COMPARATIVE ANALYSIS OF THE EFFECT OF HORMONE-INDUCED STRESS ON HEMOSTASIS IN THE CYPRINUS CARPIO AND THE OREOCHROMIS NILOTICUS

Background: The mortality of freshwater fish due to stress during various production manipulations is a severe problem, which requires a thorough understanding of the basic mechanisms involved, including the hemostasis system. Therefore, on the application level, the study of blood clotting can perform fish coagulopathies diagnostics and develop practical preventive and therapeutic anticoagulation methods for fish farming. Aim: The goal of this research was a comparative assessment of the reaction of some hemostasis parameters of two commercial fish species, carp Cyprinus carpio and tilapia Oreochromis niloticus, to the stress of different duration induced by corticosteroids. Methods: The fishes were divided into three groups: chronically stressed (induction by betamethasone), acutely stressed (induction by dexamethasone), and control animals with blood taken from the caudal hemal canal before hormone treatment(by dexamethasone and betamethasone), then 7 and 21 days after. Results and Discussion: Changes in the following parameters were studied: thrombin time, prothrombin time, activated partial thromboplastin time, the concentration of fibrinogen, soluble fibrin monomer complexes, antithrombin. It was found that both hormone-induced stress and handling stress associated with blood sampling strongly increased blood clotting ability in carps (prothrombin time decrease by 78,5-86,1%, fibrinogen increase by 12,7-100%, thrombin time decrease by 83,4-85%, and antithrombin III decreases by 15,3-21,7%), while in tilapias, acceleration of blood clotting by intrinsic and extrinsic pathways were recorded by the end of the experiment only in fishes with imitation of chronic stress (prothrombin time decrease by 76,8%, activated partial thromboplastin time decrease by 20,0%, and 2,3 multiplying soluble fibrin monomer complexes). Conclusions: It was concluded that the adaptive mechanisms of the tilapia (Oreochromis niloticus) organism allowed the clotting function to recover in most cases by the end of the experiment in all groups of fish, in contrast to carps (Cyprinus carpio).

THROMBOELASTOGRAPHY IN ASSESSING THE HEMOSTATIC SYSTEM IN INTENSIVE CARE PATIENTS

The results of thromboelastography and standard coagulogram were analyzed in 35 patients aged from 18 to 86 who were treated in the resuscitation and intensive care unit. The majority of patients (34%) were hospitalized in the department with multisystem and concomitant injuries. The remaining patients were taken to the medical institution with different diagnoses (urolithiasis, liver cirrhosis, pancreatic lesion of various types, poisoning, peptic ulcer, sepsis). The data of coagulogram and thromboelastography at different stages of treatment were compared. In patients with the development of traumatic shock, the coagulogram parameters were changed to varying degrees depending on the stage of shock. At the first stage of shock, only an increase in soluble fibrin-monomer complexes by almost 2 times and a slight increase in fibrinogen dynamics were noted in the analysis. In a patient with stage 3 traumatic shock, the coagulogram parameters were within the normal range, but according to thromboelastography (EXTEM and FIBTEM tests), hypocoagulation due to the platelet link was noted. Only the coagulogram was evaluated in dynamics, hypocoagulation was noted in the indicators of internal and external hemostasis pathways: lengthening of the activated partial thromboplastin time, a decrease in the prothrombin index and an increase in the international normalized ratio, an increase in fibrinogen A and soluble fibrin-monomer complexes. In the group of male patients with closed craniocerebral trauma, an increase in soluble fibrin-monomer complexes in the coagulogram was always combined with changes in the FIBTEM test during thromboelastography. In most patients, no changes in the classical coagulogram tests immediately after the injury are noted. At this, thromboelastography makes it possible to make up for this deficiency at an earlier time, which indicates a high sensitivity of the method.

Some indicators of hemostasis and mortality from thrombotic complications in patients with CKD stage VD who were treated by programmed hemodialysis (five-year follow-up)

