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Macromolecular assembly depends on tightly regulated pairwise binding interactions that are selectively favored at assembly sites while being disfavored in the soluble phase. This selective control can arise due to molecular density-enhanced binding, as recently found for the kinetochore scaffold protein CENP-T. When clustered, CENP-T recruits markedly more Ndc80 complexes than its monomeric counterpart, but the underlying molecular basis remains elusive. Here, we use quantitative in vitro assays to reveal two distinct mechanisms driving this behavior. First, Ndc80 binding to CENP-T is a two-step process: initially, Ndc80 molecules rapidly associate and dissociate from disordered N-terminal binding sites on CENP-T. Over time, these sites undergo maturation, resulting in stronger Ndc80 retention. Second, we find that this maturation transition is regulated by a kinetic barrier that is sensitive to the molecular environment. In the soluble phase, binding site maturation is slow, but within CENP-T clusters, this process is markedly accelerated. Notably, the two Ndc80 binding sites in human CENP-T exhibit distinct maturation rates and environmental sensitivities, which correlate with their different amino-acid content and predicted binding conformations. This clustering-induced maturation is evident in dividing human cells, suggesting a distinct regulatory entry point for controlling kinetochore assembly. We propose that the tunable acceleration of binding site maturation by molecular crowding may represent a general mechanism for promoting the formation of macromolecular structures., Competing Interests: Competing Interest Statement: Authors declare that they have no competing interests.

Background: Umbilical cord blood is used for the testing of various parameters in newborns. However, data on its applicability for hemostasis assays is insufficient., Objective: To evaluate whether umbilical cord blood can be used for standard tests, thromboelastometry and thrombodynamics for preterm and term newborns., Methods: 187 newborns were included in the study. Blood was taken from the umbilical cord and by venipuncture of the newborn. Clotting times, fibrinogen, D-dimer, thromboelastometry and thrombodynamics were measured., Results: Clotting times and fibrinogen indicated a hypocoagulable shift, while thromboelastometry and thrombodynamics showed a hypercoagulable shift in hemostasis in umbilical cord blood compared to newborn blood. D-dimer indicated an enhanced process of thrombus lysis in newborn blood compared to cord blood. Collecting blood into a tube with the addition of a contact pathway inhibitor did not significantly change the global assay parameters in either umbilical cord blood or newborn blood. In the thrombodynamics assay, spontaneous clotting was detected but suppressed by the addition of a tissue factor inhibitor., Conclusions: Hemostasis in cord and newborn blood differs for both global and standard tests. Hypercoagulability in newborns registered with the global assay thrombodynamics is associated with the presence of tissue factor in the blood., Impact Statement: 1. We found a hypercoagulation shift in newborns compared with the adult references, possibly due to the presence of tissue factor in blood. 2. Blood coagulation is enhanced in cord blood compared with blood sampled from the vein of a newborn according to thromboelastometry and thrombodynamics assays. 3. Clotting times and fibrinogen concentrations in cord blood differ from these parameters in newborn blood. 4. Studying of the (patho)physiological features of hemostasis in newborns should consider differences in cord blood and vein sampled blood., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

The hematological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important in COVID-19 pathophysiology. However, the interactions of SARS-CoV-2 with platelets and red blood cells are still poorly understood. There are conflicting data regarding the mechanisms and significance of these interactions. The aim of this review is to put together available data and discuss hypotheses, the known and suspected effects of the virus on these blood cells, their pathophysiological and diagnostic significance, and the potential role of platelets and red blood cells in the virus's transport, propagation, and clearance by the immune system. We pay particular attention to the mutual activation of platelets, the immune system, the endothelium, and blood coagulation and how this changes with the evolution of SARS-CoV-2. There is now convincing evidence that platelets, along with platelet and erythroid precursors (but not mature erythrocytes), are frequently infected by SARS-CoV-2 and functionally changed. The mechanisms of infection of these cells and their role are not yet entirely clear. Still, the changes in platelets and red blood cells in COVID-19 are significantly associated with disease severity and are likely to have prognostic and pathophysiological significance in the development of thrombotic and pulmonary complications.

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 10 5 /μL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: FIA and MAP are former employees and cofounders of Hemacore Labs LLC, OAF is employee of Hemacore Labs LLC. Hemacore Labs LLC holds several patents and patent applications that are related to the diagnostic use of spatial clot growth and thrombin generation and which has developed the assay under the trade name of Thrombodynamics®. Hemacore Labs LLC provided support in the form of reagents for Thrombodynamics-4D but did not have any additional role in the design of the study, collection and analysis of data, decision to publish or preparation of the manuscript. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. All other authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Piezo1 is a mechanosensitive cationic channel that boosts intracellular [Ca 2+ ] i . Compression of red blood cells (RBCs) during platelet-driven contraction of blood clots may cause the activation of Piezo1., Objectives: To establish relationships between Piezo1 activity and blood clot contraction., Methods: Effects of a Piezo1 agonist, Yoda1, and antagonist, GsMTx-4, on clot contraction in vitro were studied in human blood containing physiological [Ca 2+ ]. Clot contraction was induced by exogenous thrombin. Activation of Piezo1 was assessed by Ca 2+ influx in RBCs and with other functional and morphologic features., Results: Piezo1 channels in compressed RBCs are activated naturally during blood clot contraction and induce an upsurge in the intracellular [Ca 2+ ] i , followed by phosphatidylserine exposure. Adding the Piezo1 agonist Yoda1 to whole blood increased the extent of clot contraction due to Ca 2+ -dependent volumetric shrinkage of RBCs and increased platelet contractility due to their hyperactivation by the enhanced generation of endogenous thrombin on activated RBCs. Addition of rivaroxaban, the inhibitor of thrombin formation, or elimination of Ca 2+ from the extracellular space abrogated the stimulating effect of Yoda1 on clot contraction. The Piezo1 antagonist, GsMTx-4, caused a decrease in the extent of clot contraction relative to the control both in whole blood and in platelet-rich plasma. Activated Piezo1 in compressed and deformed RBCs amplified the platelet contractility as a positive feedback mechanism during clot contraction., Conclusion: The results obtained demonstrate that the Piezo1 channel expressed on RBCs comprises a mechanochemical modulator of blood clotting that may be considered a potential therapeutic target to correct hemostatic disorders., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder caused by WAS gene mutations resulting in haematopoietic/immune cell defects. Recent studies report accelerated death of WAS platelets and lymphocytes. Data on megakaryocyte (MK) maturation, viability and their possible role in thrombocytopenia development in WAS are limited. In this study we evaluate the MK viability and morphology in untreated, romiplostim-treated WAS patients compared with normal controls. The study included 32 WAS patients and 17 healthy donors. MKs were captured from bone marrow aspirates by surface-immobilized anti-GPIIb-IIIa antibody. Viability (by phosphatidylserine [PS] externalization), distribution by maturation stages and size of MK were determined by light microscopy. MK distribution by maturation stages in patients differed from controls. 40 ± 22% of WAS MKs versus 23 ± 11% of normal MKs were at maturation stage 3 (p = 0.02), whereas 24 ± 20% in WAS and 39 ± 14% in controls had megakaryoblast morphology (p = 0.05). Romiplostim treatment changed the MK maturation stages distribution close to normal. PS-positive (PS+) MK in WAS was significantly higher (21 ± 21%) than in healthy controls (2 ± 4%, p < 0.01). WAS patients with more damaging truncating mutations and higher disease score had higher PS+ MK fraction (Spearman r = 0.6, p < 0.003). We conclude that WAS MKs have increased cell death tendency and changes in maturation pattern. Both could contribute to thrombocytopenia in WAS patients., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

