Quantitative analysis of platelet αvβ3 binding to osteopontin using laser tweezers

To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin alpha(v)beta(3) for this ligand, we used laser tweezers to measure the rupture force between single alpha(v)beta(3) molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 muM 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, approximate to10% 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 E-1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the alpha(v)beta(3)-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 alpha(v)beta(3) and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of alpha(v)beta(3)-binding sites for this ligand.