Live imaging of extracellular signal‐regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer

Background Dynamic features of thrombus formation have been visualized by conventional video wide field microscopy or confocal microscopy in live mouse. However, due to technical limitations precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives By two-photon excitation microscopy of transgenic mice expressing Forster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), we visualized ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles Results When a core of densely packed platelets was developed, ERK activity was increased from the basal region close to the injured arterioles. Meanwhile, PKA was activated at the downstream side of an unstable shell that overlays the core of platelets. Intravenous administration of a MEK inhibitor PD0325901 suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. Meanwhile, a cAMP analog dbcAMP inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new window to visualization of the spatiotemporal changes of signaling molecule activities during not only thrombus formation but also other hematological disorders. This article is protected by copyright. All rights reserved.