Ca(2+)-dependent structural transitions of the platelet glycoprotein IIb-IIIa complex. Preparation of stable glycoprotein IIb and 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 Ca(2+)-dependent transitions were observed, one at about 60 microM Ca2+, where half of the complexes became dissociated, and the other at 0.1 microM 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 Ca(2+)-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.
Steiner
B
B
Gladstone Foundation Laboratories for Cardiovascular Disease, University of California, San Francisco 94140-0608.
Parise
L V
LV
Leung
B
B
Phillips
D R
DR
D002118:Calcium / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013917:Thrombin / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D013917:Thrombin; D008274:Magnesium; D002118:Calcium