Sodium Nitroprusside-Induced Activation of Vascular Smooth Muscle BK Channels Is Mediated by PKG Rather Than by a Direct Interaction with NO.

Nitric oxide (NO) is a powerful vasodilator in different vascular beds and NO-donors are widely used in clinical practice. Early data suggested that NO and NO-donors activate vascular smooth muscle high-conductance, calcium-activated potassium channels (BK channels). There exist two hypotheses explaining the effect of NO and NO-donors on BK channels-one stating that protein kinase G (PKG) mediates the effect of NO, and the other one stating that NO acts directly on the channel. Thus, the degree of contribution of PKG to the NO-induced activation of the BK channel is still not completely clear. This study tested the hypothesis that the sodium nitroprusside (SNP)-induced activation of vascular smooth muscle BK channels is fully mediated by PKG. This hypothesis was investigated using the patch-clamp technique and freshly isolated smooth muscle cells from rat tail artery. In whole-cell experiments, SNP considerably increased the outward current compared with the addition of the bath solution. SNP did not alter the current in the presence of iberiotoxin, the specific blocker of BK channels, during co-application with hydroxocobalamin, an NO-scavenger, and in the presence of Rp-8-Br-PET-cGMPS, the specific PKG-inhibitor. In inside-out patches, the activity of BK channels was increased by SNP, SNAP, and DEA-NO. However, these effects did not differ from the effect of the application of drug-free bath solution. Furthermore, a similar increase in single BK channel activity was induced by Rp-8-Br-PET-cGMPS, Rp-8-Br-PET-cGMPS together with SNP, hydroxocobalamin, and hydroxocobalamin together with SNP or DEA-NO. Finally, the activity of excised BK channels did not change in the absence of any application but was considerably increased by PKG compared with the addition of drug-free bath solution. These results suggest that NO released from NO-donors stimulates the BK current only through activation of PKG.

Kaolin, used to trigger coagulation in thrombin generation test, increases sensitivity of the method in hemophilia patients.

: Thrombin generation test (TGT) is well established tool to research blood coagulation in plasma of hemophilia patients. Traditionally coagulation in this test is triggered by a tissue factor (TF), an extrinsic coagulation pathway activator. However, it is known that disorders of the intrinsic pathway are most important for coagulation in hemophilia. In this study, we hypothesized that triggering coagulation via the intrinsic pathway could increase a sensitivity of the TGT to monitor hemophilia treatment. The aim of this study was to compare thrombin generation in hemophilia A patients with inhibitors to factor VIII before and after infusion of bypassing agent [recombinant-activated factor VIIa (rVIIa)] using standard activation of coagulation by TF or by kaolin, an activator of coagulation by intrinsic pathway. Endogenous thrombin potential (ETP) in nine patients was measured. ETP before (ETP0) and 60 min after rVIIa infusion (ETP60) were compared. It was shown that ETP0 and ETP60 were significantly different when using any coagulation activator (paired Student's t test, P = 0.017 and 3.7 × 10 for clotting activation by TF and kaolin, respectively). The ratios of ETP60/ETP0 were 1.2 ±â€Š0.2 or 30.0 ±â€Š22.4 (mean ±â€ŠSD, n = 9) for coagulation activated by TF or kaolin, respectively, and were significantly different (paired Student's t test, P < 0.005). The TGT clearly distinguished between ETP0 and ETP60 in the case of any coagulation activator, but ETP increasing after rVIIa infusion was significantly higher when activated with kaolin. This provided increased sensitivity of this method for monitoring hemophilia therapy.

The modification of the thrombin generation test for the clinical assessment of dabigatran etexilate efficiency.

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.

Application of Molecular Modeling to Development of New Factor Xa Inhibitors.

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.

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.

Thrombin inhibitors based on single-stranded DNA aptamers.

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.

New synthetic thrombin inhibitors: molecular design and experimental verification.

BACKGROUND: The development of new anticoagulants is an important goal for the improvement of thromboses treatments. OBJECTIVES: The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. METHODS: Computer-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. RESULTS: New compounds that are both effective direct thrombin inhibitors (the best K(I) was <1 nM) and strong anticoagulants in plasma (an IC(50) 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. LD(50) 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. CONCLUSIONS: The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.