The role of phosphatidylserine on the membrane in immunity and blood coagulation

The negatively charged aminophospholipid, phosphatidylserine (PtdSer), is located in the inner leaflet of the plasma membrane in normal cells, and may be exposed to the outer leaflet under some immune and blood coagulation processes. Meanwhile, Ptdser exposed to apoptotic cells can be recognized and eliminated by various immune cells, whereas on the surface of activated platelets Ptdser interacts with coagulation factors prompting enhanced production of thrombin which significantly facilitates blood coagulation. In the case where PtdSer fails in exposure or mistakenly occurs, there are occurrences of certain immunological and haematological diseases, such as the Scott syndrome and Systemic lupus erythematosus. Besides, viruses (e.g., Human Immunodeficiency Virus (HIV), Ebola virus (EBOV)) can invade host cells through binding the exposed PtdSer. Most recently, the Corona Virus Disease 2019 (COVID-19) has been similarly linked to PtdSer or its receptors. Therefore, it is essential to comprehensively understand PtdSer and its functional characteristics. Therefore, this review summarizes Ptdser, its eversion mechanism; interaction mechanism, particularly with its immune receptors and coagulation factors; recognition sites; and its function in immune and blood processes. This review illustrates the potential aspects for the underlying pathogenic mechanism of PtdSer-related diseases, and the discovery of new therapeutic strategies as well.

COVID-19, neurovascular thrombotic problem and short summary on blood coagulation disorder: a brief review

COVID-19 is the present global public health problem. This respiratory viral infection can manifest atypical presentation including neurological presentations. An important neurological problem in COVID-19 is neurovascular thrombosis. The basic pathogenesis of thrombosis in neurological system is explainable by the basic principle of thrombohemostasis. A hypercoagulability is a possible problem seen in some COVID-19 cases. In this brief review, the authors summarize venous and arterial thrombosis of neurovascular system as a complication of COVID-19. The updated pathophysiology of COVID-associated blood coagulation disorder is discussed. In addition, consideration regarding new COVID-19 vaccine related thrombotic adverse event is also raised.

Coagulation status in patients with pulmonary embolism receiving long-term anticoagulant therapy

Aim. To study the blood coagulation status by various laboratory methods in patients after pulmonary embolism (PE) receiving long-term anticoagulant therapy.Material and methods. The blood of 23 patients with pulmonary embolism, who received long-term anticoagulant therapy, was studied. The study of coagulation profile, D-dimer, thrombodynamics, thromboelastography and thrombin generation test were carried out.Results. The thrombin generation test shows a significant increase in the time of its formation, while the maximum amount of thrombin formed is half that of the reference values. There is a slightly increased median fibrin clot growth rate in the thrombodynamics test — 30,4 gm/min with a normal coagulation rate of 20-29 gm/min. The result of thromboelastography also reflects the blood hypocoagulation, in terms of R, Angle a and CI.Conclusion. Integral methods for assessing the thrombotic readiness in combination with a routine coagulation panel demonstrate a complete picture of blood coagulation potential in patients after pulmonary embolism requiring long-term anticoagulant therapy.

The effects of fructose diphosphate on routine coagulation tests in vitro

To evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0–35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, − 0.990, − 0.983, − 0.989 and − 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied.

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.

rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick Hyalomma dromedarii

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 μM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

Clot Waveform Analysis Demonstrates Low Blood Coagulation Ability in Patients with Idiopathic Thrombocytopenic Purpura

Background: Although platelets, which contain large amounts of phospholipids, play an important role in blood coagulation, there is still no routine assay to examine the effects of platelets in blood coagulation. Methods: Hemostatic abnormalities in patients with thrombocytopenia, including those with idiopathic thrombocytopenic purpura (ITP), were examined using clot wave analysis (CWA)–small-amount tissue-factor-induced FIX activation (sTF/FIXa) and thrombin time (TT). Results: Although there were no marked differences in the three parameters of activated partial thromboplastin time (APTT) between normal healthy volunteers and typical patients with ITP, the peak heights of the CWA-sTF/FIXa were markedly low in patients with ITP. The three peak times of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly longer than those in patients with a platelet count > 8.0 × 1010/L and the peak heights of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly lower than those in patients with >8.0 × 1010/L. The peak heights of the CWA-APTT in patients with ITP were significantly lower than in patients with other types of thrombocytopenia. The three peak heights of the CWA-sTF/FIXa in ITP patients were significantly lower than those in patients with other types of thrombocytopenia. The CWA-TT showed lower peak heights and longer peak times in patients with ITP in comparison to patients with other types of thrombocytopenia. Conclusions: The CWA-sTF/FIXa and CWA-TT results showed that blood coagulation is enhanced by platelets and that the blood coagulation ability in ITP patients was low in comparison to healthy volunteers and patients with other types of thrombocytopenia.