Ca²⁺ 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 α_IIbβ₃ (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y₁ and P2Y₁₂ 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 P2Y₁ was essential for ADP-evoked fibrinogen binding, whereas P2Y₁₂ and the kinase PI3K were also required for α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ activation after cellular stimulation. P2Y₁-stimulated Ca²⁺ transients coupled to α_IIbβ₃ activation only in the context of P2Y₁₂ coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.