Comparative effects on anticoagulant activity and degree of fibrin polymerization of extracts from the new peony «Ivan Gorozhankin» and "Paeonia lactiflora"

Введение. Актуальность темы исследования обусловлена проблемой борьбы с тромбозами и тромбоэмболиями безопасными для организма методами. Во многих растениях обнаружены антикоагулянты разной природы (гепариноподобные, пептиды). Цель исследования - изучение возможности проявления синергических эффектов на антикоагулянтную и фибринолитическую активность крови и процессы полимеризации фибрина экстракта из корней пиона «Иван Горожанкин» в сравнительном аспекте с действием экстракта из корней пиона «молочноцветковый». Методика. Объектом исследования служили корни пионов «Иван Горожанкин» и «молочноцветковый», произрастающих в Ботаническом саду МГУ. Пион «Иван Горожанкин» был создан скрещиванием пиона «молочноцветкового» и «лекарственного» Разработаны методы получения экстрактов из корней различных пионов. При различных разведениях экстрактов (0.1, 1, 5%) определены антикоагулянтная активность по тестам, характеризующим внутренний, внешний и общий пути свертывания крови, а также степень полимеризации фибрина плазмы крови крыс. Для сравнения был использован стандартный препарат низкомолекулярного гепарина (LMWH) животного происхождения фирмы «Celsus» (США). Проведены выделение и очистка активного начала (гепариноидов) из сухих препаратов и измерены их активности. Pезультаты. Показано, что экстракты из обоих препаратов пионов обладали антикоагулянтной и суммарной фибринолитической активностью на нестабилизированном фибрине, но в разной степени. В экстрактах из корней пиона «Иван Горожанкин» отмечались преимущественные синергические эффекты, а именно превышение антикоагулянтной активности на 20-30%, суммарной фибринолитической - на 18% по сравнению с таковыми, отмечаемыми в экстрактах из корней пиона «молочноцветковый». Подобные результаты выявлены и при изучении степени полимеризации фибрина под влиянием очищенных препаратов из пионов. Рассмотрены возможные механизмы активирующего действия экстракта из пиона «Иван Горожанкин» на антикоагулянтные свойства плазмы, суммарную фибринолитическую активность и степень полимеризации фибрина. Это связано с блокадой активности тромбина и факторов внутреннего механизма свертывания крови. При этом антикоагулянтный эффект от применения экстракта из пиона «Иван Горожанкин» по тесту APTT (activated partial thromboplastin time) превышал на 20-30% ту же активность, выявленную у пиона «молочноцветковый», которая соответствовала антикоагулянтной активности препарата сравнения LMWH. В экстракте из пиона «Иван Горожанкин» впервые обнаружено наличие антикоагулянтного гепариноподобного вещества. Заключение. Впервые установлена способность экстракта из корней пиона «Иван Горожанкин» проявлять синергические антикоагулянтные и фибриндеполимеризационные эффекты, превышающие таковые у экстракта из пиона «молочноветковый». На основе полученных данных возникает необходимость исследования пиона «Иван Горожанкин» в качестве антитромботического, а возможно, и антиатеросклеротического агента. Introduction. The research topic is relevant due to the problem of safely combating thrombosis and thromboembolism. Anticoagulants of various kinds, e.g., heparin-like and peptides, have been found in many plants. Aim. To investigate the possibility of synergistic effects on the blood anticoagulant and fibrinolytic activity and on processes of fibrin polymerization by an extract from the roots of the «Ivan Gorozhankin» peony compared with the root extract from «Paeonia lactiflora». Methods. The focus of the study was the roots of the “Ivan Gorozhankin” peony and the Paeonia lactiflora growing in the Botanical Garden of the Moscow State University. The “Ivan Gorozhankin” peony was created by crossing P. lactiflora and the “medicinal” peony. Methods for obtaining extracts from the roots of various peonies have been developed. In 1%, 3%, and 5% dilutions of the extracts, the anticoagulant activity was determined according to tests characterizing the internal, external and general blood coagulation pathways, as well as by the degree of polymerization of rat blood plasma fibrin. For comparison, we used a standard preparation of low molecular weight heparin (LMWH) of animal origin (Celsus, USA). Isolation and purification of the active substances, heparinoids, were isolated from dry preparations and purified, and their activities were measured. Results. Extracts from both peony preparations had anticoagulant and total fibrinolytic activity on unstabilized fibrin, but to different extents. In the extracts from the roots of the “Ivan Gorozhankin” peony, preferential synergistic effects were noted, namely, the anticoagulant activity was higher by 20-30%, and the total fibrinolytic activity was higher by 18% compared to those of extracts from Paeonia lactiflora roots. Similar results were obtained when studying the degree of fibrin polymerization as influenced by purified peony preparations. Possible mechanisms of the activating action of the «Ivan Gorozhankin» peony extract on the anticoagulant properties of plasma, the total fibrinolytic activity, and the degree of fibrin polymerization are considered. This action is due to the inhibition of thrombin activity and factors of the internal mechanism of blood coagulation. According to the activated partial thromboplastin time (APTT) test, the anticoagulant effect of extracts from the «Ivan Gorozhankin» peony exceeded by 20-30% the activity of Paeonia lactiflora extract, which corresponded to the anticoagulant activity of the LMWH comparator drug. Using the described biochemical methods, the presence of an anticoagulant heparin-like substance in an extract from the peony «Ivan Gorozhankin» has been discovered. Conclusion. For the first time, the ability of an extract from the roots of the «Ivan Gorozhankin» peony to exhibit synergistic anticoagulant and fibrin-depolymerization effects was demonstrated. These effects exceeded those of the Paeonia lactiflora extract. Based on these data, it appears necessary to study the «Ivan Gorozhankin» peony as an antithrombotic, and possibly as an anti-atherosclerotic agent.

