Anna N. Balandina

Improvement of spatial fibrin formation by the anti-TFPI aptamer BAX499: changing clot size by targeting extrinsic pathway initiation

Summary.  Background: Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro‐ and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high‐affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. Objective: To examine the BAX499 effect on clotting in a spatial, reaction‐diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). Methods: Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. Results: BAX499 dose‐dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m−2 by shortening lag time and increasing clot size by up to ∼2‐fold. The effect was TFPI specific as confirmed by experiments in TFPI‐depleted plasma with or without TFPI supplementation. Clotting improvement was half‐maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m−2, both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. Conclusions: BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.

Thrombin activity propagates in space during blood coagulation as an excitation wave.

Injury-induced bleeding is stopped by a hemostatic plug formation that is controlled by a complex nonlinear and spatially heterogeneous biochemical network of proteolytic enzymes called blood coagulation. We studied spatial dynamics of thrombin, the central enzyme of this network, by developing a fluorogenic substrate-based method for time- and space-resolved imaging of thrombin enzymatic activity. Clotting stimulation by immobilized tissue factor induced localized thrombin activity impulse that propagated in space and possessed all characteristic traits of a traveling excitation wave: constant spatial velocity, constant amplitude, and insensitivity to the initial stimulation once it exceeded activation threshold. The parameters of this traveling wave were controlled by the availability of phospholipids or platelets, and the wave did not form in plasmas from hemophilia A or C patients who lack factors VIII and XI, which are mediators of the two principal positive feedbacks of coagulation. Stimulation of the negative feedback of the protein C pathway with thrombomodulin produced nonstationary patterns of wave formation followed by deceleration and annihilation. This indicates that blood can function as an excitable medium that conducts traveling waves of coagulation.

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 &mgr;g/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.

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.

Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates in vitro blood coagulation similarly to thromboplastin on fibroblasts

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.

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.

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

Abstract 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.

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

BackgroundPreterm 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.MethodsCoagulation was assessed using an integral assay of thrombodynamics and standard coagulation assays, and platelet functional activity was estimated by flow cytometry.ResultsHypercoagulation 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.ConclusionsThe 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.

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.