На современном этапе остро стоит проблема разработки безопасных препаратов для борьбы с тромботическими осложнениями, возникающими при различных функциональных (сердечно-сосудистых, легочных и др.) и вирусных (развитие Covid) заболеваниях. Обычно применяют антикоагулянты гепариновой природы. В древовидных и травянистых пионах обнаружено наличие гепариноподобных веществ. Цель исследования - изучение антикоагулянтного действия и процессов деполимеризации фибрина гибридных сортов пионов («Куинджи», «Академик Садовничий» и «Коралл») и используемого для их выращивания древовидного (P. suffruticosa) в сравнительном аспекте. Методика. Объектом исследования служили корни гибридных пионов («Куинджи», «Академик Садовничий», «Коралл») и P. suffruticosa. Разработаны методы получения экстрактов из корней пионов и выявлена природа действующего начала. Определены антикоагулянтная, фибринолитическая активность и степень полимеризации фибрина после их включения в плазму крови крыс. Результаты. Экстракты из всех гибридных пионов и древовидного в концентрациях 1 и 10-3 мг/мл обладали антикоагулянтной и фибриндеполимеризационной активностью (ФДПА), но в разной степени. В них отмечалось превышение антикоагулянтной активности по тестам АЧТВ на 32-48%, тромбинового времени (ТВ) - на 8-15%, протромбинового времени - на 14-21%, и фибринолиза по тестам суммарной фибринолитической активности (СФА) - на 27-50%, ФДПА - на 49-80% , в то время как в экстракте из древовидного пиона эти активности усиливались в меньшей степени: антикоагулянтная активность повышалась на 23% (АЧТВ), на 7% (ТВ), а фибринолиз на 18-20% (СФА) и на 47% (ФДПА) по сравнению с нормой. Впервые обнаружено наличие гепариноидов в экстрактах из гибридных пионов. Заключение. Впервые установлена способность экстрактов из корней гибридных пионов проявлять синергические антикоагулянтные и фибриндеполимеризационные эффекты, превышающие таковые у экстракта из пиона древовидного. На основе полученных данных возникает необходимость исследования гибридных пионов в качестве антитромботических и антиатеросклеротических агентов. At present, developing safe drugs to combat thrombotic complications arising from various functional (cardiovascular, pulmonary, etc.) and viral (Covid-19) diseases is an acute problem. Usually, heparin-like anticoagulants are used in treatment of these diseases. These substances were found in tree and herbaceous peonies. The aim of this investigation was to compare the anticoagulant action and processes of fibrin depolymerization of hybrid peonies («Kuindzhi», «Academician Sadovnichy» and «Coral») and tree peonies (P. suffruticosa) that are used for heparin production. Roots of hybrid peonies («Kuindzhi», «Academician Sadovnichiy», «Coral») and P. suffruticosa were the object of research. Methods. Procedures to prepare peony root extractions were developed, and the nature of the active agent was determined. The anticoagulant, fibrinolytic activity, and the degree of fibrin polymerization were determined after addition to rat plasma. Results. For the first time, heparin-like substances were detected in extracts from hybrid peonies. At concentrations of 1 and 10-3 mg/ml, extracts from all hybrid peonies exhibited anticoagulant and fibrin-depolymerizing activity (FDPA), but to different degrees. Anticoagulant activity (ACTV) increased by 32-48%, thrombin time (TT) by 8-15%, prothrombin time by 14-21%, total fibrinolytic activity (TFA) by 27-50%, and FDPA by 49-80% compared to control. These activities were less in tree peony extract: ACTV increased by 23%, TT by 7%, TFA by 18-20%, and FDPA by 47% compared to control. Conclusion. For the first time the ability of hybrid peony root extract to show synergistic, anticoagulant and fibrin depolymerization effects exceeding those of extract from tree peony was established. These data demonstrate that hybrid peonies should be studied as sources of antithrombotic and anti-atherosclerotic agents.