Inhibitors of coagulation factor XIa (FXIa) are currently being investigated as potential anticoagulant therapies. We hypothesize that circulating FXIa could be a potential target for these therapies. Using previous analyses of FXIa impurities in immune globulin products involved in thrombotic adverse events, we estimated that picomolar levels of FXIa can be thrombogenic. In an in vitro clot-growth assay, 0.1-3 pM of FXIa did not, by itself, activate clotting but increased the size of growing clots. Spatio-temporal reconstruction of thrombin activity inside the clot revealed that FXIa's effect was limited to the clot-plasma interface, in which FXIa produced a taller than standard wave of thrombin. Factor-depleted plasma and a panel of selective anti-FXIa antibodies showed that exogenous FXIa effects are (1) blocked by anti-FXIa antibodies, (2) independent of FXI activation inside the clot, and (3) larger than the contribution of in situ FXIa. In a thrombin generation (TG) assay, picomolar FXIa did not initiate TG but rather promoted TG triggered by tissue factor or thrombin, suggesting that the effect of FXIa on the thrombin wave is mediated by the elevation of thrombin-triggered TG. In circulating bovine blood, low doses of human FXIa did not initiate clotting but increased the size of stenosis-triggered thrombi. FXIa injection in mice enhanced TG in plasma for at least 6 hours ex vivo, confirming the persistence of circulating FXIa. Our findings suggest that picomolar levels of circulating FXIa may not be able to initiate thrombosis but can facilitate thrombus growth through the facilitation of TG inside the clot., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)

Background: Blood coagulation abnormalities play a major role in COVID-19 pathophysiology. However, the specific details of hypercoagulation and anticoagulation treatment require investigation. The aim of this study was to investigate the status of the coagulation system by means of integral and local clotting assays in COVID-19 patients on admission to the hospital and in hospitalized COVID-19 patients receiving heparin thromboprophylaxis., Methods: Thrombodynamics (TD), thromboelastography (TEG), and standard clotting assays were performed in 153 COVID-19 patients observed in a hospital setting. All patients receiving treatment, except extracorporeal membrane oxygenation (ECMO) patients (n = 108), were administered therapeutic doses of low molecular weight heparin (LMWH) depending on body weight. The ECMO patients (n = 15) were administered unfractionated heparin (UFH)., Results: On admission, the patients (n = 30) had extreme hypercoagulation by all integral assays: TD showed hypercoagulation in ~75% of patients, while TEG showed hypercoagulation in ~50% of patients. The patients receiving treatment showed a significant heparin response based on TD; 77% of measurements were in the hypocoagulation range, 15% were normal, and 8% remained in hypercoagulation. TEG showed less of a response to heparin: 24% of measurements were in the hypocoagulation range, 59% were normal and 17% remained in hypercoagulation. While hypocoagulation is likely due to heparin treatment, remaining in significant hypercoagulation may indicate insufficient anticoagulation for some patients, which is in agreement with our clinical findings. There were 3 study patients with registered thrombosis episodes, and all were outside the target range for TD parameters typical for effective thromboprophylaxis (1 patient was in weak hypocoagulation, atypical for the LMWH dose used, and 2 patients remained in the hypercoagulation range despite therapeutic LMWH doses)., Conclusion: Patients with COVID-19 have severe hypercoagulation, which persists in some patients receiving anticoagulation treatment, while significant hypocoagulation is observed in others. The data suggest critical issues of hemostasis balance in these patients and indicate the potential importance of integral assays in its control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bulanov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used in vitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.

For the first time, the influence of COVID-19 on blood microrheology was studied. For this, the method of filtering erythrocytes through filters with pores of 3.5 μm was used. Filterability was shown to significantly decrease with the increasing severity of the patient's condition, as well as with a decrease in the ratio of hemoglobin oxygen saturation to the oxygen fraction in the inhaled air (SpO 2 /FiO 2 ). The filterability of ≤ 0.65, or its fast decrease during treatment, were indicators of a poor prognosis. Filterability increased significantly with an increase in erythrocyte count, hematocrit and blood concentrations of hemoglobin, albumin, and total protein. The effect of these parameters on the erythrocyte filterability is directly opposite to their effect on blood macrorheology, where they all increase blood viscosity, worsening the erythrocyte deformability. The erythrocyte filterability decreased with increasing oxygen supply rate, especially in patients on mechanical ventilation, apparently not due to the oxygen supplied, but to the deterioration of the patients' condition. Filterability significantly correlates with the C-reactive protein, which indicates that inflammation affects the blood microrheology in the capillaries. Thus, the filterability of erythrocytes is a good tool for studying the severity of the patient's condition and his prognosis in COVID-19.

