CA2+-DEPENDENT STRUCTURAL TRANSITIONS OF THE PLATELET GLYCOPROTEIN-IIB-IIIA COMPLEX - PREPARATION OF STABLE GLYCOPROTEIN-IIB AND GLYCOPROTEIN-IIIA MONOMERS

The platelet membrane glycoprotein (GP) IIb-IIIa complex is the receptor for adhesive proteins on activated platelets that mediates platelet aggregation. In the present study, factors affecting the structural stability of the purified GP IIb-IIIa complex and the dissociated subunits were investigated. Purified GP IIb-IIIa was incubated in various Ca2+ concentrations, and the percentage of dissociated subunits was quantitated by sucrose gradient sedimentation. Two Ca2+-dependent transitions were observed, one at about 60-mu-M Ca2+, where half of the complexes became dissociated, and the other at 0.1-mu-M Ca2+, where half of the dissociated subunits became incapable of reforming heterodimer complexes when higher Ca2+-concentrations were readded. This loss in ability to reform heterodimer complexes was caused primarily by a Ca2+-dependent transition in GP IIIa, leading to an apparent unfolding of this subunit, followed by the formation of high molecular weight aggregates. The formation of these aggregates was time- and temperature-dependent and could not be reversed by added Ca2+. Although Mg2+ prevented dissociation of GP IIb-IIIa, it failed to promote reassociation of the dissociated subunits. Based on these findings, conditions were developed for the preparation of dissociated GP IIb and GP IIIa such that 70% of the subunits remained functional in that they retained the ability to reform heterodimer complexes.