No abstract

Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan(®) in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 10⁶ and 1 × 10⁶/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 μg/ml). Without fondaparinux, 0.2 × 10⁶ ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC(50) were 0.69 ± 0.03 μg/ml for ac-M, 0.20 ± 0.03 μg/ml for MMPs, and 0.22 ± 0.02 μg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 μg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.

No abstract

Anticoagulants, including unfractionated heparin (UFH), enoxaparin and fondaparinux, are approved drugs in acute coronary syndrome (ACS). Monocytes and monocyte-derived microparticles (MMPs) play an important procoagulant role in ACS by expressing high tissue factor (TF) levels, which in turn triggers thrombin generation. The objective of our study is to compare the in-vitro inhibitory effect of UFH, enoxaparin and fondaparinux in monocytes and MMP models. Human-elutriated monocytes were activated for 5 and 18 h by lipopolysaccharide to obtain activated monocytes (ac-M) or MMPs, respectively. Thrombin generation inhibition was assessed using ac-M or MMPs mixed with platelet-poor plasma containing increased concentrations of anticoagulants. Thrombin generation inhibition was dose-dependent with a differential effect according to the drug: the highest for UFH, the lowest for fondaparinux. Rate index was the most sensitive parameter. For fondaparinux, its IC50 values (anti-Xa IU/ml) were 0.59±0.05 for ac-M and 0.17±0.03 for MMPs. For enoxaparin, rate index IC50 values were 0.27±0.03 for ac-M and 0.19±0.02 for MMPs. Our data support the notion that cell-induced thrombin generation assay may be a reliable alternative to anti-Xa assessment in determining patient anticoagulation level.

The use of chromogen substrates has permitted a new approach of the factors responsible for the regulation and equilibrium of hemostasis.Automation has allowed to have access to the problems raised by enzymatic reactions in non-purified medium. The specificity and sensibility of substrates, the specificity of activators, the role of inhibitors and physiologic substrates in different technical conditions (pH, ionic forces, temperature, dilution) modifying the maximal velocity, have been studied for a standardizing approach of enzymatic kinetics in hemostasis. Measurements of antithrombin III and fast α2 antiplasmin, factor X, prekallikrein and plasminogen and heparinemies have been analyzed by an automatic apparatus (Kern O Mat 2-Coultronics) with different chromogen substrates (Kabi-Stago).Optimum technical conditions, units in which results are expressed, interpretation of normal and pathological values are systematically studied.