Excessive ammonium blood concentration causes many serious neurological complications. The medications currently used are not very effective. To remove ammonium from the blood, erythrocyte-bioreactors containing enzymes that processing ammonium have been proposed. The most promising bioreactor contained co-encapsulated glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT). However, a low encapsulation of a commonly used bovine liver GDH (due to high aggregation), makes clinical use of such bioreactors impossible. In this study, new bioreactors containing ALT and non-aggregating GDH at higher loading were first produced using the flow dialysis method and the new bacterial GDH enzyme from Proteus sp. The efficacy of these erythrocyte-bioreactors and their properties (hemolysis, osmotic fragility, intracellular and extracellular activity of included enzymes, erythrocyte indices, and filterability) were studied and compared with native cells during 1-week storage. The ammonium removal rate in vitro by such erythrocyte-bioreactors increased linearly with an increase in encapsulated GDH activity. Alanine in vitro increased in accordance with ammonium consumption, which indicated the joint functioning of both included enzymes. Thus, novel bioreactors for ammonium removal containing GDH from Proteus sp. are promising for clinical use, since they have a more efficient GDH encapsulation and their properties are not inferior to previously obtained erythrocyte-bioreactors., (© 2022. The Author(s).)

Introduction: Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19., Materials and Methods: Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed., Results and Conclusions: All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Coagulation system is heavily involved into the process of infective endocarditis (IE) vegetation formation and can facilitate further embolization. In this study we aimed to assess the coagulation and platelet state in IE implementing a wide range of standard and global laboratory assays. We also aim to determine whether prothrombotic genetic polymorphisms play any role in embolization and mortality in IE patients., Methods: 37 patients with IE were enrolled into the study. Coagulation was assessed using standard coagulation assays (activated partial thromboplastin time (APTT), prothrombin, fibrinogen, D-dimer concentrations) and integral assays (thromboelastography (TEG) and thrombodynamics (TD)). Platelet functional activity was estimated by flow cytometry. Single nuclear polymorphisms of coagulation system genes were studied., Results: Fibrinogen concentration and fibrinogen-dependent parameters of TEG and TD were increased in patients indicating systemic inflammation. In majority of patients clot growth rate in thrombodynamics was significantly shifted towards hypercoagulation in consistency with D-dimers elevation. However, in some patients prothrombin, thromboelastography and thrombodynamics were shifted towards hypocoagulation. Resting platelets were characterized by glycoprotein IIb-IIIa activation and degranulation. In patients with fatal IE, we observed a significant decrease in fibrinogen and thrombodynamics. In patients with embolism, we observed a significant decrease in the TEG R parameter. No association of embolism or mortality with genetic polymorphisms was found in our cohort., Conclusions: Our findings suggest that coagulation in patients with infective endocarditis is characterized by general hypercoagulability and platelet pre-activation. Some patients, however, have hypocoagulant coagulation profile, which presumably can indicate progressing of hypercoagulation into consumption coagulopathy., Competing Interests: We disclose a conflict of interest for Prof. Fazoil Ataullakhanov, who is a co-founder of HemaCore LLC, which holds several patents and patent applications that are related to the diagnostic use of spatial clot growth and which has developed the assay under the trade name of Thrombodynamics® (Ataullakhanov FI, international patent applications PCT/CH2007/000543 filing date 02.11.2007 and РСТ/RU2012/000570 filing date 16.07.2012). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The even chromosome segregation between daughter cells during mitosis is crucial for genome integrity and is mostly regulated by proper attachments of spindle microtubules to kinetochores. Abnormalities in this process can lead to chromosome mis-segregation and potentially result in severe developmental disorders such as aneuploidy and cancer. Merotelic attachments when tubulin microtubules captured by the kinetochore of one chromatid originate from both spindle poles are considered as one of the key molecular processes that cause such abnormalities. In this paper, we use computer modeling and the Monte Carlo approach to reveal the reasons for retaining merotelic attachments at the end of metaphase. To this end, we varied, in small increments, the basic cell parameters within ensembles of 100, 500, and 1000 virtual cells. The analysis of configurations that ensure the preservation of the largest fraction of merotelic attachments enabled us to conclude that only a change in the size of the kinetochore corona can significantly increase the number of merotelic attachments and the angle between the centromere axis and the spindle axis. The effect of the other changes in model parameters, if any, was steadily suppressed by the end of metaphase. In addition, our computer model was validated by successfully reproducing the results of third-party theoretical studies as well as some experimental observations. We also found that the orientation of chromosomes and the number of merotelic attachments do not have an explicit correlation with each other and within some limits can change independently., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

In this work, we present a new method-Thrombodynamics-4D-for the assessment of both plasma and platelet contributions to clotting. Thrombodynamics-4D potentially allows for the determination of plasma or platelet disorders and the effects of various drugs on plasma clotting or on platelet procoagulant function. In this assay, clot formation in platelet-rich plasma or platelet-free plasma supplemented with phospholipids is activated with tissue factor immobilized on a surface. Spatial fibrin clot growth and thrombin concentration dynamics are registered by measuring light scattering of the fibrin clot and fluorescence of the product formed by cleavage of the synthetic fluorogenic substrate by thrombin, respectively. Here, we describe the preanalytical requirements, measurement methodology and calculation principles of assay parameters. Preanalytical and analytical variability and reference ranges of the assay are given. Additionally, we show some clinical examples, which determine the effect of anticoagulants, measure clotting dysfunction in patients with platelet or coagulation disorders and evaluate the effect of surgery.