Annotation. Thrombotic complications in patients with stage VD chronic kidney disease (CKD), treated by program hemodialysis is one of the causes of high mortality in this category of patients; its major pathogenetic component proved to be hemostatic system disturbances characterized by systemic activation of blood clotting process leading to the development of thrombophilia. Objective – to study the biochemical molecular markers of hemostasis in patients with stage VD CKD, treated by program hemodialysis, and to determine their long-term effect on the development of thrombotic complications and mortality. The study included 88 patients (52 males and 36 females) aged 26 to 65 years with stage VD CKD, treated by program hemodialysis. The patients were followed up for five years. Soluble fibrin (sF) level was determined by two-site enzyme-linked immune-sorbent quantitative assay; D-dimer – by enzyme immunoassay using specific monoclonal antibodies to D-dimer epitopes; protein C (pC) activity in blood plasma was estimated by its activation with copperhead snake venom followed by spectrophotometry with wavelength 405 nm. Blood plasma fibrinogen was determined using thrombin-like enzyme Antsistron-H by spectrophotometry with wavelength 280 nm. Blood plasma fibrinolytic activity was evaluated by the relationship between D-dimer and soluble fibrin. Processing of materials was carried out using the methods of variation statistics and correlation analysis. During five years of follow up there were 13 deaths (14.8%), 7 among males and 6 among females, caused by thrombotic complications. The main thrombotic complications were myocardial infarction (6), ischemic stroke (4), mesenteric thrombosis (2) and disseminated intravascular coagulation syndrome (DIC) (1) (according to pathomorphological data). The patients of general group were found to have significantly increased sF level, decreased pC as compared to the control group, as well as twofold increase of fibrinogen concentration along with decreased D-dimer/sF ratio and no response of D-dimer to increased soluble fibrin level. The tendency of D-dimer/sF ratio to increase in those who died because of thrombotic complications could be indicative of microthrombosis with formation of fibrin derivatives along with mild activation of fibrinolysis. Correlation relationships between soluble fibrin and D-dimer, fibrinogen and protein C in general group were assessed, and the following data were obtained: medium direct relationship between soluble fibrin and D-dimer (r= 0.56) (p<0.001), and absence of correlation with fibrinogen (r= -0.12) and protein C (r= -0.10) (p˃0.1). Besides, strong positive correlation was demonstrated between D-dimer and soluble fibrin (r= 0.87) (p<0.001), and moderately negative one between D-dimer and protein C (r= -0.21) (p<0.05). It should be noted that in patients with thrombotic complications, positive correlations between soluble fibrin and D-dimer become stronger (r= 0.51) (p<0.001), as well as negative ones between soluble fibrin and protein C (r= -0.22) (p<0.05). Depressive state of anticoagulant component along with activation of coagulation factors are supposed to be one of the indicators predicting thrombophilia in this category of patients.

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.

Some features of the hemostasis system in pregnant women at risk of developing preeclampsia

The objective: a study of the hemostasis system in pregnant women with a risk of preeclampsia development.Materials and methods. 100 pregnant women with the risk for preeclampsia (main group) were examined. The risk factors were determined according to the Guideline “Hypertensive Disorders During Pregnancy”, Order No. 676 of the Ministry of Health of Ukraine. The control group contained of 50 healthy women with physiological pregnancy. The groups were representative in age and reproductive history.The following indicators of hemostasis were studied: the platelet system (the number of platelets, their aggregation ability and the total platelet aggregation index (TPAI), the coagulation system (autocoagulation test, thrombin time, prothrombin index, fibrinogen concentration) and the state of the fibrinolysis system which was determined by such indicators: plasma level of free heparin, activity of antithrombin III, indicators of ethanol and protamine sulfate tests, concentration of soluble fibrin in blood plasma.Results. In pregnant women with a risk of preeclampsia, there are changes in platelet hemostasis indicators: a significant decrease in the number of platelets and a significant (p<0.05) increase in platelet aggregation ability, there is a tendency to an increase in TPAI indicators. In the main group a significant increase in the fibrinogen concentration, plasma lysis indicators and a tendency to an increase of the free heparin concentration, a decrease of antithrombin III and, in comparison with the indicators in healthy women, a 3-fold increase in the content of soluble fibrin (p<0.05) were found.Conclusions. In pregnant women with a risk of preeclampsia development, there are disorders in the vascular-platelet hemostasis, coagulation and fibrinolytic blood systems, namely, a significant tension in the platelet link of the system, an increase in thrombogenic potential, and a sharp inhibition of the fibrinolytic link of hemostasis.

