Simultaneous Assessment of Calcium Signaling and Mitochondria Activity in Human Platelets Using Continuous Flow Cytometry

ABSTRACT Increasing evidence suggests connection between mitochondrial dysfunction and platelet‐related disorders, necessitating developing of a high throughput method for assessing the functioning of mitochondria in platelets. Here we develop an approach to monitor the membrane potential of mitochondria simultaneously with intracellular calcium signaling in platelets using continuous flow cytometry. Platelets were loaded with Fura Red and DiOC 6 (3), which did not affect intracellular calcium signaling and the realization of platelet functional responses. The values were normalized using the negative controls with CCCP in order to make the signal independent of the platelet size and the number of mitochondria. Stimulation with ADP and TRAP‐6 produced transient mitochondrial hyperpolarization, whose amplitude was directly proportional to the calcium mobilization. Platelets from patients with Wiskott–Aldrich syndrome (WAS) demonstrated reduced membrane potential of mitochondria in resting platelets in comparison to healthy donors, but their ability to increase the activity of mitochondria upon stimulation was not impaired.