Mass Spectrometric Analysis of the Phosphorylation State of Human Platelet Glycoprotein IIIa

The cytoplasmic domain of platelet glycoprotein IIIa contains the structural information for the recruitment of integrin GPIIb/IIIa into focal contacts as well as the integration of extracellular ligand-binding signals into the intracellular pathway leading to kinase ppl25FAK phosphorylation. These events could be mediated by covalent modification of the GPIIIa intracellular tail. Controversial reports have been published regarding palmitoylation and phosphorylation of GPIIIa in activated platelets. Here, we have analyzed the structure of the cytoplasmic domain of GPIIIa isolated from either the RGD-binding and non-RGD-binding conformers of the Triton X-100 soluble fraction or from the insoluble fraction of plasma membrane lysates of platelets (free and cytoskeleton-attached GPIIb/IIIa, respectively). Only the unaltered GPIIIa cytoplasmic tail was found. Therefore, we conclude that the intracellular domain of GPIIIa is neither covalently modified constitutively nor is its modification required for either RGD-peptide-binding or cytoskeleton attachment.