A Data Analysis of D-Dimer & Interleukin-6 (IL-6) Test for Covid Patient

Various biomarkers, especially inflammatory markers like C-reactive protein (CRP), ferritin, fibrinogen, D-dimer and Interleukin 6 (IL-6) are associated with Covid-19 progression. Thrombosis prophylaxis with low molecular weight heparin has shown beneficial results in preventing coagulopathy a reducing risk of mortality due to thrombotic events. The COVID-19 patients highlighting the role of D-dimer, and Interleukin-6 (IL-6). During plasma coagulation soluble fibrin is generated by the influence of thrombin on fibrinogen. The soluble fibrin is crosslinked to the vessel walls by factor XIIIa. When splitting this cross linked fibrin, characteristic products called D-dimers are released. Increased D-dimer concentrations are found in thrombotic diseases and microthrombotic events (e.g. in case of disseminated intravascular coagulation, DIC). D-dimer determination is mainly used to exclude deep vein thrombosis of the leg and pulmonary embolism. D-Dimer levels rise during pregnancy and high levels are associated with complications. D-dimer is a fibrin degradation product that is often used to measure and assess clot formation. Amid the COVID-19 pandemic, elevated D-dimer levels have been associated with disease severity and mortality trends. Interleukin-6 (IL-6) is a pro-inflammatory cytokine secreted by T cells and macrophages to stimulate immune response to trauma, especially burns or other tissue damage leading to inflammation. IL-6 is also secreted by macrophages in response to specific microbial molecules, referred to as Pathogen Associated Molecular Patterns (PAMPs), which trigger the innate immune response and initiate inflammatory cytokine production. IL-6 is one of the most important mediators of fever and of the acute phase response. IL-6 is also called a “myokine”, a cytokine produced from muscle that increases in response to muscle contraction. Additionally, osteoblasts secrete IL-6 to stimulate osteoclast formation. The detection and control of pro-inflammatory response is crucial in the early stages of viral infection. COVID-19 is an emerging viral disease of global concern and optimal treatment has yet to be determined. Unknown response of treatment of COVID-19 is important during patient monitoring. IL-6 is one of the key cytokines after activated macrophages. Here we will present a laboratory data analysis of COVID-19 patients in different age group highlighting the role of positivity D-dimer and interleukin-6 (IL-6).

Dynamics of hemostasiological parameters in cows during pregnancy, after birth and in obstetric, gynecological and orthopedic pathology

It is established that the physiological course of the gestational process occurs with the intensification of coagulation processes in the hemostasis system with thrombophilia, which has a two–phase nature – from the first month of pregnancy to the seventh and in the period of 2–3 days to calving with a peak of hypercoagulation by 2–5 that day after birth. The activation of hemostasis components is correlated with a high level of natural anticoagulant protein C and increased fibrinolysis activity through tissue plasminogen activator, which is controlled by a high level of its type I inhibitor. The main hemostasiological criteria are the levels: in phase I – fibrinogen 4,9–6,1 g/l, soluble fibrin about 0,04 g/l, activated partial thromboplastin time 38,1–39,7 s, functionally inactive forms prothrombin 0,88 ± 0,2 mg/ml in the first month of pregnancy; in phase II – fibrinogen 5,1–7,0 g/l, soluble fibrin about 0,01 g/l, functionally inactive forms of prothrombin 0,17 ± 0,05 mg/ml and protein C 72,0 ± 1,4 % after childbirth. In obstetric and gynecological pathology, the hypercoagulable state is aggravated to consumption coagulopathy, which is reflected in high plasma levels of soluble fibrin – 0,064 ± 0,005 g/l, fibrin / fibrinogen cleavage products – 7,5 ± 0,4 μg/ml, functionally inactive forms of prothrombi. – 1,90 ± 0,34 mg/ml and inhibitor of tissue plasminogen activator type I – 45,8 ± 0,9 IU/ml at the lowest level of tissue plasminogen activator – 0,38 ± 0,08 IU/ml (according to the norms 1,1–1,3 IU/ml) and protein C deficiency – <70%. Hypercoagulation in pregnant cows with purulent– necrotic lesions of the extremities is exacerbated by more pronounced hyperfibrinogenemia, an increase in its metabolites by 2,7 times (p<0,05) and a decrease in the activity of F XIII by 1,3 times (p<0,05) and the amount in the blood of proteinase inhibitor α2–M – 1,6 times (p<0,05). At the same time, the hypercoagulation syndrome in cows with obstetric and gynecological pathology due to its comorbidity with orthopedic not only intensifies, which is expressed in an increase in the level of hyperfibrinogenemia by 1,2 times (p<0,05), reduction of general coagulation tests by 1,3–2,1 times (p<0,001) and a decrease in the activity of FXIII in 1,2–1,3 (p<0,05), but is complicated by a decrease in the antiproteinase potential of the blood in 1,6–1,8 times. Key words: cows, pregnancy, obstetric, gynecological pathology, purulent-necrotic lesions in the area of fingers, hemostasiological indicators.