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 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 microM 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, approximately 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 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.
Highlights
Integrin-mediated cell adhesion plays a fundamental role in processes as diverse as wound repair, cancer cell metastasis, organogenesis, implantation, and thrombosis [1]
Because laser tweezers can measure the force of interaction between individual receptor-ligand pairs [7], they can potentially differentiate between agonist-stimulated changes in integrin avidity versus changes in integrin affinity
To verify the specificity of the interaction of OPN with the latter group of platelets, rupture forces were measured in the presence of a cyclic RGD-peptide, a competitive inhibitor of OPN binding to ␣v3
Summary
Integrin-mediated cell adhesion plays a fundamental role in processes as diverse as wound repair, cancer cell metastasis, organogenesis, implantation, and thrombosis [1]. To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin ␣v3 for this ligand, we used laser tweezers to measure the rupture force between single ␣v3 molecules on the platelet surface and osteopontin-coated beads.
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