Phosphorylation sites in the integrin β3 cytoplasmic domain in intact platelets

Abstract

Protein seryl/threonyl phosphatase inhibitors such as calyculin A block inside-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinogen, responses that depend on integrin α(IIb)β3 signaling. We hypothesized that this reflects a change in α(IIb)β3 structure caused by a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the β3 subunit, and phosphoamino acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 became stoichiometrically phosphorylated during inhibition of platelet phosphatases by calyculin A. This region of β3 is linked to outside-in signaling such as platelet spreading responses. The effect of calyculin A on platelet adhesion and spreading and on the phosphorylation of T-753 in β3 is reversed by the calcium ionophore A23187, demonstrating that these effects of calyculin A are not generally toxic ones. We propose that phosphorylation of β3 on threonine 753, a region of β3 linked to outside-in signaling, may be a mechanism by which integrin α(IIb)β3 function is regulated.