Spatiotemporal pattern of calpain activation, PS exposure, and reactive oxygen species generation in the ballooning platelet. Extended focus images obtained at 20 minutes after platelet adherence to fibrillar collagen show the spatial location of exposed PS indicated in red (as monitored by membrane annexin-V accumulation), calpain indicated in cyan, and reactive oxygen species (ROS) indicated in green. Calpain activity was monitored by the 7-amino-4-chloromethylcoumarin (CMAC)-based substrate, fluorogenic t-BOC-Leu-Met-CMAC substrate, which yields fluorescent peptidase products with improved retention in live platelets. The generation of ROS during membrane ballooning was followed in real time by means of MitoSox (ThermoFisher Scientific), which is rapidly oxidized by superoxide to produce highly fluorescent products. The chart shows the temporal profile of calpain activation, PS exposure, and ROS generation in a ballooning human platelet. Written informed consent was obtained in accordance with the Declaration of Helsinki. Human blood was obtained from healthy, drug-free volunteers under the University of Bristol, United Kingdom, Research Ethics approval (E5736). Live cell imaging was performed at 25°C using a spinning-disk confocal system as previously described.^8,18  Scale bar represents 3 µm. Data are representative of 4 independent experiments. F/F₀, relative fluorescence intensity over time, where F₀ is the background-subtracted fluorescence intensity before platelet activation.