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

Endothelial- and Platelet-Derived Microparticles Are Generated During Liver Resection in Humans

BACKGROUND Cell-derived plasma microparticles (<1.5 μm) originating from various cell types have the potential to regulate thrombogenesis and inflammatory responses. The aim of this study was to test the hypothesis that microparticles generated during hepatic surgery co-regulate postoperative procoagulant and proinflammatory events. METHODS In 30 patients undergoing liver resection, plasma microparticles were isolated, quantitated, and characterized as endothelial (CD31+, CD41-), platelet (CD41+), or leukocyte (CD11b+) origin by flow cytometry and their procoagulant and proinflammatory activity was measured by immunoassays. RESULTS During liver resection, the total numbers of microparticles increased with significantly more Annexin V-positive, endothelial and platelet-derived microparticles following extended hepatectomy compared to standard and minor liver resections. After liver resection, microparticle tissue factor and procoagulant activity increased along with overall coagulation as assessed by thrombelastography. Levels of leukocyte-derived microparticles specifically increased in patients with systemic inflammation as assessed by C-reactive protein but are independent of the extent of liver resection. CONCLUSIONS Endothelial and platelet-derived microparticles are specifically elevated during liver resection, accompanied by increased procoagulant activity. Leukocyte-derived microparticles are a potential marker for systemic inflammation. Plasma microparticles may represent a specific response to surgical stress and may be an important mediator of postoperative coagulation and inflammation

Microparticles from apoptotic platelets promote resident macrophage differentiation

Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PMap). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PMap formation. Flow cytometry and scanning electron microscopy demonstrated that PMap display activated integrins and interact to form microparticle aggregates. PMap were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PMap promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin αMβ2), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PMap polarized the cells into resident M2 monocytes. Cells treated with PMap actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PMap stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PMap with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH2. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH2, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a Ki of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes

Molecular interactions at the surface of extracellular vesicles

Extracellular vesicles such as exosomes, microvesicles, apoptotic bodies, and large oncosomes have been shown to participate in a wide variety of biological processes and are currently under intense investigation in many different fields of biomedicine. One of the key features of extracellular vesicles is that they have relatively large surface compared to their volume. Some extracellular vesicle surface molecules are shared with those of the plasma membrane of the releasing cell, while other molecules are characteristic for extracellular vesicular surfaces. Besides proteins, lipids, glycans, and nucleic acids are also players of extracellular vesicle surface interactions. Being secreted and present in high number in biological samples, collectively extracellular vesicles represent a uniquely large interactive surface area which can establish contacts both with cells and with molecules in the extracellular microenvironment. Here, we provide a brief overview of known components of the extracellular vesicle surface interactome and highlight some already established roles of the extracellular vesicle surface interactions in different biological processes in health and disease

International seminar on the red blood cells as vehicles for drugs.

The first human transfusion was performed by the pioneer Dr Jean-Baptiste Denis in France in 1667 and now, three centuries later, around 50 millions blood units are transfused every year, saving millions of lives. Today, there is a new application for red blood cells (RBCs) in cellular therapy: the effective use of erythrocytes as vehicles for chemical or biological drugs. Using this approach, the therapeutic index of RBC-entrapped molecules can be significantly improved with increased efficacy and reduced side effects. This cell-based medicinal product can be manufactured at an industrial scale and is now used in the clinic for different therapeutic applications. A seminar dedicated to this field of research, debating on this inventive formulation for drugs, was held in Lyon (France) on 28 January 2011. Drs KC Gunter and Y Godfrin co-chaired the meeting and international experts working on the encapsulation of drugs within erythrocytes met to exchange knowledge on the topic ‘The Red Blood Cells as Vehicles for Drugs’. The meeting was composed of oral presentations providing the latest knowledge and experience on the preclinical and clinical applications of this technology. This Meeting Highlights article presents the most relevant messages given by the speakers and is a joint effort by international experts who share an interest in studying erythrocyte as a drug delivery vehicle. The aim is to provide an overview of the applications, particularly for clinical use, of this innovative formulation. Indeed, due to the intrinsic properties of erythrocytes, their use as a drug carrier is one of the most promising drug delivery systems investigated in recent decades. Of the different methods developed to encapsulate therapeutic agents into RBCs [1,2,] the most widely used method is the lysis of the RBCs under tightly controlled hypotonic conditions in the presence of the drug to be encapsulated, followed by resealing and annealing under normotonic conditions (Figure 1). This results in uniform encapsulation of the material into the cells and a final product with good stability, reproducibility and viability. This process, which has now been developed to an industrial scale, is the technique chosen by the majority of the experts presenting their work in this seminar (by R Franco)