BackgroundThe development of new anticoagulants is an important goal for the improvement of thromboses treatments.ObjectivesThe design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration.MethodsComputer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured.ResultsNew compounds that are both effective direct thrombin inhibitors (the best KI was <1 nM) and strong anticoagulants in plasma (an IC50 in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD50 values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C.ConclusionsThe high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

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.

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.

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.

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Investigation of inhibitory effect of two single-stranded DNA thrombin-inhibiting aptamers (15TBA and 31TBA) on fibrin polymerization in fibrinogen solutions and comparison of anticoagulant properties of these aptamers by a new global coagulation test of thrombodynamics. Measurement of aptamers' functional stability in human plasma and blood in vitro in order to investigate the involvement of 3'-exonuclease in fast decrease of aptamers' functional activity in vivo. Thrombin inhibition activity was measured in a buffer system in vitro as effects of aptamers on fibrin polymerization. Anticoagulant activity was investigated by measuring the spatial clot growth rate in the presence of aptamers. The stability of aptamers during incubation in human plasma was investigated in vitro by measuring activated partial thromboplastin time. Both aptamers dose-dependently inhibit fibrin polymerization in a buffer solution (IC50=10 nm for 15TBA and 3 nm for 31TBA) and are effective anticoagulants in human plasma (IC50 for spatial clot growth rate decreasing are 9.5 μmol/l and 4.0 μmol/l for 15TBA and 31TBA, correspondingly). Both aptamers remain stable in plasma or whole blood in vitro for at least 4 h. It was shown that 31TBA was 2-3 times more effective than 15TBA. Both aptamers were stable in human plasma and whole blood in vitro. So, the 3'-exonuclease could not be the reason for fast decrease of aptamers' functional activity in vivo. The main role in the removal of oligonucleotides from the circulation is played obviously by the liver.