Исследование гепаринов и гепариноидов в качестве антитромботических агентов актуально для физиологии и медицины. Многие растения включают гепариноподобные компоненты (гепариноиды), которые препятствуют тромбообразованию. Цель - установление влияния экстракта из корней пиона «Марьин корень» (Paeonia anomala), содержащего гепариноид, на полимеризацию фибрина при процессах тромбообразования ex vivo и определение возможных механизмов его антитромботического действия. Методика. Исследовано влияние гепариноида из пиона (Paeonia anomala) на процессы растворения фибрина в условиях тромбообразования ex vivo. Разработана модель тромбоза (МТ) ex vivo. К плазме крови крыс (объем 0,2 мл) добавляли 2 NIH ед. тромбина (0,05 мл), фибриновый сгусток образовывался в течение 2-3 мин. Экстракт гепариноида из пиона (0,1 мл 0.5%-й) добавляли к предобразованному сгустку через 12 мин после моделирования тромбоза (опыт А), или одновременно с добавлением тромбина к плазме крови (опыт Б). Использовали венозную (из v. jugularis) кровь крыс-самцов Wistar. Полимеризацию фибрина выявляли по тесту фибриндеполимеризационной активности (ФДПА) плазмы крови на нестабилизированном фибрине. В продуктах растворения фибрина под влиянием гепариноида оценивали активность тромбина (по тесту тромбинового времени), cвертывающего фактора XIIIa (по определению активности фактора XIIIa) и ФДПА. Результаты. В опыте А спустя 10 мин после добавления гепариноида к предобразованному сгустку отмечалось появление в нем жидкой фазы, что свидетельствовало о способности исследуемого гепариноида растворять фибрин. В опыте Б сгусток или не образовывался, или же был рыхлым. Полученные данные свидетельствовали об ингибировании процесса полимеризации фибрина под влиянием гепариноида. Выявлены антитромбиновые и антифибринстабилизирующие эффекты гепариноида из пиона при добавлении к фибриновому сгустку. Рассматриваются возможные механизмы действия гепариноида на блокаду полимеризации фибрина. Заключение. Растительный гепариноид препятствовал процессам полимеризации фибрина или растворял образующиеся фибриновые сгустки, что связано с его антитромбиновым и антифибринстабилизирующим действием. Studying heparins and heparinoids as antithrombotic agents is relevant for physiology and medicine. Many plants contain heparin-like components (heparinoids) that prevent thrombosis. The aim of the study was to identify effects of a heparinoid obtained from peony (Maryin root, P. anomala) roots on polymerization of fibrin and fibrinolytic activity of blood plasma and to suggest possible mechanisms of these effects in experimentally induced ex vivo thrombosis in rats. Methods. The effect of peony (Paeonia anomala) root heparinoid on fibrin dissolution was studied in the ex vivo conditions of thrombosis. For ex vivo modeling of thrombosis (MT), 2 NIH units of thrombin (0.05 ml) were added to 0.2 ml of rat plasma. A fibrin clot formed within 2-3 minutes. The peony heparinoid extract (0.5%, 0.1 ml) was added either to the pre-formed clot at 12 min after MT induction (experiment A) or simultaneously with the addition of thrombin to plasma (experiment B). Jugular vein blood from Wistar male rats was used. Fibrin polymerization was detected using a plasma fibrin-depolymerization activity (FDPA) test on non-stabilized fibrin. Thrombin activity (thrombin time test), coagulation factor XIIIa activity, and FDPA were evaluated in products of fibrin dissolution induced by the heparinoid. Results. In experiment A, at 10 min after the addition of heparinoid to the pre-formed clot, a liquid phase emerged, which indicated an ability of the pion heparinoid to dissolve fibrin. In experiment B, the clot either did not form or was liquid. These results indicated inhibition of fibrin polymerization under the action of the heparinoid. Therefore, the peony heparinoid added to the fibrin clot antagonized thrombin and fibrin stabilization. The article addresses possible mechanisms of the heparinoid inhibition of fibrin polymerization. Conclusion. The studied plant heparinoid prevented processes of fibrin polymerization or dissolved formed fibrin clots due to depression of thrombin activity and fibrin stabilization.

Studying heparins and heparinoids as antithrombotic agents is relevant for physiology and medicine. Many plants contain heparin-like components (heparinoids) that prevent thrombosis. The aim of the study was to identify effects of a heparinoid obtained from peony (Maryin root, P. anomala) roots on polymerization of fibrin and fibrinolytic activity of blood plasma and to suggest possible mechanisms of these effects in experimentally induced ex vivo thrombosis in rats. Methods. The effect of peony (Paeonia anomala) root heparinoid on fibrin dissolution was studied in the ex vivo conditions of thrombosis. For ex vivo modeling of thrombosis (MT), 2 NIH units of thrombin (0.05 ml) were added to 0.2 ml of rat plasma. A fibrin clot formed within 2-3 minutes. The peony heparinoid extract (0.5%, 0.1 ml) was added either to the pre-formed clot at 12 min after MT induction (experiment A) or simultaneously with the addition of thrombin to plasma (experiment B). Jugular vein blood from Wistar male rats was used. Fibrin polymerization was detected using a plasma fibrin-depolymerization activity (FDPA) test on non-stabilized fibrin. Thrombin activity (thrombin time test), coagulation factor XIIIa activity, and FDPA were evaluated in products of fibrin dissolution induced by the heparinoid. Results. In experiment A, at 10 min after the addition of heparinoid to the pre-formed clot, a liquid phase emerged, which indicated an ability of the pion heparinoid to dissolve fibrin. In experiment B, the clot either did not form or was liquid. These results indicated inhibition of fibrin polymerization under the action of the heparinoid. Therefore, the peony heparinoid added to the fibrin clot antagonized thrombin and fibrin stabilization. The article addresses possible mechanisms of the heparinoid inhibition of fibrin polymerization. Conclusion. The studied plant heparinoid prevented processes of fibrin polymerization or dissolved formed fibrin clots due to depression of thrombin activity and fibrin stabilization.

