Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

It is widely accepted that glycoprotein (GP) Ib contains one Iba and one IbR subunit that are connected by a disulfide bond. It is unclear which Cys residue in lba, C484 or C485, forms the disulfide bond with Ib beta. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ib beta. In the context of isolated peptides containing the lba or Ib beta transmembrane domain and nearby Cys residue, C484 and C485 in the Ib alpha peptide were both capable of forming a disulfide bond with the Ib beta peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ib beta subunits but only 1 Ib alpha and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ib alpha, not the number of Ib beta subunits therein. Overall, these results demonstrate that Ib alpha is covalently connected to 2 Ib beta subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The alpha beta(2) composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.