Expression and Purification of a Soluble Functional form of the Platelet αIIbβ3 Integrin

Platelet glycoproteins αIIb and β3 are membrane proteins that associate to form a Ca2+-dependent heterodimer which constitutes an inducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, αIIb and β3 were both deleted of their transmembrane and cytoplasmic domains and were expressed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and compared to those of wild-type αIIbβ3. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that observed for the wild-type αIIbβ3 in COS cells or in human megakaryocytes. The truncated β3 subunit associated with the Pro-truncated form of the αIIb subunit. This precursor form was not secreted. After proteolytic cleavage of the Pro-truncated αIIb, the mature heterodimer was secreted into the culture supernatant. To quantify the molar ratio of the various secreted soluble forms, an immunocapture assay was designed. All secreted tr-αIIb subunits associated with tr-β3. In contrast, tr-β3 was produced and secreted in excess as the free form. Immunoreactivity of the wild-type and soluble truncated complexes was identical since all the monoclonal antibodies used reacted with surface-located epitopes on both complexes. This indicated that the soluble truncated heterodimer adopted a native conformation. To purify this soluble heterodimer, tr-αIIbβ3-containing culture supernatant was adsorbed on an RGDW-affinity column and eluted with a solution of the free peptide RGDW. In the RGD-eluted material, the amount of each subunit was stoichiometric, suggesting that the complex was not disrupted during purification. The capacity of the wild-type and truncated RGD-eluted complexes to interact with soluble fibrinogen was compared using a solid-phase immunocapture assay. tr-αIIbβ3 and platelet αIIbβ3 exhibited similar fibrinogen-binding capacity. For both complexes, these interactions were mediated by RGD and γ fibrinogen signals.