Effect of new chalcone analogues on hemostasis in animals with experimental «сytokine storm»

Введение. «Цитокиновый шторм» представляет собой расстройство иммунной системы с выраженной гиперцитокинемией, характеризующееся развитием коагуляционных нарушений с высоким уровнем летальности. Цель исследования: оценить влияние новых аналогов халкона на изменение реакций гемостаза у крыс в условиях экспериментального «цитокинового шторма». Материалы и методы. Исследование было выполнено на 80 крысах- самцах линии Wistar, разделенных на 8 равных групп по 10 особей. «Цитокиновый шторм» моделировали путем внутрибрюшинного введения липополисахарида в дозе 10 мг/кг. Исследуемые соединения в дозе 20 мг/кг интраперитонеально и препарат сравнения — гепарин (20 ЕД/кг, подкожно) вводили через 60 мин после моделирования патологии. Через 24 ч в сыворотке крови у крыс оценивали содержание фибриногена, D-димера, растворимых фибрин-мономерных комплексов (РФМК), активность антитромбина III (АТ-III), тромбиновое время (ТВ) и степень АДФ-стимулированной агрегации тромбоцитов. Результаты. Применение аналогов халкона способствовало восстановлению гемостатических реакций, что выражалось в снижении концентраций фибриногена, D-димера, РФМК, степени агрегации тромбоцитов и повышении активности АТ-III и ТВ. При этом в ряду изучаемых веществ соединение, содержащее гидроксил во 2-м положении и метильную группу в 5-м положении, проявляло несколько больший уровень фармакологической активности, нежели остальные исследуемые соединения. Заключение. На основании полученных данных можно предположить актуальность дальнейшего изучения аналогов халкона как средств, нормализующих гемостаз при гиперцитокиновых расстройствах. Background. «Cytokine storm» is a disorder of the immune system with severe hypecytokinemia, characterized by the development of coagulation disorders with a high level of mortality. Objectives: to evaluate the effect of new chalcone analogues on changes of hemostasis reactions in rats under the conditions of an experimental «cytokine storm». Materials/Methods. The study was performed on 80 male Wistar rats divided into 8 equal groups of 10 individuals. The «cytokine storm» was modeled in animals by intraperitoneal injection of lipopolysaccharide at a dose of 10 mg/kg. The test-compounds at a dose of 20 mg/kg intraperitoneally and the reference drug — heparin (20 U/kg, subcutaneously) were administered 60 minutes after the pathology simulation. After 24 hours, the serum levels of fi brinogen, D-dimer, soluble fibrin-monomer complexes, antithrombin III activity, thrombin time, and the degree of ADP-stimulated platelet aggregation were evaluated in rats. Results. The study showed that the use of chalcone analogues contributed to the restoration of hemostasis reactions, which was expressed in a decrease in theconcentration of fibrinogen, D-dimer, soluble fibrin-monomer complexes, the degree of platelet aggregation, and an increase in antithrombin III activity and thrombin time. At the same time, among the studied substances, the compound containing hydroxyl in the 2nd position and the methyl group in the 5th position showed a slightly higher level of pharmacological activity than the other test compounds. Conclusions. Based on the obtained data, it is actuality to assume the relevance of further study of chalcone analogues as agents that normalize hemostasis in hypercytokine disorders.

Validation of a Prospective Urinalysis-Based Prediction Model for ICU Resources and Outcome of COVID-19 Disease: A Multicenter Cohort Study

In COVID-19, guidelines recommend a urinalysis on hospital admission as SARS-CoV-2 renal tropism, post-mortem, was associated with disease severity and mortality. Following the hypothesis from our pilot study, we now validate an algorithm harnessing urinalysis to predict the outcome and the need for ICU resources on admission to hospital. Patients were screened for urinalysis, serum albumin (SA) and antithrombin III activity (AT-III) obtained prospectively on admission. The risk for an unfavorable course was categorized as (1) “low”, (2) “intermediate” or (3) “high”, depending on (1) normal urinalysis, (2) abnormal urinalysis with SA ≥ 2 g/dL and AT-III ≥ 70%, or (3) abnormal urinalysis with SA or AT-III abnormality. Time to ICU admission or death served as the primary endpoint. Among 223 screened patients, 145 were eligible for enrollment, 43 falling into the low, 84 intermediate, and 18 into high-risk categories. An abnormal urinalysis significantly elevated the risk for ICU admission or death (63.7% vs. 27.9%; HR 2.6; 95%-CI 1.4 to 4.9; p = 0.0020) and was 100% in the high-risk group. Having an abnormal urinalysis was associated with mortality, a need for mechanical ventilation, extra-corporeal membrane oxygenation or renal replacement therapy. In conclusion, our data confirm that COVID-19-associated urine abnormalities on admission predict disease aggravation and the need for ICU (ClinicalTrials.gov number NCT04347824).