Coagulation profile in two nephropathic dogs

Coagulatory abnormalities are common in renal dysfunction in humans. The studies on coagulatory abnormalities in renal failure in dogs are limited. The present paper deals with coagulation profile in acute and chronic kidney disease in dogs. The haemostatic defects observed in acute renal dysfunction included thrombocytopaenia, prolonged capillary bleeding time (CBT), elevated D-Dimer and hypoantithrombinemia which indicated a hypercoagulable state. Prolongation of prothrombin time (PT), activated partial thromboplastin time (aPTT), elevated D-Dimer concentration and hypoantithrombinemia in chronic kidney disease indicated the presence of hypocoagulable state

Effect of Time and Haemolysis on Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) Measurement on Blood Samples

Background: Coagulation, also known as blood clotting, is the process by which blood convert from a liquid to a gel, forming a blood clot. It referred to haemostasis, the stopping of blood loss from a damaged vessel, followed by repair. Material and methods: This was cross sectional study conducted at the albawasla medical laboratory, Khartoum, Sudan during the period August to November, 2021 and to evaluate the effect of time and hemolysis on prothrombin time and activated partial thromboplastin time tests. 50 samples (case group) were collected from the patients attending police teaching hospital and requested to the PT and APTT test in addition to that,50 apparently healthy donors with no history of any coagulation problems or any chronic disease were selected as control group. Three ml of venous blood samples were collected in container with Tri Sodium Citrate anticoagulant. The coagulation tests (PT and APTT) were performed using semiautomatic device (coagulometer machine MI). Results: The result of this study revealed that; when compared the measurement of PT and APTT immediately and after one hour there was insignificant differences (p. v.>0.05). also when compared the measurement of PT and APTT between hemolyzed and non-hemolyzed samples there was significant differences ( p. v.<0.05) in addition when compared case and control for the PT and APTT immediately, after one hour, hemolyzed and non-hemolyzed sample there was significant differences ( p. v.<0.05) except the APTT hemolyzed samples and insignificant differences with age and gender ( p. v.>0.05). For the correlation there was significant correlation in the case group for the PT and APTT immediately, after one hour, and hemolyzed samples. Conclusion: In the cases group results showed insignificant differences in the results of PT and APTT between immediate sample and after 1 hour in and significant differences in the results of PT and APTT between hemolyzed and non-hemolyzed samples, also there was insignificant differences between age and gender, immediately, after one hr. and hemolyzed sample in PT and APTT. Keywords: Homeostasis, hemolyzed sample, PT and APTT

