Crossed immunoelectrophoresis of human platelet membranes. Effect of charge on association and dissociation of the glycoprotein GPIIb-GPIIIa membrane complex.

作者：Simon Karpatkin, R Ferziger, David M. Dorfman · 发表于：Journal of Biological Chemistry · 年份：1986 · DOI：10.1016/s0021-9258(18)67013-9 · 被引用次数：15 · 研究领域：Protein Interaction Studies and Fluorescence Analysis、Antibiotics Pharmacokinetics and Efficacy、Platelet Disorders and Treatments

The mechanism of association of the human platelet membrane GPIIb-GPIIIa-Ca2+ complex was studied by treating solubilized membranes with various enzymes and cationic peptides and by studying the binding of 45Ca2+ and 125I-fibrinogen before and after dissociation with EGTA and association with Ca2+. Neuraminidase shifted the complex cathodally (presumably due to cleavage of negatively charged domains), whereas trypsin had no such effect. The EGTA-dissociated complex was almost completely reassociated with neuraminidase or the cationic peptide, tetralysine. The monoclonal antibody 10E5, which specifically binds to the Ca2+-associated complex (not to its dissociated components), also bound to the neuraminidase-associated complex. Thus, Ca2+ is not necessary for the association of the complex. Neuraminidase treatment of washed intact platelets resulted in a cathodal shift of the membrane Triton X-100-extracted associated complex with no effect on its ability to dissociate in the presence of EGTA. Neuraminidase treatment of ADP-perturbed washed platelets also resulted in a cathodal shift of the associated complex; however, dissociation with EGTA was inhibited. Thus, critical neuraminidase-sensitive components of the complex (sialic acid residues) are not exposed on the surface of the platelet membrane of resting platelets, but do become accessible following platelet stimulation with ADP or membrane solubilization with Triton X-100. 45Ca2+ bound to the associated complex, to GPIIb ...