Сoagulation hemostasis in experimental light desynchronosis

Введение. Патологическое повышение активности свертывающей системы вызывает нарушение реологических свойствкрови, является фактором риска развития тромбоза крупных сосудов и возникновения ишемической болезни сердца, инфаркта миокарда, тромбофилии и ДВС-синдрома. Цель исследования: изучение влияния светового десинхроноза на функциональную активность коагуляционного механизма гемостаза в эксперименте. Материалы и методы. Экспериментальное исследование было выполнено на 36 нелинейных самцах белых крыс, разделенных на 3 группы: контрольную группу, не подвергавшуюся воздействию светового десинхроноза, и 2 опытных, находившихся в условиях искусственного освещения на протяжении 10 и 21 суток, соответственно. Состояние коагуляционного звена системы гемостаза оценивали по следующим показателям: активированное частичное тромбопластиновое время, протромбиновое время и концентрация фибриногена. Антикоагулянтную активность крови оценивали путем определения активности в плазме крови антитромбина III. Состояние фибринолитической системы оценивали по концентрации в плазме растворимых фибрин-мономерных комплексов и D-димеров. Для динамической оценки активности тромбина применяли тест генерации тромбина. Результаты. Установлено негативное влияние светового десинхроноза на состояние гемостаза. На 10-е сутки эксперимента выявлено компенсаторное повышение активности противосвертывающей и фибринолитической систем. Нахождение подопытных животных в условиях длительной световой депривации на протяжении 21 суток способствовало развитию стресса и сдвигу системы гемостаза в сторону гиперкоагуляции. Заключение. Результаты проведенного экспериментального исследования позволяют сделать вывод о возникновении стресс-реакции у подопытных животных под воздействием светового десинхроноза, а также о гиперкоагуляционных нарушениях равновесия в системе гемостаза, что создает предпосылки для формирования тромбинемии. Background. A pathological increasing of coagulation activity causes disorders of blood rheological properties, that is a risk factor for large vessels thrombosis and coronary heart disease, myocardial infarction, thrombophilia and disseminated intravascular coagulation. Objectives: to study the effect of light desynchronosis on the functional activity of hemostasis coagulation mechanism in experiment. Materials/Methods. The experimental study was carried out on 36 nonlinear male white rats, divided into 3 groups: a control group that was not exposed to light desynchronosis, and 2 experimental groups under artificial lighting for 10 and 21 days, respectively. Coagulation hemostasis was assessed by following parameters: activated partial thromboplastin time, prothrombin time, and fibrinogen content. Anticoagulant blood activity was assessed by antithrombin III activity. Plasma levels of soluble fibrin-monomeric complexes and D-dimers were used for fibrinolytic system assessment. The thrombin generation assay was used for thrombin activity assessment. Results. Light desynchronosis negatively impact hemostasis. On the 10th day of the experiment we revealed a compensatory increasing of anticoagulant and fibrinolytic activities. Exposure of experimental animals to prolonged light deprivation for 21 days caused stress development and hemostasis shift towards hypercoagulation. Conclusions. Light desynchronosis leads to stress reaction in experimental animals as well as to hypercoagulable hemostasis disorders that creates the preconditions for thrombinemia.

Hemostatic system parameters and level of magnesium in patients with beta-thalassemiа

