of monolayer and transition most of the endothelial cells in G0/G1 phase of cell cycle. Cells were cultivated in the presence of 300 pM of C-145 during 48 h. The cell viability after treatment with C-145 was evaluated by the MTT assay. It was shown that the level of apoptotic cells in the presence of C-145 decreased to 19 ± 1% against 24 ± 2.1 in control. The population of proliferative pool of cells (G2/M+S) in the presence of C-145 was increased in 4 times (Table) compared to control probes.
Thus, any activating action of C-145 on endothelium in vivo was observed. However cell culture studies demonstrated antiapoptotic and proliferation-stimulating effects of C-145 on PAE that could be a new promising feature of C-145 that has to be studied more precisely.
It is known that some medications injected intravenously could provoke decrease of platelet count that is known as drug-induced thrombocytopenia [14]. In most cases they
Density of apoptotic cells and distribution of endothelial cells in the presence of C-145 (300 pM)
Apoptotic cells, % Cell cycle stages
Gc/Gi G2/M S
Control 24 ± 2.1 85.71 ± 2.3 4.85 ± 0.5 9.71 ± 0.7
C-145 19 ± 1 34.13 ± 1.4 33.19 ± 1.8 32.68 ± 1.3
0 4 24
Time after C-145 injection, hour
Fig. 3. The level of tPA in rabbit plasma following intravenous administration of 46 pM of C-145
induce the production of antibodies specific to surface molecules of platelets which are active only in the presence of drug [15]. Thrombocytopenia is common for the use of heparin and low-molecular weight heparin (HIT, heparin-induced thrombocytopenia
[16]) and antagonists of GPIIbIIIa-receptors
[17]. Last ones could induce the appearance of ligand-induced binding sites on the surface of platelet receptor GPIIbIIIa that are the targets to auto-antibodies [18].
Another reason of thrombocytopenia could be the disorder of platelet production by megakaryocytes [19] or fast degradation of platelets [20]. Recently demonstrated anti-platelet effect of C-145 in vivo could not be explained by the affection of platelet production as far as it was observed only after 2 hours of C-145 administration. In the same time the period of life of platelet in the bloodstream could last to 100 hours [21].
Fast decrease of platelets count by C-145 could provide additional anticoagulant effect but on the other hand it made obligatory the instant control of platelet viability during treatment with C-145 and, possibly, concomitant therapy targeted to platelet restoration.
Some anticoagulant medication can act on endothelial cells mainly by increasing or inhibiting their proliferation [22], but
sometimes drug-induced activation of endothelium leading to bleeding is also observed [23].
Intravenous administration of C-145 did not affect endothelial cells that was confirmed by determining the level of tPA which is the marker of endothelial activation. However the study of C-145 effects on endothelial cells culture allowed to conclude anti-apoptotic and proliferation-stimulating effects of C-145 that could be promising during revascularization of tissues after ischemia.
So we have analyzed the effects of intravenously administrated calix[4]arene C-145 (46 pM) on cellular haemostasis in vivo. Our findings (composed with data on C-145 effects on protein haemostasis) allow us to conclude the possibility of its use in anticoagulant therapy. Anti-platelet action of C-145 can significantly increase its anticoagulant action. Instant monitoring of platelet functionality could minimize risk of possible by-effects of C-145 administration.
The research was a part of Project "Study of calix[4]arenes as haemostatic, antifibrinolytic and antithrombotic agents" according to the State scientific-technical program "Fundamental issues of new nanomaterials and nanotechnologies creation".
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Д1Я КАЛ1КС[4]АРЕНУ С-145 НА КЛ1ТИННУ ЛАНКУ СИСТЕМИ ГЕМОСТАЗУ
В. О.Чернишенко1, Д. С. Корольова1, Т. В. Школаенко2, В. G. Досенко3, Д. О. Пашевш3, В. I. Кальченко4, С. О. Черенок4, Н. М. Храновська5, Л. В. Гарманчук , Е. В. Луговськой1, С. В. КомЬсаренко1
Институт 6ioxiMiï iM. О. В. Палладша НАН Украши, Кшв 21нститут 6ioopraHi4Hoï xîmîï та нафтохiмiï
НАН Укрaïни, Киïв 31нститут фiзiологiï iM. О. О. Богомольця НАН Украши, Киïв 4ННЦ "1нститут б^логп"
Киïвського нaцiонaльного унiверситету iMeHi Тараса Шевченка 5Нaцiонaльний 1нститут раку МОЗ Украши, Киïв
Метою роботи було вивчення дп потенцш-ного антитромботичного агента — калшс[4] aрен-метилен-бiс-фосфоновоï кислоти — ка-лiкс[4]aрену С-145 на активащю та aгрегaцiю тромбоцитiв in vivo, а також на пролiферaцiю та апоптоз ендотелмциив у тканиннш культурi.
