Determination of fibrin clot growth and spatial thrombin propagation in the presence of different types of phospholipid surfaces.

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.

Impaired platelet activity and hypercoagulation in healthy term and moderately preterm newborns during the early neonatal period.

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.

Sensitivity and Robustness of Spatially Dependent Thrombin Generation and Fibrin Clot Propagation.

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/m2, 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.

The laboratory control of anticoagulant thromboprophylaxis during the early postpartum period after cesarean delivery.

INTRODUCTION: The incidence of venous thromboembolism (VTE) after cesarean section is up to 0.6%, and the widespread use of cesarean section draws attention to this group. The dosage and duration of low-molecular-weight heparin (LMWH) prophylaxis after delivery is estimated by anamnestic risk-scales; however, the predictive potency for an individual patient's risk can be low. Laboratory hemostasis assays are expected to solve this problem. The aim of this study was to estimate the potency of tests to reflect the coagulation state of patients receiving LMWH in the early postpartum period. MATERIALS AND METHODS: We conducted an observational study on 97 women undergoing cesarean section. Standard coagulation tests (Fg, APTT, prothrombin, D-dimer), an anti-Xa assay, rotation thromboelastometry and thrombodynamics/thrombodynamics-4D were performed. Coagulation assay parameters were compared in groups formed in the presence or absence of LMWH to estimate the laboratory assays' sensitivity to anticoagulation. RESULTS: Coagulation assays revealed hypercoagulation after delivery and a tendency toward normalization of coagulation during early postpartum. The thromboprophylaxis results revealed a higher percentage of coagulation parameters within the normal range in the LMWH group. CONCLUSION: This research is potentially beneficial for the application of thrombodynamics and thrombodynamics-4D in monitoring coagulation among patients with high VTE risk who receive thromboprophylaxis with heparin.

Classic and Global Hemostasis Testing in Pregnancy and during Pregnancy Complications.

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.

Platelets provide robustness of spatial blood coagulation to the variation of initial conditions.

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 × 105/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.

Hypercoagulation detected by routine and global laboratory hemostasis assays in patients with infective endocarditis.

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.

Task-oriented modular decomposition of biological networks: trigger mechanism in blood coagulation.

Analysis of complex time-dependent biological networks is an important challenge in the current postgenomic era. We propose a middle-out approach for decomposition and analysis of complex time-dependent biological networks based on: 1), creation of a detailed mechanism-driven mathematical model of the network; 2), network response decomposition into several physiologically relevant subtasks; and 3), subsequent decomposition of the model, with the help of task-oriented necessity and sensitivity analysis into several modules that each control a single specific subtask, which is followed by further simplification employing temporal hierarchy reduction. The technique is tested and illustrated by studying blood coagulation. Five subtasks (threshold, triggering, control by blood flow velocity, spatial propagation, and localization), together with responsible modules, can be identified for the coagulation network. We show that the task of coagulation triggering is completely regulated by a two-step pathway containing a single positive feedback of factor V activation by thrombin. These theoretical predictions are experimentally confirmed by studies of fibrin generation in normal, factor V-, and factor VIII-deficient plasmas. The function of the factor V-dependent feedback is to minimize temporal and parametrical intervals of fibrin clot instability. We speculate that this pathway serves to lessen possibility of fibrin clot disruption by flow and subsequent thromboembolism.

Thrombodynamics-A new global hemostasis assay for heparin monitoring in patients under the anticoagulant treatment.

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.

Thromboelastography, thrombin generation test and thrombodynamics reveal hypercoagulability in patients with multiple myeloma.

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.

Drug-drug interaction of the anti-TFPI aptamer BAX499 and factor VIII: studies of spatial dynamics of fibrin clot formation in hemophilia A.

BACKGROUND: In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. OBJECTIVE: To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro. METHODS: Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%). CONCLUSIONS: The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.

Laboratory tests for coagulation system monitoring in a patient with ß-thalassemia.

Sensitive methods for assessment of the hemostatic state are essential for providing adequate therapy to patients with ß-thalassemia. The present study was designed to monitor the changes in the hemostatic state of a patient with ß-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.

Investigation of the phenotype heterogeneity in severe hemophilia A using thromboelastography, thrombin generation, and thrombodynamics.

BACKGROUND: Hemophilia A (HA) patients with similar factor VIII levels can demonstrate varying bleeding tendencies. In particular, 10-15% of all severe HA patients (FVIII:C<1IUdL(-1)) do not require regular replacement therapy. Modern global coagulation assays can help to detect and study this "mild" bleeding phenotype. Here, we investigated the coagulation status of different bleeding phenotypes using various types of global coagulation assays. MATERIALS AND METHODS: Ten HA patients with severe phenotype and eleven patients with mild phenotypes were included in the study. For each patient, thromboelastography (TE), thrombodynamics (TD), and kaolin- or tissue factor-induced thrombin generation (TG) were measured. TG in platelet-rich plasma (PRP) was investigated using our original modification when the thrombin generation curve showed two peaks, previously shown to depend on platelet activity. We also utilized TG and TD with the addition of thrombomodulin. RESULTS: The second peak amplitude and ETP of PRP TG were the only parameters that were significantly higher in mild bleeders (peak 41.6 ± 3.5 nM, ETP 1966 ± 169 nM*min) than in patients with severe bleeding (peak 28.3 ± 3.3 nM, ETP 1359 ± 130 nM*min). CONCLUSIONS: Our results suggest that severe and mild HA phenotypes could be distiguished by TG assay in PRP suggesting that difference in platelet activity can be involved in the phenotype formation. According to our previous results we can suppose that the mechanism of the phenotypic heterogeneity is linked with TG mediated by PS-expressing platelets.

Predicting prothrombotic tendencies in sepsis using spatial clot growth dynamics.

Inflammation in sepsis is associated with hypercoagulation that may lead to thrombosis and disseminated intravascular coagulation. Conventional diagnostic assays are poorly sensitive to procoagulant changes in sepsis. Objectives of the article is to study changes in hemostatic state of septic patients using spatial clot growth assay (currently being developed under the trademark of thrombodynamics) and to compare the sensitivity of this method with the sensitivity of conventional methods. Sixteen patients with hematological malignancies and sepsis were enrolled in the study. All patients had been surveyed for a month following the infection onset. Spatial clot growth assay monitors fibrin clot development in a nonstirred thin layer of platelet-free plasma activated by immobilized tissue factor. Clotting time tests, thromboelastography, D-dimer assays were also performed. Spatial clot growth revealed hypercoagulation in six patients. D-dimer levels increase (with vein thrombosis in one case) was subsequently observed in five of them. D-dimer levels did not increase when spatial clot growth was normal. At the next time point, after spatial clot growth assay showed hypercoagulation, the mean D-dimer concentration was significantly higher than after a normal analysis (457 versus 234 µg/l; P < 0.05); there was no such correlation for other assays. The remaining 10 patients had elevated D-dimer levels on the first day; this either decreased gradually or remained elevated. Spatial clot growth showed normalization in survivors and growing hypocoagulation in nonsurvivors. Measuring spatial clot growth dynamics has potential diagnostic utility for the evaluation of thrombotic risk.