Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation

Background and objective: For many pathological states, microparticles are supposed to be one of the causes of hyper-coagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test.Results: Monocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0-200-10(3)/ulrange of microparticles concentrations. However, at concentrations greater than 500.10(3)/mu l, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine.Conclusion: Microparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.</div

Blood coagulation dynamics: mathematical modeling and stability results

The hemostatic system is a highly complex multicomponent biosystem that under normal physiologic conditions maintains the fluidity of blood. Coagulation is initiated in response to endothelial surface vascular injury or certain biochemical stimuli, by the exposure of plasma to Tissue Factor (TF), that activates platelets and the coagulation cascade, inducing clot formation, growth and lysis. In recent years considerable advances have contributed to understand this highly complex process and some mathematical and numerical models have been developed. However, mathematical models that are both rigorous and comprehensive in terms of meaningful experimental data, are not available yet. In this paper a mathematical model of coagulation and fibrinolysis in flowing blood that integrates biochemical, physiologic and rheological factors, is revisited. Three-dimensional numerical simulations are performed in an idealized stenosed blood vessel where clot formation and growth are initialized through appropriate boundary conditions on a prescribed region of the vessel wall. Stability results are obtained for a simplified version of the clot model in quiescent plasma, involving some of the most relevant enzymatic reactions that follow Michaelis-Menten kinetics, and having a continuum of equilibria.CEMAT/IST through FCT [PTDC/MAT/68166/2006]; Czech Science Foundation [201/09/0917]; Grant Agency of the Academy of Sciences of the CR [IAA100190804]; Ministry of Education of Czech Republic [6840770010]info:eu-repo/semantics/publishedVersio

ДИАГНОСТИКА НАРУШЕНИЙ В СИСТЕМЕ ГЕМОСТАЗА ПРИ ПРИМЕНЕНИИ ВАРФАРИНА У БОЛЬНЫХ КАРДИОХИРУРГИЧЕСКОГО ПРОФИЛЯ

Objective: to reveal early changes in the hemostatic system during warfarin therapy in cardiac surgical patients, by comprehensively evaluating their hemostatic status.Subjects and methods. Seventyfive patients receiving cardiac surgical treatment were examined. All the patients took warfarin for 5±1.5 days. Laboratory studies involving the determination of routine coagulogram readings and thrombodynamic indicators (lag time (Tlag) and rate (Vs) of clot growth, and concentrations of individual Factors II, VI, IX, and X) were used to evaluate the patients' hemostatic status.Results. 28% of the patients were found to have an international normalized ratio (INR) of above 3.0. There was a correlation of Tlag with INR (R2=0.66). Both indicators were  comparatively highly correlated with Factor II and Factor X concentrations (R2=0.50 and 0.40 for Tlag; R2=0.53 and 0.48 for INR) and were uncorrelated with Factor IX levels (R2=0.20 for Tlag and 0.34 for INR). However, there was a difference in Factor VII concentrations: no correlation for Tlag (R2=0.20) whereas it for INR was rather high (R2=0.42). The index Vs was uncorrelated with INR (R2=0.24) and the concentration of blood coagulation factors (R2&lt;0.1). There was a high correlation between Factor II and Factor X concentrations (R2=0.87); the correlation between the concentrations of all other pairs of coagulation factors was substantially lower (R20.45). The lack of correlation of a thrombodynamic indicator, such as clot growth rate, with the concentration of coagulation factors points to the fact that warfarin acts mainly on the phase of coagulation activation rather than that of clot propagation.Conclusion. The weak correlation between coagulation factors (except that of a pair of Factor II and Factor X) is indicative of the individual response of the patients to warfarin treatment and the need to monitor the hemostatic status by global hemostatic tests rather than by individual proteins. The thrombodynamic indicator Tlag reflects the effect of warfarin in proportion to INR. Warfarin virtually fails to affect the rate of clot growth so this indicator may be used to evaluate the patient's procoagulant status uncompensated for with warfarin intake. Цель работы. Выявление ранних изменений в системе гемостаза при терапии варфарином у кардиохирургических больных путем комплексной оценки гемостатического статуса пациентов.Материалы и методы. Исследовано 75 пациентов, проходивших кардиохирургическое лечение. Все пациенты получали варфарин (продолжительность лечения больных — 5±1,5 суток). Для оценки гемостатического статуса больных использовали лабораторные методы, включающие определение рутинных показателей коагулограммы, тромбодинамики (время задержки Tlag и скорости роста сгустка (Vs), исследования концентрации II, VII, IX, X факторов).Результаты. Установлено, что у 28%больных показатель МНО находился в области свыше 3.0. Установлена корреляция Tlag с  MНО (R2=0,66). Оба показателя имеют сравнительно высокую корреляцию с концентрацией FII и FX (R2=0,50 и 0,40 для Tlag, R2=0,53 и 0,48 для МНО) и не коррелируют с концентрацией FIX (R2=0,20 для Tlag и 0,34 для МНО). Однако наблюдается расхождение в случае концентрации FVII: корреляция для Tlag отсутствует (R2=0,20), тогда как для МНО она достаточно высока (R2=0,42). Параметр Vs не коррелирует с МНО (R2=0,24) и концентрацией факторов свертывания (R2&lt;0,1). Обнаружена высокая корреляция между концентрациями факторов FII и FX (R2=0,87), корреляция между концентрациями всех других пар факторов свертывания существенно ниже (R20,45). Отсутствие корреляции такого показателя тромбодинамики как скорость роста сгустка от концентрации факторов указывает на то, что варфарин действует преимущественно на фазу активации свертывания, но не на фазу распространения сгустка.Заключение. Слабая корреляция факторов свертывания между собой (за исключением пары фактор II — фактор X) указывает на индивидуальный ответ больных на лечение варфарином и необходимость мониторинга состояния гемостаза не по отдельным белкам, а глобальными тестами. Параметр Tlag в тромбодинамике отражает эффект варфарина пропорционально МНО. Варфарин практически не влияет на скорость роста сгустка Vs, поэтому данный параметр можно использовать для оценки прокоагулянтного состояния пациента, не компенсируемого приемом варфарина

