Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase

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Abstract
We have developed a method for measuring the local concentration of ATP at the extracellular surface of live cells. This method relies on the specific attachment to the cell surface of a chimeric protein that consists of the IgG-binding domain ofStaphylococcus aureus protein A fused in-frame with the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP during its secretion from activated platelets. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.