Ca 2+ waves coordinate purinergic receptor-evoked integrin activation and polarization

Cells sense extracellular nucleotides through the P2Y class of purinergic G protein-coupled receptors (GPCRs), which stimulate integrin activation through signaling events, including intracellular Ca mobilization. We investigated the relationship between P2Y-stimulated repetitive Ca waves and fibrinogen binding to the platelet integrin α β (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y and P2Y generated a series of Ca transients that each induced a rapid, discrete increase in fibrinogen binding. The peak and net increase of individual fibrinogen binding events correlated with the Ca transient amplitude and frequency, respectively. Using BAPTA loading and selective receptor antagonists, we found that Ca mobilization downstream of P2Y was essential for ADP-evoked fibrinogen binding, whereas P2Y and the kinase PI3K were also required for α β activation and enhanced the number of Ca transients. ADP-evoked fibrinogen binding was initially uniform over the cell periphery but subsequently redistributed with a polarity that correlated with the direction of the Ca waves. Polarization of α β may be mediated by the actin cytoskeleton, because surface-bound fibrinogen is highly immobile, and its motility was enhanced by cytoskeletal disruption. In conclusion, spatial and temporal patterns of Ca increase enable fine control of α β activation after cellular stimulation. P2Y -stimulated Ca transients coupled to α β activation only in the context of P2Y coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.