Особенности развития и течения синдрома диссеминированного внутрисосудистого свертывания при хирургических вмешательствах у детей с онкологическими заболеваниями

Coagulopathy always accompanies blood loss, and its transformation into disseminated intravascular coagulation syndrome (DIC) is associated with increased morbidity and mortality.Objective: to characterize the features of the development and course of DIC during bleeding, as well as identify the main predictors of its formation during surgical interventions in children with oncological diseases.Material and Methods. A retrospective study of children under 18 years of age with oncological pathology who received surgical treatment for the period from 2017 to 2019 years. Children who received blood transfusion and hemostatic therapy with intraoperative bleeding were selected. The resulting cohort (n=207) was divided into two groups using the modified ISTH assessment system: children with DIC (n=59), without DIC (n=148). Demographic, clinical, and laboratory factors were compared between groups. The final model of multivariate logistic regression included signs that were before the development of DIC on the second day after the operation and were selected as a result of univariate analysis (P&lt;0.05), had less than 10% missing data and were clinically plausible. The prediction accuracy of the multivariate model was checked by analyzing the area under the ROC curve.Results. DIC was found to develop often in children with cancer during surgical operations in the retroperitoneal space (OR=2.09 [1.07; 4.05]; P=0.03) and liver (OR=3.86 [1.72; 8.67]; P=0.001). Multiple organ failure (MOF) was more severe and was represented by pulmonary, hepatic and renal failure in the group with identified DIC. The development of MOF was accompanied by a decrease in tissue perfusion and an increase in D-dimer. The probability of detecting acute thrombosis after surgery was 4.5 times higher in the group of patients with DIC than in the group without DIC (OR=4.5 [1.4; 14.3]; P=0.01). 90-daily survival was 84.41±6.49% [71.69%; 97.13%] in the group of patients with DIC, and 96.22±3.12 [90.1%; 100%] in the group without DIC. Multivariate analysis showed that age less than 8 years, platelet count less than 150X109/l, hypocalcemia less than 1 mmol/l and the period of intraoperative critical hypotension for more than 25 minutes are predictors of the development of DIC after surgery. ROC analysis showed excellent quality of the obtained predictive model (AUC=0,94 [0,9; 0,97]).Conclusion. In children with oncological diseases, in the presence of bleeding, coagulopathy in the postoperative period is transformed into a DIC-syndrome, proceeding clinically with the development of organ failure. Age less than 8 years, platelet count less than 150X109/l, hypocalcemia less than 1 mmol/L and a period of intraoperative critical hypotension of more than 25 minutes are predictors of the development of DIC. The extreme expression of the «organ» type DIC is the progression of thrombotic syndrome to life threatening complications, which reduces the 90-day survival by 12%.Кровопотере всегда сопутствует коагулопатия, а ее трансформация в синдром диссеминированного внутрисосудистого свертывания (ДВС-синдром) связана с повышенным уровнем заболеваемости и смертности.Цель исследования. Охарактеризовать особенности развития и течения ДВС-синдрома при кровотечениях, а также выявить основные предикторы его формирования при оперативных вмешательствах у детей с онкологическими заболеваниями.Материалы и методы. Ретроспективное исследование у детей в возрасте до 18 лет с онкологическими заболеваниями, получавших хирургическое лечение в период с 2017 по 2019 годы. Отобрали детей, получавших гемотрансфузии и гемостатическую терапию при интраоперационном кровотечение. Полученную когорту (n=207) разделили на две группы с использованием модифицированной системы оценки ISTH: дети с ДВС-синдромом (n=59), без ДВС-синдрома (n=148). Провели сравнение демографических, клинических и лабораторных факторов между группами. В окончательную модель многофакторной логистической регрессии включили признаки, которые были до развития ДВС-син-дрома на 2-е сутки после операции и были отобраны в результате однофакторного анализа (p&lt;0,05), имели менее 10% пропущенных данных и были клинически правдоподобными. Точность прогнозирования многофакторной модели проверили по анализу площади под кривой ROC.Результаты. Установили, что ДВС-синдром у детей с онкологическими заболеваниями часто развивается при операциях в области забрюшинного пространства (OR=2,09 [1,07; 4,05]; p=0,03) и печени (OR=3,86 [1,72; 8,67]; p=0,001). Полиорганная недостаточность (ПОН) была более тяжелой и была представлена легочной, печеночной и почечной недостаточностью в группе с выявленным ДВС-синдро-мом. Развитие ПОН сопровождалось снижением показателей тканевой перфузии и ростом D-димера. Вероятность выявления острого тромбоза после операции была в 4,5 раза выше в группе пациентов с ДВС-синдромом, чем в группе без ДВС-синдрома (OR=4,5 [1,4; 14,3]; p=0,01). 90-дневная выживаемость составила в группе пациентов с ДВС-синдромом — 84,41±6,49% [71,69%; 97,13%], а в группе без ДВС-синдрома — 96,22±3,12% [90,1%; 100%]. Многофакторный анализ показал, что возраст менее 8 лет, количество тромбоцитов менее 150Х109/л, гипокальциемия менее 1 ммоль/л и период интраоперационной критической гипотонии более 25 минут являются предикторами развития ДВС-синдрома после операции. ROC-анализ показал превосходное качество полученной прогностической модели (AUC=0,94 [0,9; 0,97]).Заключение. У детей с онкологическими заболеваниями, при наличии кровотечения, коагуло-патия в послеоперационном периоде трансформируется в ДВС-синдром, протекающий клинически с развитием органной недостаточности. Возраст менее 8 лет, количество тромбоцитов менее 150Х109/л, гипокальциемия менее 1 ммоль/л и период интраоперационной критической гипотонии более 25 минут являются предикторами развития ДВС-синдрома. Крайним выражением ДВС-синдрома «органного типа» является прогрессирование тромботического синдрома до реализации осложнений, угрожающих жизни, что и уменьшает 90-дневную выживаемость на 12%

