Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood

© 2016 by The American Society of Hematology. Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmuscle my osin II, red blood cells (RBCs), fibrin(ogen), factor XIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca2+, the integrin αIIbβ3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders

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

A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost

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%

Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation

On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

Segregation of Flowing Blood: Mathematical Description

Blood rheology is completely determined by its major corpuscles which are erythrocytes, or red blood cells (RBCs). That is why understanding and correct mathematical description of RBCs behavior in blood is a critical step in modelling the blood dynamics. Various phenomena provided by RBCs such as aggregation, deformation, shear-induced diffusion and non-uniform radial distribution affect the passage of blood through the vessels. Hence, they have to be taken into account while modelling the blood dynamics. Other important blood corpuscles are platelets, which are crucial for blood clotting. RBCs strongly affect the platelet transport in blood expelling them to the vessel walls and increasing their dispersion, which has to be considered in models of clotting. In this article we give a brief review of basic modern approaches in mathematical description of these phenomena, discuss their applicability to real flow conditions and propose further pathways for developing the theory of blood flow