Validation of the calibrated thrombin generation test (cTGT) as the reference assay to evaluate the procoagulant activity of nanomaterials

Abstract

We validated a preclinical toxicological screening assay and provided guidelines to evaluate the potential impact of nanoparticles (NPs) on blood coagulation. Five NPs with various physicochemical properties were studied using several existing methods of clotting times and thrombin generation assays in human normal pool plasma. In both recalcification clotting time (RCT) and calibrated thrombin generation test (cTGT), the NPs exhibited procoagulant activity (SiO₂ ≥ SiC ≥ TiC > CuO > CB) but cTGT was more sensitive and relevant than RCT. Thus, the cTGT appears as a reference assay to investigate the nanoparticle (NP) procoagulant activity in human plasma. It should be used as the reference toxicity test for evaluating the effects of nanomaterials on coagulation cascade. In addition, we also showed that the use of the Pluronic F-108 dispersant and/or the sonication for the NP suspension preparation may mask their procoagulant activity and thus should be avoided.

Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines

“…Activation and cleavage of mouse plasma kallikrein by superparamagnetic iron oxide nanoparticles in vitro was shown to be related to kallikrein activation in vivo [90]. A study of five nanomaterials (carbon black, silicon dioxide, silicon carbide, titanium carbide and copper oxide) for which various degrees of thrombogenicity had been demonstrated in vivo by previous studies revealed corresponding procoagulant activity in vitro in APTT and calibrated thrombin generation assays [91]. This study also concluded that although both assays detected procoagulant activity of the tested nanomaterials, the calibrated thrombin generation assay appeared to be more sensitive [91].…”

The euglobulin clot lysis time to assess the impact of nanoparticles on fibrinolysis

“…Consequently, these NPs did not prevent the formation of a solid fibrin scaffold as observed with the control. As demonstrated in previous studies, SiO 2 and Ag NPs are also procoagulants (Laloy et al 2012(Laloy et al , 2014a. One potential hypothesis of the underlying mechanism is that the charges of SiO 2 and Ag NPs induce a catalytic surface for coagulation and fibrinolytic factors, disturbing the hemostatic balance.…”

“…A spectrophotometric (890 nm) microtiter plate reader measures the optical density changes in euglobulin clot of plasma over time. It is known that NPs interfere with methods using light absorbance detection (Laloy et al 2012). Although ECLT is based on an optical density, there is no light interference with this assay.…”

“…The physicochemical characterization of the nanopowders was previously reported (Laloy et al 2012). The average diameter measured by FEG-SEM/ TEM, the specific surface area by BET, the bulk composition by EDX, and the surface composition by XPS are presented in Table 1.…”

“…Some studies report the hemocompatibility of NPs, in particular on red blood cells, platelets, and coagulation process (Dobrovolskaia et al 2008b;Krajewski et al 2013;Laloy et al 2012Laloy et al , 2014aMocan 2013). More specifically, our team assessed and validated some specific techniques for the study of NPs on red blood cell hemolysis, primary hemostasis (platelet adhesion, activation, and aggregation), and secondary hemostasis (blood coagulation) (Laloy et al 2012(Laloy et al , 2014a. No data are currently available on the impact of NPs on fibrinolysis.…”