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 Ca2+ mobilization. We investigated the relationship between P2Y-stimulated repetitive Ca2+ waves and fibrinogen binding to the platelet integrin 640 3 (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y(1) and P2Y(12) generated a series of Ca2+ transients that each induced a rapid, discrete increase in fibrinogen binding. The peak and net increase of individual fibrinogen binding events correlated with the Ca2+ transient amplitude and frequency, respectively. Using BAPTA loading and selective receptor antagonists, we found that Ca2+ mobilization downstream of P2Y(1) was essential for ADP-evoked fibrinogen binding, whereas P2Y(12) and the kinase PI3K were also required for alpha IIb beta(3) activation and enhanced the number of Ca2+ 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 Ca2+ waves. Polarization of alpha IIb beta(3) 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 Ca2+ increase enable fine control of alpha IIb beta(3) activation after cellular stimulation. P2Y(1)-stimulated Ca2+ transients coupled to alpha IIb beta(3) activation only in the context of P2Y(12) coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.