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

Theoretical Analysis of the Built-in Metabolic Pathway Effect on the Metabolism of Erythrocyte-Bioreactors That Neutralize Ammonium

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

Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study

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

Antiplatelet Agents Can Promote Two-Peaked Thrombin Generation in Platelet Rich Plasma: Mechanism and Possible Applications

<div><h3>Background</h3><p>Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks.</p> <h3>Objective</h3><p>We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve.</p> <h3>Methods</h3><p>Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers.</p> <h3>Results</h3><p>The addition of the P₂Y₁₂ receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E₁ (PGE₁), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE₁ or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE₁ mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one.</p> <h3>Conclusions</h3><p>Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types - plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.</p> </div

Contribution of phospholipid surfaces to the first and the second thrombin generation peaks.

<p>Plasma phospholipids, α-granules and preactivated platelets form the first peak. PS expression on the platelets' surface can mediate the second one formation.</p

Analysis of thrombin generation peak parameters.

<p>For approximation of TGCs with one peak, a type I Gumbel's distribution function was used (A). The TGCs with two peaks were described by superposition of two extreme distribution Gumbel's functions (B). In some cases, it was impossible to determine how many peaks the TGC contained (C). Fitting was performed until the thrombin concentration began to be comparable with the experimental error (see Fig. S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055688#pone.0055688.s001" target="_blank">File S1</a>).</p

Kinetics of platelet activation in the presence of PGE₁, DMSO, and MeS-AMP.

<p>A suspension of gel-filtered platelets (100·10³ cells per µl) labeled with annexin V-RPE and anti-CD62P-FITC was activated with human α-thrombin solution (35 nM final concentration) containing PGE₁ (A,B), DMSO (C,D), or MeS-AMP (E,F). The mean RPE (A,C,E) fluorescence and the percentage of CD62P-positive platelets (B,D,F) after different incubation times is presented.</p

Influence of PGE₁ on TG in PRP.

<p>A: TGCs in PRP supplemented with 1.6% DMSO in the presence of different PGE₁ concentrations. PRP without PGE₁ was used as a control. B, C: The mean values of areas (B) and amplitudes (C) of the first peak in PRP, PRP with 830 nM PGE₁ addition, and PPP. Coagulation was activated with 2 pM of TF. The platelet concentration was 100·10³ platelets per µl. Mean values and SD are presented; n = 18, 8, and 7 for PRP, PRP+PGE₁, and PPP, correspondingly. Student's t-test with P values equal to 0.05 and 0.01 was used to obtain statistics. The difference is not significant in all the bars where P-value is not presented.</p

Criteria for determination of the quantity of TGC peaks^*.

*<p>The first row displays the ranges of coefficients of correlation between the TGC and the two- or one-peak fitting (R₂ and R₁, respectively) and the ranges of the R₂/R₁ ratio for 19 TGCs known to contain one peak. The second row shows the same parameters for 30 curves known to contain two peaks.</p