A live cell micro-imaging technique to examine platelet calcium signaling dynamics under blood flow.

The platelet is a specialized adhesive cell that plays a key role in thrombus formation under both physiological and pathological blood flow conditions. Platelet adhesion and activation are dynamic processes associated with rapid morphological and functional changes, with the earliest signaling events occurring over a subsecond time-scale. The relatively small size of platelets combined with the dynamic nature of platelet adhesion under blood flow means that the investigation of platelet signaling events requires techniques with both high spatial discrimination and rapid temporal resolution. Unraveling the complex signaling processes governing platelet adhesive function under conditions of hemodynamic shear stress has been a longstanding goal in platelet research and has been greatly influenced by the development and application of microimaging-based techniques. Advances in the area of epi-fluorescence and confocal-based platelet calcium (Ca(2+)) imaging have facilitated the in vitro and in vivo elucidation of the early signaling events regulating platelet adhesion and activation. These studies have identified distinct Ca(2+) signaling mechanisms that serve to dynamically regulate activation of the major platelet integrin α(IIb)β(3) and associated adhesion and aggregation processes under flow. This chapter describes in detail a ratiometric calcium imaging protocol and associated troubleshooting procedures developed in our laboratory to examine live platelet Ca(2+) signaling dynamics. This technique provides a method for high-resolution imaging of the Ca(2+) dynamics underpinning platelet adhesion and thrombus formation under conditions of pathophysiological shear stress.