A szerzett haemophilia A sikeres kezelése

Összefoglaló. A szerzett haemophilia A ritka autoimmun betegség, melyben gátlótest képződik a VIII. véralvadási faktor ellen. Az inhibitor véralvadásra gyakorolt hatása súlyos, életet veszélyeztető vérzéses állapotot idéz elő. A beteg élete a gyors diagnózison múlik: a jellemző klinikai kép mellett a megnyúlt, normálplazmával nem korrigálható aktivált parciális tromboplasztinidő megléte esetén a kórkép alapos gyanúja merül fel. Egy súlyos vérszegénység miatt kórházunkba beutalt nőbeteg esetében a szerzett haemophilia A a felvételt követő napon már diagnosztizálásra került. A vérzés megszüntetésére aktivált protrombinkomplex-koncentrátumot alkalmaztunk, valamint immunszuppresszív terápiát vezettünk be. A kórkép korai felismerése és a megfelelő kezelés azonnali megkezdése a beteg gyógyulását eredményezte. Esetünkkel arra szeretnénk felhívni a figyelmet, hogy a szerzett haemophilia A gyors diagnózisa egyszerű, könnyen hozzáférhető véralvadási paraméter, az aktivált parciális tromboplasztinidő meghatározásán és nem korrigálható megnyúlásának felismerésén múlik. Orv Hetil. 2021; 162(49): 1977–1981. Summary. Acquired haemophilia A is a rare autoimmune disorder, in which antibodies are formed against coagulation factor VIII. The effect of the inhibitor on blood clotting results in severe, life-threatening bleeding diathesis. The patient’s life depends on the rapid diagnosis: besides the characteristic clinical presentation, a prolonged activated partial thromboplastin time, which is not corrigible with normal plasma, suggests the existence of the disorder. In the case of the female patient who was referred to our hospital due to severe anaemia, acquired haemophilia A was diagnosed rapidly, the day after her admission. We used activated prothrombin complex concentrate to stop the bleeding, and introduced immunosuppressive therapy. The early recognition of the disease and immediate initiation of adequate treatment resulted in the patient’s full recovery. With our case presentation, we would like to draw attention to the fact that the rapid diagnosis of acquired haemophilia A depends on the determination of a simple, easily accessible coagulation parameter, the activated partial thromboplastin time and on the immediate recognition of its incorrigible prolongation. Orv Hetil. 2021; 162(49): 1977–1981.

Antiphospholipid Antibodies and Acquired Thrombophilia: A Biological Marker for Recurrent Miscarriage

Background: Repeated miscarriage can cause tissue injury can lead to the formation of antibodies to the phospholipids. Recurrent miscarriage (RM) is considering the one of the most common cause of sterility. Which has received more attention in recent years as a result of an increase in the number of reproductive-aged women. Materials and Methods: Plasma samples were tested for antiphospholipid antibodies using ELISA, and platelet count using Sysmex (KX21) Heamatology analyzer and Activated Partial Thromboplastin Time using semi-automated machine (STAGO PT31039352 (for coagulation). Results: The prevalence of Anti phospholipid antibodies (APL) was 30.5% in Sudanese patients with recurrent miscarriage, the prevalence of (Anti phospholipid Antibodies-IgM and IgG) was found to be 23.6% in patients with recurrent miscarriage compared to (Anti phospholipid Antibodies-IgG) was found to be 11.1% ((P value≤0.001), low platelets count (<50×109/l) observed in 10 (13.5%), as well as prolongation of activated partial thromboplastin time (APTT) among studied group were detected among 19 (26.1%). Conclusion: Higher prevalence of antiphospolidids antibodies, and acquired thrombophilia was detected among Sudanese women with recurrent abortion; The findings are concerning because they link an increased risk of thrombosis and a hypercoagulable state lead to recurrent miscarriage in pregnant women.

High Perceived Stress May Shorten Activated Partial Thromboplastin Time and Lead to Worse Clinical Outcomes in Patients With Coronary Heart Disease

Objective: To determine the association of perceived stress with coagulation function and their predictive values for clinical outcomes.Methods: This prospective cohort study derived from a cross-sectional study for investigating the psychological status of inpatients with suspicious coronary heart disease (CHD). In this study, the 10-item Perceived Stress Scale (PSS-10) as an optional questionnaire was used to assess the severity of perceived stress. Coagulation function tests, such as activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen were measured within 1 h after admission. Furthermore, 241 patients with CHD out of 705 consecutive inpatients were included in the analyses and followed with a median of 26 months for the clinical outcomes.Results: The patients in high perceived stress status (PSS-10 score &gt; 16) were with shorter APTT (36.71 vs. 38.45 s, p = 0.009). Shortened APTT ( ≤ 35.0 s) correlated with higher PSS-10 score (14.67 vs. 11.22, p = 0.003). The association of APTT with depression or anxiety was not found. Multiple linear models adjusting for PT estimated that every single point increase in PSS-10 was relevant to approximately 0.13 s decrease in APTT (p = 0.001) regardless of the type of CHD. APTT (every 5 s increase: hazard ratio (HR) 0.68 [0.47–0.99], p = 0.041) and perceived stress (every 5 points increase: HR 1.31 [1.09–1.58], p = 0.005) could predict the cardiovascular outcomes. However, both predictive values would decrease when they were simultaneously adjusted. After adjusting for the physical clinical features, the associated of perceived stress on cardiac (HR 1.25 [1.04–1.51], p = 0.020) and composite clinical outcomes (HR 1.24 [1.05–1.47], p = 0.011) persisted.Conclusions: For the patients with CHD, perceived stress strongly correlates with APTT. The activation of the intrinsic coagulation pathway is one of the mechanisms that high perceived stress causes cardiovascular events. This hints at an important role of the interaction of mental stress and coagulation function on cardiovascular prognosis. More attention needs to be paid to the patients with CHD with high perceived stress.

