Integrin αIIbβ3 activation in Chinese hamster ovary cells and platelets increases clustering rather than affinity

Abstract

Integrin αIIbβ3 affinity regulation by talin binding to the cytoplasmic tail of β3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of αIIbβ3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn2+, a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of β3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and fertebrate systems and suggest new models for talin regulation of integrin activity in human platelets. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.