The limitations of the efficiency of ammonium-neutralizing erythrocyte-bioreactors based on glutamate dehydrogenase and alanine aminotransferase reactions were analyzed using a mathematical model. At low pyruvate concentrations in the external medium (below about 0.3 mM), the main limiting factor is the rate of pyruvate influx into the erythrocyte from the outside, and at higher concentrations, it is the disappearance of a steady state in glycolysis if the rate of ammonium processing is higher than the critical value (about 12 mM/h). This rate corresponds to different values of glutamate dehydrogenase activity at different concentrations of pyruvate in plasma. Oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by glutamate dehydrogenase decreases the fraction of NADPH in the constant pool of nicotinamide adenine dinucleotide phosphates (NADP + NADPH). This, in turn, activates the pentose phosphate pathway, where NADP reduces to NADPH. Due to the increase in flux through the pentose phosphate pathway, stabilization of the ATP concentration becomes impossible; its value increases until almost the entire pool of adenylates transforms into the ATP form. As the pool of adenylates is constant, the ADP concentration decreases dramatically. This slows the pyruvate kinase reaction, leading to the disappearance of the steady state in glycolysis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with thrombotic complications. To elucidate pathogenic mechanisms, hemostatic disorders in RA were correlated with other laboratory and clinical manifestations. Hemostasis was assessed using relatively new complementary tests, the spatial growth of a plasma clot (Thrombodynamics assay), and contraction of whole blood clots. Platelet functionality was assessed with flow cytometry that quantified the expression of P-selectin and the fibrinogen-binding capacity of platelets before and after activation with a thrombin receptor-activating peptide. Parameters of fibrin clot growth and the kinetics of contraction of blood clots were significantly altered in patients with RA compared to the control group. In Thrombodynamics measurements, an increase in the clot growth rate, size, and optical density of plasma clots altogether indicated chronic hypercoagulability. The rate and extent of blood clot contraction in patients with RA was significantly reduced and associated with platelet dysfunction revealed by an impaired response to activation. Changes in the parameters of clot growth and contraction correlated with the laboratory signs of systemic inflammation, including hyperfibrinogenemia. These results confirm the pathogenic role of hemostatic disorders in RA and support the validity of fibrin clot growth and the blood clot contraction assay as indicators of a (pro)thrombotic state.

Thrombotic and inflammatory complications induced by vascular implants remain a challenge to treat cardiovascular disease due to the lack of self-adaption and functional integrity of implants. Inspired by the dynamic remodeling of the extracellular matrix (ECM), we constructed a bio-mimic ECM with a dual-layer nano-architecture on the implant surface to render the surface adaptive to inflammatory stimuli and remodelable possessing long-term anti-inflammatory and anti-thrombotic capability. The inner layer consists of PCL-PEG-PCL [triblock copolymer of polyethylene glycol and poly(ε-caprolactone)]/Au-heparin electrospun fibers encapsulated with indomethacin while the outer layer is composed of polyvinyl alcohol (PVA) and ROS-responsive poly(2-(4-((2,6-dimethoxy-4-methylphenoxy)methyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane) (PBA) fibers. In response to acute inflammation after vascular injury, the outer layer reduces ROS rapidly by PBA degradation for inflammation suppression. The degraded outer layer facilitates inner layer reconstruction with enhanced hemocompatibility through the H-bond between PVA and PCL-PEG-PCL. Furthermore, chronic inflammation is effectively depressed with the sustained release of indomethacin from the inner layer. The substantial enhancement of the functional integrity of implants and reduction of thrombotic and inflammatory complications with the self-adaptive ECM are demonstrated both in vitro and in vivo. Our work paves a new way to develop long-term anti-thrombotic and anti-inflammatory implants with self-adaption and self-regulation properties.

Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs 3.9% in controls; P <0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P <0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia., (Copyright© 2020 Ferrata Storti Foundation.)

Background:  Congenital and acquired hemostatic disorders are among the pathogenic factors of pregnancy loss. Studying mechanistic relations between impaired hemostasis and fetal losses is important for the prognosis and prophylaxis of obstetric complications., Objective:  This article aims to establish latent hemostatic disorders in nonpregnant women as an important premorbid risk factor of pregnancy loss., Methods and Results:  Hemostasis was characterized using two relatively new in vitro assays, namely thrombodynamics (spatial clot growth) and kinetics of blood clot contraction, which together reflect the hemostatic or thrombotic potential. In addition, platelet functionality was assessed using flow cytometry. Our study included 50 women with a history of pregnancy loss and 30 parous women without previous obstetric complications. In patients with pregnancy loss, hypercoagulability was observed along with significant impairment of blood clot contraction associated with chronic platelet activation and dysfunction. Both hypercoagulability and defective clot contraction were significantly more pronounced in patients with a history of three or more miscarriages compared with patients with a history of one or two miscarriages. In addition, a significant inhibition of clot contraction was found in patients with miscarriage occurring after 10 weeks of gestation compared with those who lost a fetus earlier in pregnancy., Conclusion:  These results indicate that chronic hypercoagulability and impaired clot contraction constitute a premorbid status in patients with pregnancy loss. The data confirm a significant pathogenic role of hemostatic disorders in pregnancy loss and suggest the predictive value of thrombodynamics and blood clot contraction assays in evaluating the risk of pregnancy loss., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Thirteen tubulin protofilaments, made of αβ-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of β-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αβ-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis., Competing Interests: The authors have declared that no competing interests exist.

Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbβ3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbβ3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Folate metabolism in mammalian cells is essential for multiple vital processes, including purine and pyrimidine synthesis, histidine catabolism, methionine recycling, and utilization of formic acid. It remains unknown, however, whether these processes affect each other via folate metabolism or can function independently based on cellular needs. We addressed this question using a quantitative mathematical model of folate metabolism in rat liver cytoplasm. Variation in the rates of metabolic processes associated with folate metabolism (i.e., purine and pyrimidine synthesis, histidine catabolism, and influxes of formate and methionine) in the model revealed that folate metabolism is organized in a striking manner that enables activation or inhibition of each individual process independently of the metabolic fluxes in others. In mechanistic terms, this independence is based on the high activities of a group of enzymes involved in folate metabolism, which efficiently maintain close-to-equilibrium ratios between substrates and products of enzymatic reactions.

