Quantitative Analysis of Platelet αvβ3 Binding to Osteopontin Using Laser Tweezers

Abstract

To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin αvβ3 for this ligand, we used laser tweezers to measure the rupture force between single αvβ 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 μM ADP bound strongly to osteopontin, producing rupture forces as great as 100 piconewtons (pN) with a peak at 45-50 pN. By contrast, 90% of unstimulated, resting non-reactive platelets bound weakly to osteopontin, with rupture forces rarely exceeding 30-35 pN. However, ≈10% of unstimulated platelets, resting reactive platelets, exhibited rupture force distributions similar to stimulated platelets. Moreover, ADP stimulation resulted in a 12-fold increase in the probability of detecting rupture forces >30 pN compared with resting non-reactive platelets. Pre-incubating stimulated platelets with the inhibitory prostaglandin E1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the αvβ3-specific cyclic peptide XJ735 returned force histograms to those of non-reactive platelets. These experiments demonstrate that ADP stimulation increases the strength of the interaction between platelet αvβ3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of α vβ3-binding sites for this ligand.