Aim. To study the hemostatic system parameters and magnesium levels in patients with beta-thalassemia. Methods. The object of the study was the blood serum of 96 women with beta-thalassemia: 46 patients with intermediate beta-thalassemia and 50 patients with beta-thalassemia minor, without clinical manifestations of hypercoagulation. The blood serum of 30 healthy donors was used as the control group. It was studied hemostasis system parameters: platelet count activated partial thromboplastin time (aPTT), prothrombin time, plasma fibrinogen level, D-dimer level, euglobulin clot lysis time, antithrombin III activity. The serum magnesium level and risk of deficiency were determined using the MDQ questionnaire. Results. In patients with intermediate beta-thalassemia, an increase in the level of thrombinemia marker D-dimer (500 ng/ml) was revealed. Patients with intermediate beta-thalassemia were divided into two groups according to the revealed level of D-dimer: 14 (30.46.8%) patients with latent hypercoagulation in group 1 and 32 (69.66.8%) patients without latent hypercoagulation in group 2. It was found that in the group with a high levels D-dimer, fibrinogen level was increased (p 0.05), fibrinolysis time was prolonged (p 0.05), activated partial thromboplastin time was shortened (p 0.05), and antithrombin III activity was slightly reduced (p 0.05). The serum magnesium level in patients of the first group was lower (t=7.3; p 0.001), and the risk of deficiency in the questionnaire was higher than in patients of the second group (r=0.785, p 0.05). Hemostasis and magnesium levels in patients with beta-thalassemia minor did not differ from the control group (p 0.05). Conclusion. One-third of patients with intermediate beta-thalassemia have a pre-thrombotic state for hemostasis latent hypercoagulation and magnesium deficiency which can be predictors of clinical signs of thrombosis.

Prevention of massive intraoperative bleedings associated with heparin with the systemic use of fibrin monomer in the experiment

Цель исследования - изучение способности фибрин-мономера предупреждать тяжелую интраоперационную кровопотерю, ассоциированную с введением нефракционированного гепарина, при дозированной травме печени. Методика. На кроликах «Шиншилла» индуцировали гипокоагуляцию нефракционированным гепарином (150 ед/кг). Профилактику интраоперационных кровотечений осуществляли внутривенным введением фибрин-мономера (0,25 мг/кг) за 1 ч до травмы или протамина сульфата (1,5 мг/кг) за 10 мин до травмы. После нанесения стандартной травмы печени оценивали объем (в % ОЦК) и темп (мг/с) кровопотери. Анализировали число тромбоцитов, активированное парциальное тромбопластиновое время, протромбиновое и тромбиновое время свертывания, уровень фибриногена и активность антитромбина III, параметры ротационной тромбоэластометрии крови. Результаты. Объем кровопотери в группах животных после в/в введения фибрин-мономера и протамина сульфата на фоне гепаринизации был, соответственно, в 5,1 и 4,0 раза меньше по сравнению с группой плацебо, получавшей тот же антикоагулянт. Вместе с тем, фибрин-мономер не влиял на параметры коагулограммы (отсутствие видимого гемостазиологического эффекта) и тромбоэластограммы, тогда как применение протамина сульфата в качестве антидота гепарина сопровождалось нормализацией данных тромбоэластометрии и коррекцией гипокоагуляционного сдвига по активированному парциальному тромбопластиновому времени, протромбиновому и тромбиновому времени. Заключение. Установлено, что фибрин-мономер (0,25 мг/кг) снижает посттравматическое кровотечение в условиях блокады свертывания крови гепарином без видимых признаков восстановления гемостатического равновесия. The research objective was to study the ability of fibrin monomer to prevent severe intraoperative blood loss associated with administration of unfractionated heparin in controlled liver injury. Methods. Hypocoagulation was induced in chinchilla rabbits with unfractionated heparin (150 U/kg). Intraoperative bleeding was prevented by administration of fibrin monomer (FM, 0.25 mg/kg, i.v.) one hour prior to the injury and of protamine sulfate (PS, 1.5 mg/kg, i.v.) 10 min prior to the injury. Following the liver injury, blood loss was assessed as percentage of circulating blood volume and the blood loss rate (mg/s). Platelet counts, aPTT, PT, TT, fibrinogen level, antithrombin III activity, and parameters of blood rotation thromboelastometry were analyzed. Results. The volume of blood loss was 5.1 times and 4.0 times less, respectively, after the FM and PS administration during heparinization compared to the placebo group treated with the same anticoagulant. However, FM affected neither coagulogram indexes (no visible hemostasiological effect) nor thromboelastogram while the use of PS as an antidote for heparin was associated with normalization of thromboelastometric data and correction of hypercoagulative changes in aPTT, PT, TT. Conclusion. FM at a dose of 0.25 mg/kg reduced severity of posttraumatic bleeding induced by heparin inhibition of coagulation with no visible signs of hemostatic balance recovery.

