Ca2+ waves coordinate purinergic receptor–evoked integrin activation and polarization

• Published in: Science signaling 2020-01, Vol.13 (615)
• Main Authors: Bye, Alexander P, Gibbins, Jonathan M, Mahaut-Smith, Martyn P
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; Actin; Activation; Adenosine diphosphate; Binding; Calcium (extracellular); Calcium (intracellular); Calcium antagonists; Calcium ions; Calcium signalling; Cytoskeleton; Disruption; Fibrinogen; Fluorescence; Intracellular; Intracellular signalling; Kinases; Megakaryocytes; Nucleotides; Platelets; Polarity; Polarization; Purine P2Y receptors; Receptors; Signaling
• ISSN: 1945-0877; 1937-9145
• DOI: 10.1126/scisignal.aav7354

Ca2+ waves and integrin activationThe integrin GPIIb/IIIa is highly abundant on the surface of platelets and can be activated by intracellular Ca2+ signaling in an “inside-out” manner to bind to the adhesive ligand fibrinogen. Bye et al. imaged intracellular Ca2+ signaling and fibrinogen binding events in primary rat megakaryocytes activated through the ADP-stimulated receptors P2Y1 and P2Y12. The authors found that signaling by both receptors was required for full integrin activation, which depended on P2Y1-stimulated Ca2+ signaling and P2Y12-stimulated activation of the kinase PI3K. In addition, fibrinogen binding became polarized in these cells in a manner dependent on the direction of ADP-stimulated Ca2+ waves.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 αIIbβ3 (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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 activation after cellular stimulation. P2Y1-stimulated Ca2+ transients coupled to αIIbβ3 activation only in the context of P2Y12 coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.