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Thrombocytopenia is one of the most commonly observed drug-induced adverse hematologic toxicities in the clinic. Therefore, in vitro and in vivo evaluations of effect of drugs on platelets are an important component in preclinical safety assessment in drug development. To date, a number of mechanisms have been identified to be associated with drug-mediated thrombocytopenia. Amongst these, some are conserved across species whereas others are not. Therefore, a case-by-case approach is needed to assess drug-induced thrombocytopenia at preclinical stages to understand translatability to humans. The present chapter reviews mechanisms in drug-mediated thrombocytopenia with a focus on nonimmune (direct myelotoxicity) as well as immune-mediated thrombocytopenia by both small and large molecule therapeutics. Several in vitro and in vivo models as well as challenges in assessing drug-mediated thrombocytopenia in preclinical stages will also be discussed.

One of the objectives of studying endothelial cells in vitro is to evaluate neutrophil-endothelial cell interactions including potential consequences of oxidant-mediated damage to the endothelial cell. Current under-standing of endothelial cell oxidative function is derived primarily from the measurement of extracellular products. We utilized 2 dyes, 2′,7′-dichlorofluorescin diacetate (DGFH-DA) and hydroethidine (HE), which measure hydrogen peroxide (H2O2) and superoxide anion (O2-) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH-DA and stimulated with H2O2 exhibited an increase in the fluorescent product 2′,7′-dichlorofluorescein (DCF) (measure of intracellular H2O2) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H2O2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O2-) which lasted for approximately 60 min. Superoxide dismutase increased DCF fluorescence in endothelial cells stimulated with H2O2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduced DCF and EB fluorescence by 48% and 37% respectively in endothelial cells stimulated with H2O2. Catalase completely inhibited an increase in DCF or EB fluorescence in endothelial cells stimulated with H2O2. There was a direct correlation between mean DCF and EB fluorescence intensity and the concentration of H2O2 or the number of phorbol 12-myristate 13-acetate-activated neutrophils added to endothelial cells. We conclude from these studies that DCFH-DA and HE can be used to measure intracellular H2O2 and O2- in endothelial cells and that the xanthine oxidase pathway for intracellular O2- production accounts for approximately 40% of the total intracellular O2- generated in endothelial cells after stimulation with H2O2. The combination of image cytometry and flow cytometry will be important for future evaluations of endothelial cell function. J. Leukoc. Biol. 55: 253–258; 1994.