Exogenous supplementation of antithrombin III in adult and pediatric patients receiving extracorporeal membrane oxygenation

Background: Antithrombin III deficiency can occur with heparin anticoagulation during extracorporeal membrane oxygenation leading to heparin resistance. Antithrombin III supplementation has been shown to improve anticoagulation; however, there is no consensus on appropriate administration. We described the effect of antithrombin III supplementation on coagulation parameters in adult and pediatric extracorporeal membrane oxygenation patients. Methods: We conducted a retrospective cohort study using electronic medical records of patients who received ⩾1 dose of antithrombin III during extracorporeal membrane oxygenation while on continuous heparin. Endpoints included the change in anti-Xa levels and antithrombin III activity at −6 versus 6 h relative to antithrombin III supplementation, and heparin infusion rates at 6 versus 12 h after antithrombin III supplementation. Results: Eighteen patients receiving 36 antithrombin III administrations were analyzed. Mean (standard deviation) anti-Xa values at −6 versus 6 h were 0.15 (0.07) versus 0.24 (0.15) IU/mL ( p-value: 0.250) for pediatrics and 0.19 (0.22) versus 0.31 (0.27) IU/mL ( p-value: 0.052) for adults. Mean (standard deviation) plasma antithrombin III activity at the same intervals were 32% (14.2%) versus 66.8% (25.1%; p-value: 0.062) for pediatrics and 30.3% (14%) versus 52.8% (8.1%; p-value: 0.094) for adults. Mean (standard deviation) heparin rates at 6 versus 12 h after antithrombin III for pediatrics were 23.6 (6) versus 23.5 (6.5) units/kg/h ( p-value: 0.728), and 15.3 (6.6) versus 13.5 (8) units/kg/h ( p-value: 0.188) for adults. Conclusion: Administration of antithrombin III improved anti-Xa levels in both populations, however, did not significantly reduce heparin rates. Our findings suggest that the use of antithrombin III restores heparin responsiveness in patients with low antithrombin III activity and low anti-Xa activity.

Antithrombin III in the prevention of thrombotic complications in high risk patients undergoing liver transplantation

Introduction. Recent studies have indicated an increased incidence of thrombotic vascular complications after liver transplantation. The reasons may be associated with surgical technique and “unbalanced” hemostasis in patients with diffuse liver diseases. The imbalance is determined by the deficiency of physiological procoagulants and anticoagulants due to a reduced protein-synthesis function of the liver in chronic hepatocyte injury. At the same time, 90% of all spontaneous antithrombin activity is associated with antithrombin III.Aim. The aim of the study was to evaluate the efficacy of using antithrombin III concentrate in liver transplant patients.Material and methods. A retrospective study included 46 patients undergoing liver transplantation who had nonocclusive thrombosis in the portal vein system prior to surgery and postoperative venous or arterial thrombosis.Results. The treatment results were compared between the group with antithrombin III concentrate and the control group in patients with portal vein thrombosis before surgery and postoperative venous or arterial thrombosis; the antithrombin III activity dynamics in the early postoperative period was assessed; the incidence of infectious, and vascular complications and the mortality rates were analyzed.Conclusion. The antithrombin III concentrate administration during liver transplantation and in the postoperative period contributes to a rapid normalization of antithrombin III activity in blood, the decrease in mortality and in the incidence of infectious and thrombotic complication rates.