Integrated laboratory coagulation tests in hypercoagulation diagnosis and thrombosis risk assessment. Part I. The pathophysiology of thrombosis and hypercoagulation

Thrombosis is a fatal hemostatic disorders occurring in various conditions ranging from pregnancy and surgery to cancer, sepsis and heart attack. Despite the availability of different anticoagulants and accumulated clinical experience, proving their effectiveness, thrombosis remains a major cause of morbidity and mortality. This is largely due to the fact that conventional laboratory coagulation tests are not sufficiently sensitive to the hypercoagulable state, and they are difficult to use for assessing the risk of thrombosis. Specific molecular markers (D-dimers, fibrinopeptide, thrombin-antithrombin complex) are more effective, but also have a large number of disadvantages. A possible solution is the use of integrated test, which simulate in vitro the majority of the physiological coagulation processes. In the first part of this paper the biochemical processes that cause the risk of thrombosis were discussed

Integrated laboratory coagulation tests in hypercoagulation diagnosis and thrombosis risk assessment. Part II. The sensitivity of integral tests to hypercoagulable states

In the second part we present a review of the existing data about ability of integrated tests, as already introduced in clinical practice, and the new (test of thrombin generation, thromboelastography, thrombodynamics, perfusion chamber) to assess the risk of thrombosis in different pathologies. We can conclude that the existing integrated tests can be an important tool in the diagnosis of hypercoagulation. However, lack of standardization prevents their use: various tests and modifications of each test are different in sensitivity and specificity for each pathological condition. Furthermore, even in situations where the tests can reliably identify a group of patients with different degrees of thrombosis risk, their use in clinical practice is often difficult, since the differences between these groups were statistically significant, but the normal range and patients significantly overlap

Significance of two transmembrane ion gradients for human erythrocyte volume stabilization.

Functional effectiveness of erythrocytes depends on their high deformability that allows them to pass through narrow tissue capillaries. The erythrocytes can deform easily due to discoid shape provided by the stabilization of an optimal cell volume at a given cell surface area. We used mathematical simulation to study the role of transport Na/K-ATPase and transmembrane Na+ and K+ gradients in human erythrocyte volume stabilization at non-selective increase in cell membrane permeability to cations. The model included Na/K-ATPase activated by intracellular Na+, Na+ and K+ transmembrane gradients, and took into account contribution of glycolytic metabolites and adenine nucleotides to cytoplasm osmotic pressure. We found that this model provides the best stabilization of the erythrocyte volume at non-selective increase in the permeability of the cell membrane, which can be caused by an oxidation of the membrane components or mechanical stress during circulation. The volume of the erythrocyte deviates from the optimal value by no more than 10% with a change in the non-selective permeability of the cell membrane to cations from 50 to 200% of the normal value. If only one transmembrane ion gradient is present (Na+), the cell loses the ability to stabilize volume and even small changes in membrane permeability cause dramatic changes in the cell volume. Our results reveal that the presence of two oppositely directed transmembrane ion gradients is fundamentally important for robust stabilization of cellular volume in human erythrocytes