EXPRESSION AND PURIFICATION OF A SOLUBLE FUNCTIONAL FORM OF THE PLATELET ALPHA-IIB-BETA-3 INTEGRIN

Platelet glycoproteins alpha IIb and beta 3 are membrane proteins that associate to form a Ca2+-dependent heterodimer which constitutes an i nducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, alpha IIb and beta 3 were both deleted of their transmembrane and cytoplasmic domains and were expre ssed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and comp ared to those of wild-type alpha IIb beta 3. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that o bserved for the wild-type alpha IIb beta 3 in COS cells or in human me gakaryocytes. The truncated beta 3 subunit associated with the Pro-tru ncated form of the alpha IIb subunit. This precursor form was not secr eted. After proteolytic cleavage of the Pro-truncated alpha IIb, the m ature heterodimer was secreted into the culture supernatant. To quanti fy the molar ratio of the various secreted soluble forms, an immunocap ture assay was designed. All secreted tr-alpha IIb subunits associated with tr-beta 3. In contrast, tr-beta 3 was produced and secreted in e xcess 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 ind icated that the soluble truncated heterodimer adopted a native conform ation. To purify this soluble heterodimer, tr-alpha IIb beta 3-contain ing culture supernatant was adsorbed on an RGDW-affinity column and el uted with a solution of the free peptide RGDW. In the RGD-eluted mater ial, the amount of each subunit was stoichiometric, suggesting that th e complex was not disrupted during purification. The capacity of the w ild-type and truncated RGD-eluted complexes to interact with soluble f ibrinogen was compared using a solid-phase immunocapture assay. tr-alp ha IIb beta 3 and platelet alpha IIb beta 3 exhibited similar fibrinog en-binding capacity. For both complexes, these interactions were media ted by RGD and gamma fibrinogen signals.