Alternative Method for Evaluating Mitochondrial Permeability Transition using Platelet Activation

Abstract

Drug-induced liver injury (DILI) is a major cause of drug attrition and market withdrawal. Compounds associated with DILI typically exhibit some degree of mitochondrial liability, as empirically observed in multiple previous studies. Among the various mitochondrial toxicities, mitochondrial permeability transition (MPT) has been implicated in severe DILI. Conventional MPT assessments rely on swelling assays using mitochondria isolated from fresh animal tissues, which limits their applicability to human-based systems and high-throughput screening. To address this, we developed and validated a platelet-based approach to detect MPT by leveraging their known responsiveness to CypD-dependent mitochondrial perturbation. We evaluated 22 compounds using both a conventional mitochondrial swelling assay with mouse liver mitochondria and platelet-based activation assay. A correlation was observed between the two methods (r = 0.848), suggesting general concordance. Furthermore, we applied this assay to human platelets obtained from transfusion-ineligible platelet products and observed MPT-dependent activation by compounds such as diclofenac. However, lot-to-lot variability highlighted the challenges in terms of reproducibility. These findings suggest that platelets can serve as mechanistically relevant and accessible tools for detecting MPT induction and offer a potential alternative to animal-derived mitochondrial assays. This approach may contribute to the early identification of mitochondrial liabilities during drug development.