Ефекти кaлiкс[4]aрену С-145 оцшювали in vitro пiсля додавання у збагачену тромбоцитами плазму кров^ а також in vivo тсля вну-трiшньовенного введення у кровоток кролика в еквiвaлентних кiлькостях (46 дМ). Агрегaцiю тромбоцитiв iндукувaли ADP i встановлювали за допомогою агрегометра Solar AP2110. Форму та грануляршсть цитоплазми тромбоцитiв визначали протоковим цитометром COULTER EPICS XL. Рiвень активатора плазмшогену тканинного типу — tPA — вимiрювaли методом iмуноензимного aнaлiзу ELISA. Ефекти калшс[4]арену C-145 на культуру ендотель альних кл^ин визначали з використанням 3-(4,5-диметилмазол-24л)-2,5-диметилтетра-золiю бромiду — МТТ-тесту. Популящю проль феративного пулу (G2/M+S) клггин ендотелiю визначали за допомогою цитометри.
Методами aгрегaтометрiï та цитометрiï встановлено, що кaлiкс[4]aрен С-145 не акти-вуе тромбоцити та не впливае на ïx aгрегaцiю in vitro. Водночас вже через двi години тсля введення калшс[4]арену С-145 здоровим лаборатор-ним кролям тромбоцити втрачали здатшсть до aгрегaцiï, а пул штактних тромбоцитiв зменшу-вався бшьш нiж удвiчi. Викиду в плазму кров1 маркера aктивaцiï едндотелмцимв tPA in vivo, а також шпбування пролiферaцiï ендотелiоцитiв у культурi клiтин не спостерiгaли.
Таким чином, калшс[4]арен С-145 виявляе значний антитромбоцитарний ефект in vivo, не дiючи безпосередньо на тромбоцити та ендо-тел^цити in vitro. Тaкi влaстивостi калшс[4] арену уможливлюють його використання як ефективного антитромботичного агента.
Ключовi слова: калшс[4]арени, гемостаз, антитромботичш препарати, полiмеризaцiя фiбрину.
ДЕЙСТВИЕ КАЛИКС[4]АРЕНА С-145
НА КЛЕТОЧНОЕ ЗВЕНО СИСТЕМЫ ГЕМОСТАЗА
В. А. Чернышенко1, Д. С. Королева1, Т. В. Николаенко4, В. Е. Досенко3, Д. А Пашевин3, В.И. Кальченко4, С.А Черенок4, Н.Н.Храновская , Л. В. Гарманчук , Э. В. Луговской1, С. В. Комисаренко1
1Иститут биохимии им. А. В. Палладина НАН Украины, Киев 2ОНЦ "Институт биологии"
Киевского национального университета имени Тараса Шевченко 3Институт физиологии им. А. А. Богомольца
НАН Украины, Киев 4Институт биоорганической химии и нефтехимии
НАН Украины, Киев 5Национальный Институт рака МЗ Украины, Киев
Целью работы было изучение действия потенциального антитромботического агента — натриевой соли каликс[4]арен-метилен-бис-фосфоновой кислоты — каликс[4]арена С-145 на активацию и агрегацию тромбоцитов in vivo, а также на пролиферацию и апоптоз эндотелио-цитов в тканевой культуре.
Эффекты каликс[4]арена С-145 оценивали in vitro после добавления в плазму крови, богатую тромбоцитами, а также in vivo после внутривенного введения в кровоток кролика в эквивалентных количествах (46 дМ). Агрегацию тромбоцитов индуцировали ADP и определяли с помощью агрегометра Solar AP2110. Форму и гранулярность цитоплазмы тромбоцитов устанавливали проточным цитометром COULTER EPICS XL. Уровень активатора плазминогена тканевого типа — tPA — измеряли методом иммуно-энзимного анализа ELISA. Эффекты каликс[4] арена C-145 на культуру эндотелиоцитов изучали с помощью 3-(4,5-диметилтиазол-2-ил)-2,5-диметилтетразолия бромида — МТТ-теста. Популяцию пролиферативного пула (G2/M+S) клеток эндотелия определяли с применением цитометрии.
Методами агрегатометрии и цитометрии установлено, что каликс[4]арен С-145 не активирует тромбоциты и не влияет на их агрегацию in vitro. Однако уже через два часа после введения каликс[4]арена С-145 здоровым лабораторным кролям тромбоциты теряли способность агрегировать, а пул интактных тромбоцитов уменьшался в два раза. В то же время не наблюдали выброса в плазму крови маркера активации эндотелиоцитов tPA in vivo, а также ингибирования пролиферации эндотелиоцитов в культуре клеток.
Таким образом, каликс[4]арен С-145 обладает значительным антитромбоцитарным эффектом in vivo, не действуя непосредственно на тромбоциты и эндотелиоциты in vitro. Такие свойства каликсарена позволяют рассматривать возможность его применения в качестве эффективного антитромботического агента.
Ключевые слова: каликс[4]арены, гемостаз, антитромботические препараты, полимеризация фибрина.