Package inserts for C1-esterase inhibitor (C1INH) products include warnings for an elevated risk of possible thrombosis in certain individuals, referring to thromboembolic events (TEEs) that were reported to occur after C1INH infusions. However, the mechanism(s) that could explain possible development of TEEs due to C1INH remains unknown. In this work, we evaluated plausible impact of C1INH on the protein C (PC) anticoagulant system. We performed thrombin generation (TG) assays (TGA) and analyzed spatial fibrin clot propagation using thrombodynamics in plasma of individual donors after the addition of thrombomodulin (TM) and C1INH. The addition of C1INH was consistent with the plasma concentrations resulting from doses currently approved for the HAE treatment up to ones consistent with off-label use in patients with risk of inflammation. 16 IU/ml of C1INH significantly enhanced thrombin peak (TP) generation in the presence of 12 and 15 nM TM. TG enhancement was observed by the addition of C1INH to make concentrations equal to 2 and 4 IU/ml in some donor plasmas. C1INH addition in the presence of TM enhanced the stop time of spatial clot growth in Thrombodynamics assay. A chromogenic activity assay demonstrated that C1INH inhibited PC activation by thrombin in the presence of TM. Substitution of TM with APC in TGA attenuated the TP enhancing effect of C1INH. The collective results of the present study suggest a concentration dependent C1INH interaction with the PC system. This study introduces a plausible TM-dependent mechanism, that may explain reported TEEs via suppressed production of APC in the presence of C1INH., (Copyright © 2019. Published by Elsevier Ltd.)

Increased blood ammonium concentrations cause neurological complications. Existing drugs are not always sufficiently effective. Alternatively, erythrocytes-bioreactors (EBRs) loaded with enzymes utilizing ammonium, were suggested for ammonium removal from blood. However all they worked only for a short period of time. The reasons for this were not investigated. In this study, EBR mathematical models were developed and analysed based on the reactions of glycolysis and different enzymes utilizing ammonium, which showed that the efficiency and duration of EBRs' functioning could be limited due to low permeability of the cell membrane for some key substrates and products. A new enzyme system including glutamate dehydrogenase and alanine aminotransferase was proposed and realised experimentally, which was not limited by cell membrane permeability for glutamate and α-ketoglutarate due to creating metabolic pathway where these metabolites were produced and consumed cyclically. New bioreactors removed ammonium in vitro at the rate of 1.5 mmol/h × l RBCs (for human bioreactors) and in vivo in a model of hyperammoniemia in mice at the rate of 2.0 mmol/h × l RBCs (for mouse bioreactors), which correlated with model calculations. Experimental studies proved the proposed mathematical models are correct. Mathematical simulation of erythrocyte-bioreactors opens new opportunities for analysing the efficiency of any enzyme included in erythrocytes.

Background: Preterm newborns are at thrombohemorrhagic risk during the early neonatal period. Taking into account the lack of informative tools for the laboratory diagnosis of hemostasis disorders in newborns, our goal was to determine the baseline values of thrombodynamics and platelet functional activity in healthy term and moderately preterm newborns during the early neonatal period future potential clinical use of these tests., Methods: Coagulation was assessed using an integral assay of thrombodynamics and standard coagulation assays, and platelet functional activity was estimated by flow cytometry., Results: Hypercoagulation of newborns, represented by a significantly higher clot growth velocity and the presence of spontaneous clots in the thrombodynamics, was combined with platelet hypoactivity. Granule release, phosphatidylserine exposure, and the ability to change shape upon activation were decreased in the platelets of moderately preterm newborns. The platelet function remained at the same level over the first four days of life, whereas the hypercoagulation became less pronounced., Conclusions: The hemostasis of newborns is characterized by hypercoagulation combined with reduced platelet functional activity. Moderately preterm and term newborns do not differ in the parameters of coagulation, while some of the functional responses of platelets are lower in moderately preterm newborns than in term.

Objective- After activation at the site of vascular injury, platelets differentiate into 2 subpopulations, exhibiting either proaggregatory or procoagulant phenotype. Although the functional role of proaggregatory platelets is well established, the physiological significance of procoagulant platelets, the dynamics of their formation, and spatial distribution in thrombus remain elusive. Approach and Results- Using transmission electron microscopy and fluorescence microscopy of arterial thrombi formed in vivo after ferric chloride-induced injury of carotid artery or mechanical injury of abdominal aorta in mice, we demonstrate that procoagulant platelets are located at the periphery of the formed thrombi. Real-time cell tracking during thrombus formation ex vivo revealed that procoagulant platelets originate from different locations within the thrombus and subsequently translocate towards its periphery. Such redistribution of procoagulant platelets was followed by generation of fibrin at thrombus surface. Using in silico model, we show that the outward translocation of procoagulant platelets can be driven by the contraction of the forming thrombi, which mechanically expels these nonaggregating cells to thrombus periphery. In line with the suggested mechanism, procoagulant platelets failed to translocate and remained inside the thrombi formed ex vivo in blood derived from nonmuscle myosin ( MYH9)-deficient mice. Ring-like distribution of procoagulant platelets and fibrin around the thrombus observed with blood of humans and wild-type mice was not present in thrombi of MYH9-knockout mice, confirming a major role of thrombus contraction in this phenomenon. Conclusions- Contraction of arterial thrombus is responsible for the mechanical extrusion of procoagulant platelets to its periphery, leading to heterogeneous structure of thrombus exterior.

