Calcium oscillation and phosphatidylinositol 3-kinase positively regulate integrin α_IIbβ₃-mediated outside-in signaling

The frequency of calcium oscillation reveals the platelet activation status, however, the biological significance of the periodic calcium responses and methods of communication with other integrin-mediated signals are not clear. RGD-containing disintegrin rhodostomin coated substrates were employed to enhance platelet spreading and calcium oscillation through direct binding and clustering of the receptor integrin α_IIbβ₃. The results showed that the activation of phosphatidylinositol 3-kinase (PI3-K) and internal calcium pathways were crucial for α_IIbβ ₃ outside-in signaling. PI3-K antagonists wortmannin and LY294002 inhibited disintegrin substrates and induced platelet spreading and calcium oscillation. At the same time, pretreatment of platelets with the microsomal calcium-ATPase inhibitor thapsigargin to deplete internal calcium stores severely impaired the calcium oscillation as well as PI3-K activation and spreading on disintegrin substrates. Because inhibition of one pathway could inhibit the other, our data indicates that PI3-K and calcium oscillation are synergistically operated and form a positive-feedback regulation in integrin α_IIbβ₃-mediated outside-in signaling.