Bax translocation to the mitochondria is a key event that regulates the release of proteins like cytochrome C and Smac/Diablo from the mitochondria, which leads to downstream apoptotic events [40]. Thus, to further explore whether hyperthermia could induce mitochondrial translocation of Bax and cytochrome C release, platelets were pretreated across a range of temperatures and subjected to isolation and analysis of mitochondrial and cytosolic fractions (Figure 4A and 4B). We found that hyperthermia significantly promoted mitochondrial translocation of Bax and cytochrome C release. In order to determine whether Mito-TEMPO affected mitochondrial translocation of Bax and cytochrome C release in hyperthermia-treated platelets, washed platelets were pre-incubated with Mito-TEMPO or solvent control before hyperthermia treatment (Figure 4C and 4D). We showed that Mito-TEMPO significantly inhibited hyperthermia-induced mitochondrial translocation of Bax and cytochrome C release. These observations suggested that mitochondrial ROS play pivotal roles in hyperthermia-induced mitochondrial translocation of Bax and cytochrome C release.