Blood coagulation is a delicately regulated space- and time-dependent process that leads to the formation of fibrin clots preventing blood loss upon vascular injury. The sensitivity of the coagulation network was previously investigated without accounting for transport processes. To investigate its sensitivity to coagulation factor deficiencies in a spatial reaction-diffusion system, we combined an in vitro experimental design with a computational systems biology model. Clot formation in platelet-free plasma supplemented with phospholipids was activated with identical amounts of tissue factor (TF) either homogeneously distributed (concentration 5 pM, homogeneous model) or immobilized on the surface (surface density 100 pmole/m 2 , spatially heterogeneous model). Fibrin clot growth and thrombin concentration dynamic in space were observed using video microscopy in plasma of healthy donors or patients with deficiencies in factors (F) II, FV, FVII, FVIII, FIX, FX, or FXI. In the spatially heterogeneous model, near-activator thrombin generation was decreased in FV-, FVII-, and FX-deficient plasma. In the homogeneous model, clotting was not registered in these samples. The simulation and experiment data showed that the coagulation threshold depended on the TF concentration. Our data indicate that the velocity of spatial clot propagation correlates linearly with the concentration of thrombin at the clot wave front but not with the overall thrombin wave amplitude. Spatial clot growth in normal plasma at early stages was neither reaction nor diffusion limited but became diffusion limited later. In contrast, clot growth was always diffusion limited in FV-, FVII-, and FX-deficient plasma and reaction limited in FVIII-, FIX-, and FXI-deficient plasma. We conclude that robustness of the spatially heterogeneous coagulation system was achieved because of the combination of 1) a local high TF surface density that overcomes activation thresholds, 2) diffusion control being shared between different active factors, and 3) an early saturated stimulus-response dependence of fibrin clot formation by thrombin., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Heparin therapy and prophylaxis may be accompanied by bleeding and thrombotic complications due to individual responses to treatment. Dosage control based on standard laboratory assays poorly reflects the effect of the therapy. The aim of our work was to compare the heparin sensitivity of new thrombodynamics (TD) assay with sensitivity of other standard and global coagulation tests available to date., Study Population and Methods: A total of 296 patients with high risk of venous thromboembolism (deep vein thrombosis (DVT), early postoperative period, hemoblastosis) were enrolled in the study. We used a case-crossover design to evaluate the sensitivity of new thrombodynamics assay (TD) to the hemostatic state before and after unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) therapy/prophylaxis and to compare it with the activated partial thromboplastin time (APTT), anti-Xa activity test, thrombin generation test (TGT) and thromboelastography (TEG). A receiver operating characteristic (ROC) curve analysis was used to evaluate changes before and after heparin prophylaxis and therapy. Blood was sampled before heparin injection, at the time of maximal blood heparin concentration and before the next injection., Results: Hypercoagulation before the start of heparin treatment was detected by TD, TGT and TEG but not by APTT. The area under the ROC curve (AUC) was maximal for TD and anti-Xa, intermediate for TGT and TEG and minimal for APTT., Conclusions: These results indicate that TD has a high sensitivity to the effects of UFH and LMWH after both prophylactic and therapeutic regimes and may be used for heparin monitoring., Competing Interests: We disclose a conflict of interest for Prof. Fazoil Ataullakhanov, who is a co-founder of HemaCore LLC, which holds several patents and patent applications that are related to the diagnostic use of spatial clot growth and which has developed the assay under the trade name of Thrombodynamics® (Ataullakhanov FI, international patent applications PCT/CH2007/000543 filing date 02.11.2007 and РСТ/RU2012/000570 filing date 16.07.2012). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The actual coagulation status may be reliably measured using only highly sensitive global functional tests; however, they are not numerous and all of them have disadvantages. Thrombodynamics (TD), a novel global coagulation test, is sensitive to hypo- and hypercoagulable states. The main properties of this test were investigated, and its capabilities for hemostasis analysis were verified through pharmacodynamic monitoring of the most widely used anticoagulants, heparins. The anticoagulant effects in the plasma of donors (n = 20) and patients after hip replacement (n = 20) spiked with unfractionated heparin or enoxaparin were measured in vitro to eliminate the influence of pharmacokinetic factors. Sensitivity for heparins was compared for activated partial thromboplastin time, thrombin generation tests and TD. TD was shown to reliably characterize the pharmacodynamics of any heparin in the entire range of its prophylactic and therapeutic concentrations. Inter-individual variability for the anticoagulant action of heparins was also calculated using the TD data. This variability did not differ between the investigated groups and did not exceed 12% and 20% for the stationary clot growth rate in the presence of unfractionated heparin and enoxaparin, respectively. That finding was in accordance with the values determined earlier using the thrombin generation test. The study results showed that TD has advantages over the other global methods of coagulation analysis. These advantages are good standardization, high reproducibility, independence of the parameter values from patient age and gender, and a narrower parameter distribution in a normal population. These results indicate that TD is a promising universal assessment method that improves the quality of hemostasis analysis because it more reliably detects deviations from the parameters' reference values., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Fibrinolysis is a cascade of proteolytic reactions occurring in blood and soft tissues, which functions to disintegrate fibrin clots when they are no more needed. In order to elucidate its regulation in space and time, fibrinolysis was investigated using an in vitro reaction-diffusion experimental model of blood clot formation and dissolution. Clotting was activated by a surface with immobilized tissue factor in a thin layer of recalcified blood plasma supplemented with tissue plasminogen activator (TPA), urokinase plasminogen activator or streptokinase. Formation and dissolution of fibrin clot was monitored by videomicroscopy. Computer systems biology model of clot formation and lysis was developed for data analysis and experimental planning. Fibrin clot front propagated in space from tissue factor, followed by a front of clot dissolution propagating from the same source. Velocity of lysis front propagation linearly depended on the velocity clotting front propagation (correlation r2 = 0.91). Computer model revealed that fibrin formation was indeed the rate-limiting step in the fibrinolysis front propagation. The phenomenon of two fronts which switched the state of blood plasma from liquid to solid and then back to liquid did not depend on the fibrinolysis activator. Interestingly, TPA at high concentrations began to increase lysis onset time and to decrease lysis propagation velocity, presumably due to plasminogen depletion. Spatially non-uniform lysis occurred simultaneously with clot formation and detached the clot from the procoagulant surface. These patterns of spatial fibrinolysis provide insights into its regulation and might explain clinical phenomena associated with thrombolytic therapy.

Artificial plasma expanders (PEs) are widely used in modern transfusion medicine. PEs do not contain components of the coagulation system, so their infusion in large volumes causes haemodilution and affects haemostasis. However, the existing information on this effect is contradictory. We studied the effect of the very process of plasma dilution on coagulation and tested the hypothesis that moderate dilution with a PE should accelerate clotting owing to a decrease in concentration of coagulation inhibitors. The standard clotting times, a thrombin generation test, and the spatial rate of clot growth (test of thrombodynamics) were used to assess donor plasma diluted in vitro with various PEs. The pH value and Ca +2 concentration were maintained strictly constant in all samples. The effect of thrombin inhibitors on dilution-induced hypercoagulation was also examined. It was shown that coagulation was enhanced in plasma diluted up to 2.0-2.5-fold with any PE. This enhancement was due to the dilution of coagulation inhibitors in plasma. Their addition to plasma or PE could partially prevent the hypercoagulation shift.