Hemostasis in rats with different levels of motor activity after vital stress

Введение. Психоэмоциональный стресс, связанный с риском для жизни и здоровья (витальный стресс), вызывает комплексную ответную реакцию всего организма. Система гемостаза, обеспечивающая жидкостные характеристики циркулирующей крови, играет существенную роль в формировании процессов адаптации или дезадаптации. Нарушения равновесия в процессах свертывания и противосвертывания вместе с изменениями микроциркуляции являются основой патогенеза острых и хронических заболеваний с развитием тромботических либо геморрагических осложнений. Цель исследования: оценить состояние системы гемостаза у крыс с разным уровнем двигательной активности после острой психогенной травмы в виде витального стресса. Материалы и методы. Исследования выполнены на 44 лабораторных половозрелых крысахсамцах линии Wistar, которые составили 2 экспериментальные группы с низкой (n 15) и высокой (n 15) двигательной активностью и контрольную группу интактных животных (n 14). Спонтанную двигательную активность оценивали с помощью теста открытое поле . В качестве острого психотравмирующего воздействия использовали модель психической травмы у крыс в виде витального стресса, вызванного переживанием гибели партнера от действий хищника. Результаты. Острое психотравмирующее воздействие у животных с низкой двигательной активностью вызывало угнетение агрегации тромбоцитов. В группе животных с высокой двигательной активностью была выявлена гиперкоагуляция по внешнему пути активации плазменного гемостаза, а также на конечных этапах коагуляции. В обеих экспериментальных группах наблюдали укорочение времени полимеризации фибринмономера, снижение уровня фибриногена, а также активности антитромбина III на фоне активации фибринолиза. Заключение. Изменения состояния системы гемостаза у крыс с разным уровнем двигательной активности после острого психоэмоционального стресса имели одинаковую направленность, но различную степень выраженности ответной реакции. Полученные результаты позволяют охарактеризовать однократное психоэмоциональное воздействие как не выходящее за рамки эустресса (по данным коагулограммы). Introduction. Psychoemotional stress associated with the risk to life and health (vital stress) causes a complex total body response. Hemostasis supports fluid characteristics of circulating blood and plays a significant role in the formation of adaptation or disadaptation processes. Imbalance in the processes of coagulation and anticoagulation with microcirculation changes are the basis of pathogenesis of acute and chronic diseases with the development of thrombotic or hemorrhagic complications. Aim: to assess hemostasis state in rats with different levels of motor activity after acute psychogenic trauma (vital stress). Materials and methods. The studies were performed on 44 laboratory matured Wistar male rats that were divided into 2 experimental groups with low (n 15) and high (n 15) motor activity and a control group of intact animals (n 14). Spontaneous motor activity was assessed using the open field test. A model of mental trauma was used for the formation of acute psychotraumatic effect in rats in the form of vital stress caused by the experience of partner death from a predator. Results. Acute psychotraumatic effect in animals with low motor activity caused inhibition of platelet aggregation. In animals with high motor activity, hypercoagulation was revealed in the external pathway of plasma hemostasis activation, as well as at the final stages of coagulation. Shortening of fibrin monomer polymerization time, decreasing of fibrinogen level and antithrombin III activity with fibrinolysis activation were observed in both experimental groups. Conclusion. After acute psychoemotional stress hemostasis changes in rats with different levels of motor activity had the same direction, but different intensity of response. The obtained results allow to characterize a single psychoemotional effect as not exceeding the limits of eustress (according to the coagulogram data).

Особливості змін показників системи гемостазу та рівня с-реактивного протеїну у хворих на гострий холецистит

Introduction. Acute cholecystitis, which is requently complicated by the bile ducts mechanical obstruction, is characterized by a variety of symptoms – from local inflammation to significant changes in hepatocytes and cholestatic intoxication. These symptoms often manifest violation of the liver functions, which is involved in the synthesis of most proteins, as the system of hemostasis, as well as the so-called «acute phase proteins».The aim of the study – to determine and conduct comparative analysis of hemostatic parameters and level of C-reactive protein (CRP) in patients with acute calculous cholecystitis (ACC).Methods of the research. 67 people were examined: 20 of them – healthy, 25 – patients with ACC without complications (1 group) and 22 – patients with ACC complicated with obstructive jaundice (2 group ). The study of hemostasis was performed by determining platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration, the content of soluble fibrin-monomeric complexes (SFMC), antithrombin III activity (ATIII) and Hagemann-dependent fibrinolysis. Determination of CRP level in blood serum was also performed.Results and Discussion. We found a significant elongation of the PT, APTT, increased concentration of fibrinogen, SFMC content, Hagemann-dependent fibrinolysis and the level of CRP in patients with ACC in comparison with the control group (p<0.05, p<0.001). In addition, a significant decrease in ATIII level and reduction of the number of platelets in patients with ACC in comparison with the practically healthy people (p<0.05) was found.Conclusions. The basis of the identified changes in parameters of hemostasis system most likely lies in hepatocellular insufficiency as the cause of the reduced synthesis of many components of haemostasis on the background of inflammatory liver damage. Disorders of primary and secondary hemostasis with insufficient anticoagulants activity can lead to multiple lesions in hemodynamics and microcirculation. The increase in the content of SFMC in plasma suggests the presence of DIC syndrome. A prolongation of PT and APTT will characterize DIC syndrome as the use factors of a phase hypocoagulation.A significant increase in the level of CRP in patients with ACC complicated with obstructive jaundice indicates its involvement in the progress of the inflammatory process.