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).

Use of Bivalirudin-Specific Monitoring Assays in Ventricular Assist Device Patients

Increasing numbers of pediatric ventricular assist device (VAD) patients are being anticoagulated with the parenteral direct thrombin inhibitor bivalirudin because it is reportedly associated with fewer bleeding and thrombotic events. With expanded use, management is shifting from a handful of experts to a wider pool of clinicians and trainees, increasing the importance of identifying broadly acceptable, standardized monitoring assays. The pharmacokinetics of bivalirudin have not been well-studied in the pediatric population and drug monitoring in all ages has been problematic for critically ill patients who require intermediate or longer-term therapeutic anticoagulation. The dilute thrombin time (dTT), available in many clinical laboratories, has been suggested as a potentially superior alternative to the activated partial thromboplastin time (aPTT), but results have been inconsistent. As clinical use of the dTT (c-dTT) for monitoring bivalirudin increased at our institution, we sought to evaluate the performance of commercially available, "research only" functional bivalirudin assays with calibrators and controls to measure bivalirudin's anticoagulation effect, utilizing residual plasmas and clinical data from VAD patients treated with bivalirudin. Residual citrated, platelet poor plasma samples from clinically ordered laboratory tests in VAD patients were collected and stored frozen at -70 oC from February 8, 2018, to January 4, 2021. With IRB approval, the samples were analyzed in conjunction with medical record review. Two experimental assay kits were utilized, ex-dTT: a dilute thrombin time assay (Hemoclot, Hyphen-Biomed, FR) and ex-anti FIIa: a chromogenic anti-factor IIa assay (BiophenDTI, Hyphen-Biomed, FR). Bivalirudin calibrators (Biophen, Hyphen-Biomed, FR) were used to develop a standard curve for the assays. Controls of low and high (1.5 and 4 microgram/mL) bivalirudin (Biophen, Hyphen-Biomed, FR) were used for quality control of the assays. Results from the two experimental assays were compared with available standard laboratory monitoring when results were available. In total, 115 residual plasma samples from 11 different patients (up to 16 samples per patient) were analyzed. Subjects included 1 adult (37 yr.) and 10 pediatric patients (0-18 yr.). There was excellent correlation between the two experimental assays (Fig. 1A). Correlation was good between the c-dTT and each of the experimental assays; however, with a clinical laboratory platform change (instrument, reagents) midway through sample collection, the c-dTT results shifted substantially in their corresponding estimate of bivalirudin concentration (Figs 1B and C). Activated partial thromboplastin time (aPTT) had poor correlation with the c-dTT and with each of the experimental assays (Fig. 1D). Here we report results from two commercially available kits that estimate bivalirudin concentration using dTT or chromogenic anti-factor IIa assays, in comparison with clinically generated results from VAD patients treated with bivalirudin. Our findings agree with previous observations that the aPTT shows poor correlation with dTT assays (clinically used and experimental) over days and weeks of anticoagulation. The two experimental assays had excellent correlation with each other and good correlation with the c-dTT; however, the fact that the c-dTT results shifted dramatically with a clinical laboratory platform change is illustrative of the need for a bivalirudin-specific monitoring assay as an essential tool for improving outcomes at our center and across centers. More research is needed to understand which type of monitoring assay (dTT or anti-FIIa) may be better suited to particular clinical circumstances. Figure 1 Figure 1. Disclosures Lorts: Abbott: Consultancy; Medtronic: Consultancy; Berlin Heart: Consultancy; Syncardia: Consultancy; Abiomed: Consultancy.