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD + ) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD + and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD + , and pyruvate metabolized ethanol 20-40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD + in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

Pregnancy is associated with a significant procoagulant shift in the hemostatic system balance as well as other metabolic changes. Pregnancy can thereby provoke manifestation of otherwise dormant disorders of hemostasis (e.g., thrombophilia), or even cause new, pregnancy-specific disorders (e.g., HELLP syndrome). Application and interpretation of laboratory assays of hemostasis in pregnancy is particularly challenging, because normal physiological ranges are no longer applicable, and because the most dangerous and complex changes are not detected by classic routine coagulation/platelet assays. New global assays of coagulation and of platelet-dependent hemostasis appear to be promising in this respect, but are still far from clinical practice and rarely appear in current patient management guidelines. These global assays require a high level of research to identify their relationship to clinically significant outcomes. Here, we review the state-of-the-art knowledge of the molecular changes in the hemostatic system in normal pregnancy and during pregnancy-related complications (preeclampsia, thrombotic microangiopathies, antiphospholipid syndrome, etc.). We also discuss the sensitivity of various classic and innovative assays to these pregnancy-associated changes, and describe current and potential future applications of these assays in meeting specific clinical needs., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

A new oral anticoagulant, dabigatran etexilate (DE, a prodrug of direct thrombin inhibitor (DTI) dabigatran), has been used clinically to prevent thrombosis. The assessment of dabigatran efficiency is necessary in some clinical cases, such as renal insufficiency, risk of bleeding, and drug interactions. However, a specific thrombin generation test (TGT) that is one of the most informative and sensitive to anticoagulant therapy (calibrated automated thrombinography (САТ)) shows a paradoxical increase of test parameters, such as endogenous thrombin potential (ETP) and peak thrombin, in patients receiving DE. The paradoxical behaviour of ETP and peak thrombin in these patients in the presence of DTIs is mostly caused by a decrease in the activity of thrombin in the α2-macroglobulin-thrombin complex that is used as a calibrator in CAT. For a correct estimation of the TGT parameters in patient's plasma containing DTIs we proposed to use our previously described alternative calibration method that is based on the measurement of the fluorescence signal of a well-known concentration of the reaction product (7-amino-4-methylcoumarin). In this study, the validity of such approach was demonstrated in an ex vivo study in patients with knee replacement and two special patients with multiple myeloma, who received DE for thrombosis prophylaxis.

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 myosin 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, Ca(2+), 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., (© 2016 by The American Society of Hematology.)

Tubulin polymers, microtubules, can switch abruptly from the assembly to shortening. These infrequent transitions, termed "catastrophes", affect numerous cellular processes but the underlying mechanisms are elusive. We approached this complex stochastic system using advanced coarse-grained molecular dynamics modeling of tubulin-tubulin interactions. Unlike in previous simplified models of dynamic microtubules, the catastrophes in this model arise owing to fluctuations in the composition and conformation of a growing microtubule tip, most notably in the number of protofilament curls. In our model, dynamic evolution of the stochastic microtubule tip configurations over a long timescale, known as the system's "aging", gives rise to the nonexponential distribution of microtubule lifetimes, consistent with experiment. We show that aging takes place in the absence of visible changes in the microtubule wall or tip, as this complex molecular-mechanical system evolves slowly and asymptotically toward the steady-state level of the catastrophe-promoting configurations. This new, to our knowledge, theoretical basis will assist detailed mechanistic investigations of the mechanisms of action of different microtubule-binding proteins and drugs, thereby enabling accurate control over the microtubule dynamics to treat various pathologies., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.

In this study, we considered a continuous model of platelet thrombus growth in an arteriole. A special model describing the adhesion of platelets in terms of their concentration was derived. The applications of the derived model are not restricted to only describing arterial platelet thrombus formation; the model can also be applied to other similar adhesion processes. The model reproduces an auto-wave solution in the one-dimensional case; in the two-dimensional case, in which the surrounding flow is taken into account, the typical torch-like thrombus is reproduced. The thrombus shape and the growth velocity are determined by the model parameters. We demonstrate that the model captures the main properties of the thrombus growth behavior and provides us a better understanding of which mechanisms are important in the mechanical nature of the arterial thrombus growth.

An important trend in the modern concept of blood coagulation is the growing agreement that, in order to understand regulation of coagulation in vivo and disorders of its function, it is essential to take into account its spatial heterogeneity, diffusion, and flow. In a way, this suggests that the idea of the "coagulation cascade" itself becomes increasingly misleading because there is no such place in an organism where reactions of this cascade really co-exist: activation, propagation and termination of coagulation are regulated by different subsets of chemical reactions that have different spatial localization and depend on cofactors expressed by different cell types in different tissues, so that only diffusion and flow can link these distinct "compartments" together into the one functional system. Here we review the last two decades of evidence obtained from in vitro, in vivo and computational systems biology approaches. When combined, the data comprise into an adequately comprehensive picture of the spatial regulation and organization of blood coagulation. In addition to the basic insights into the regulatory mechanisms, these approaches provided interesting results in the fields of coagulation diagnostics and other applications. Finally, the remaining unresolved and conflicting issues in the spatiotemporal regulation of coagulation are discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Blood platelets need to undergo activation to carry out their function of stopping bleeding. Different activation degrees lead to a stepped hierarchy of responses: ability to aggregate, granule release, and, in a fraction of platelets, phosphatidylserine (PS) exposure. This suggests the existence of decision-making mechanisms in the platelet intracellular signaling network. To identify and investigate them, we developed a computational model of PAR1-stimulated platelet signal transduction that included a minimal set of major players in the calcium signaling network. The model comprised three intracellular compartments: cytosol, dense tubular system (DTS) and mitochondria and extracellular space. Computer simulations showed that the stable resting state of platelets is maintained via a balance between calcium pumps and leaks through the DTS and plasma membranes. Stimulation of PAR1 induced oscillations in the cytosolic calcium concentrations, in good agreement with experimental observations. Further increase in the agonist level activated the mitochondrial uniporter leading to calcium uptake by mitochondria, which caused the collapse of mitochondrial membrane potential in a fraction of platelets leading to the PS exposure. The formation of this subpopulation was shown to be a stochastic process determined by the small number of activated PAR1 receptors and by heterogeneity in the number of ion pumps. These results demonstrate how a gradual increase of the activation degree can be converted into a stepped response hierarchy ultimately leading to formation of two distinct subpopulations from an initially homogeneous population.