The search and development of direct and rapid anticoagulants used per os, is an urgent problem in physiological and medical science. A number of plants contain heparin-like components with a positive effect on the hemostatic system, both within normal and in some pathological conditions of the body. The aim of the work was to study the complex effect of fibrin, a heparin-like substance (heparinoid) from the roots of Paeonia lactiflora, on fibrinolytic, anticoagulant systems of the body and polymerization processes, when it is administered per os in animals within normal conditions and when modeling the state of prethrombosis. Materials and methods. To carry out the research, the roots of Paeonia lactiflora growing in the Botanical Garden of Moscow State University, and laboratory animals – male Wistar rats – were used. To study the antithrombotic effects of the extract from roots containing heparinoid, the state of prethrombosis was modeled in rats. The determined parameters of hemostasis were: anticoagulant activity according to the tests of activated partial thromboplastin time and thrombin time, fibrinolytic activity according to the test of total fibrinolytic activity, fibrin polymerization according to the test of fibrindepolymerization activity of blood plasma. Results. With repeated (every 24 hours within 3 days) oral administration of the extract containing heparinoid, in animals within normal conditions and with prethrombosis, the following anticoagulant effects were established in the blood: an increase in anticoagulant, fibrindepolymerization and fibrinolytic plasma activity. Possible mechanisms of the activating effect of heparinoid on fibrinolysis and anticoagulant properties of plasma due to the excretion of tissue plasminogen activator into the bloodstream from the endothelium, thrombin inhibition, and fibrin polymerization are described. Moreover, the anticoagulant effect of the use of the extract from the peony roots was equivalent to that of the reference drug of low molecular weight heparin from Celsus (USA). For the first time, it was revealed that when modeling experimental prethrombosis, the administration of heparinoid in rats at the dose of 37.5 IU/kg МЕ/кг body weight restored impaired hemostasis, which requires a further study. Conclusion. The ability of heparinoid from peony roots to normalize the functional state of the anticoagulant system during the development of prethrombosis in animals has been established. The restriction of fibrin polymerization during oral administration of heparinoid from peony in animals by increasing the enzymatic fibrinolytic and fibrindepolymerization activity of blood plasma was revealed. In the future, heparinoid can be used as an antithrombotic agent.

Experimental data on the effect of the natural immunopeptide tuftsin and its synthetic counterpart on hemostasis parameters in vitro and in vivo when administered intranasally to animals (white laboratory rats) are given. It has been shown that in vitro both preparations induced a fibrin-depolymerization effect on fibrin that is not stabilized by factor XIIIa. For each preparation, it was noted that, when administered to rats, simultaneous antiplatelet, anticoagulant, and fibrin-depolymerization effects were observed in the blood plasma, with Selank having a more pronounced effect.

Experimental data on the involvement of the blood anticoagulant system components (heparin complex compounds, short regulatory peptides) in fibrin formation processes are presented. Inhibition of thrombin activity and interactions of short glyproline peptides and their heparin complexes with fibrin monomer molecules are demonstrated. Peptides and their heparin complexes prevent the formation of primary polymeric fibrin and exhibit fibrin-depolymerizing activity on the formed polymeric fibrin. The mechanisms underlying the effects of heparin complexes on degradation of fibrin not stabilized with factor XIIIa is discussed on the basis of the results of spectral analysis and coagulation tests.

In the present work, the formation of the proline-heparin complex has been established. The way to synthesize the complex in vitro has been developed. The complex synthesized with the molar ratio of proline to heparin of 3: 1 extended the time of formation of a fibrin clot, reduced platelet aggregation, and showed a lytic effect towards nonstabilized fibrin in vitro conditions. Ten minutes after intravenous administration of the proline-heparin complex, there was an increase in levels of fibrin-depolymerization, anticoagulant, antifibrin-stabilizing and antiplatelet activity in blood of animals, whereas its components, proline and heparin, did not have such effects.