Thrombosis is a deadly malfunctioning of the hemostatic system occurring in numerous conditions and states, from surgery and pregnancy to cancer, sepsis and infarction. Despite availability of antithrombotic agents and vast clinical experience justifying their use, thrombosis is still responsible for a lion's share of mortality and morbidity in the modern world. One of the key reasons behind this is notorious insensitivity of traditional coagulation assays to hypercoagulation and their inability to evaluate thrombotic risks; specific molecular markers are more successful but suffer from numerous disadvantages. A possible solution is proposed by use of global, or integral, assays that aim to mimic and reflect the major physiological aspects of hemostasis process in vitro. Here we review the existing evidence regarding the ability of both established and novel global assays (thrombin generation, thrombelastography, thrombodynamics, flow perfusion chambers) to evaluate thrombotic risk in specific disorders. The biochemical nature of this risk and its detectability by analysis of blood state in principle are also discussed. We conclude that existing global assays have a potential to be an important tool of hypercoagulation diagnostics. However, their lack of standardization currently impedes their application: different assays and different modifications of each assay vary in their sensitivity and specificity for each specific pathology. In addition, it remains to be seen how their sensitivity to hypercoagulation (even when they can reliably detect groups with different risk of thrombosis) can be used for clinical decisions: the risk difference between such groups is statistically significant, but not large.

Patients with multiple myeloma (MM) are at increased risk of venous thromboembolism. Therefore, adequate laboratory control of hemostasis and subsequent adjustments of anticoagulant therapy are necessary. We studied hemostasis changes using thromboelastography (TEG), thrombin generation test (TGT) and thrombodynamics (TD) in primary MM patients (PMMpt, n=25) and patients in remission (RMMpt, n=34) during blood stem cell (BSC) mobilization. TD and TEG reveal hypercoagulability in PMMpt (*p<0.05) in relation to healthy volunteers. There was no difference in any of the tests between PMMpt and RMMpt. We detected no heparin effect in 22% of patients one day after the onset of the prophylactic heparin treatment (500 IU/h) during BSC mobilization; tests shifted toward the hypercoagulability in 75% of patients one day after cyclophosphamide (4 g/m2) chemotherapy. Global hemostasis tests were in good agreement with each other, revealed hypercoagulability and heparin "resistance" in patients with MM and may be useful for therapy individualization.

In consequence of the key role of factor Xa in the clotting cascade and absence of its activity in the processes that do not affect coagulation, this protein is an attractive target for development of new blood coagulation inhibitors. Factor Xa is more effective and convenient target for creation of anticoagulants than thrombin, inhibition of which may cause some side effects. This study is aimed at finding new inhibitors of factor Xa by molecular computer modeling including docking SOL and postdocking optimization DISCORE programs. After validation of molecular modeling methods on well-known factor Xa inhibitors the virtual screening of NCI Diversity and Voronezh State University databases of ready-made low molecular weight species has been carried out. Seventeen compounds selected on the basis of modeling results have been tested experimentally in vitro. It has been found that 12 of them showed activity against factor Xa (IC50 = 1.8-40 μM). Based on analysis of the results, the new original compound was synthesized and experimentally verified. It shows activity against factor Xa with IC50 value of 0.7 μM.

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Standardization of pre-analytical conditions is the obligatory step for all potential diagnostic tests. Spatial clot growth (Thrombodynamics) is a new global hemostasis assay that considers spatial organization of coagulation. The principal parameter is rate of fibrin clot growth from the tissue-factor coated surface. In this work we studied the pre-analytical variables of Thrombodynamics assay that include conditions of blood collection, sample preparation and storage., Materials and Methods: Blood of apparently healthy volunteers was used. Eight types of citrate blood collection tubes were tested, centrifugation conditions for plasma preparation were evaluated and impact of plasma freezing/thawing was tested., Results: Among the blood collection tubes tested, BD Vacutainer glass tubes showed a significantly higher clot growth rate compared to plastic tubes. There was no difference between 3.2% and 3.8% of sodium citrate. For plasma preparation, a single 15 min centrifugation at 1,600 g shows significantly increased clot growth rate compared to plasma obtained by two sequential centrifugations (15 min 1 600 g, 5min 10,000 g). There was no significant difference between 1,600 g and 2,100 g if the second centrifugation was performed. For the second centrifugation there was no difference between 20 min at 1 600 g and 5 min at 10,000g. Frozen-thawed plasma showed increased clot growth rate compared to fresh plasma., Conclusion: The data represent the necessary steps for the standardization of Thrombodynamics assay and for the formulation of the operating guide., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Sensitive methods for assessment of the hemostatic state are essential for providing adequate therapy to patients with β-thalassemia. The present study was designed to monitor the changes in the hemostatic state of a patient with β-thalassemia at the primary stage and under heparin treatment following splenectomy. The hemostatic state of the patient was assessed using conventional tests (activated partial thromboplastin time, prothrombin index, thrombin time), fibrinogen and D-dimer assays, thromboelastography (TEG), thrombin generation test, and a novel thrombodynamics clot growth assay. Thrombodynamics parameters indicated the hypercoagulation state on the primary evaluation which progressed after splenectomy: stationary clot growth velocity increased from 32 to 38 μm/min (normal range 20-30 μm/min). Hypercoagulation state was confirmed by Doppler echocardiography, which detected portal vein thrombosis on day 23 after surgery. The results of the other tests' parameters were in the normal ranges before splenectomy. The TEG parameters were sensitive to low molecular weight heparin (LMWH) injections; but the values were close to the normal ranges before and after injections. The thrombodynamics assay demonstrated a high sensitivity to LMWH injections, and registered a decrease of the hypercoagulability in the course of therapy (P < 0.05). TGT was not performed during LMWH therapy. This clinical case demonstrates the potential of the thrombodynamics assay to serve as a sensitive method for coagulation system monitoring and prediction of prothrombotic tendencies in patients with hemolytic anemias.