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 ␣ v ␤ 3 for this ligand, we used laser tweezers to measure the rupture force between single ␣ v ␤ 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 M 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, Ϸ10% 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 ␣ v ␤ 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 ␣ v ␤ 3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of ␣ v ␤ 3 -binding sites for this ligand.Integrin-mediated cell adhesion plays a fundamental role in processes as diverse as wound repair, cancer cell metastasis, organogenesis, implantation, and thrombosis (1). The ability of integrins to interact with ligands can be regulated, to a greater or lesser extent, by cellular metabolism, a process known as "inside-out signaling" (2). However, it is often not possible to determine experimentally whether inside-out signaling augments cell adhesion by increasing integrin avidity for ligands via integrin clustering or whether it actually increases the affinity of integrins for ligands. Agonist-induced changes in integrin avidity are thought to be largely responsible for the increase in ␣ L ␤ 2 -and ␣ 4 ␤ 1 -mediated adhesion of lymphocytes to ICAM-1 and VCAM-1, respectively, although agonist-stimulated changes in the affinity of these integrins for their ligands have also been reported (3, 4). On the other hand, the ability of the platelet integrin ␣ IIb ␤ 3 to support platelet aggregation is due to an agonist-induced increase in the affinity of ␣ IIb ␤ 3 for soluble ligands (5).Laser tweezers are an optical system in which external forces applied to a particle trapped by a laser can be accurately measured, because the angular deflection of the laser beam is directly proportional to the lateral force applied to the particle. Laser tweezers are sensitive and accurate at the lower end of the force spectrum (0 -150 pN) 1 (6) and therefore are a suitable system with which to study integrin-ligand interactions (7). Moreover, because laser tweezers can measure the force of interac...

A model for the role of integrins in flow induced mechanotransduction in osteocytes

“…5. In contrast, experimental measurements revealed rupture strength for single ␤ 3 integrin-ligand pairs on the order of 50-100 pN at a loading rate of 10,000 pN/s (24,25). Physiological loading rates are much lower.…”

Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level

“…On the basis of several criteria that have been proposed to test whether the observed ruptures were due to the attachments of single or multiple pairs of molecules, 25,29 our data indicate that under the experimental conditions studied the rupture events were due to single bimolecular attachments. First, the rupture of multiple attachments should occur in a sequence of multiple steps, whereas the rupture of a single attachment should always occur in one step.…”

“…The results suggest that forces ranging from 0 to approximately 40 pN are required to rupture nonspecific protein-protein binding, consistent with nonspecific rupture forces reported previously for a number of biologically inert protein pairs. 25,29 Although shown in the subsequent experimental histograms, nonspecific forces less than 40 pN were excluded from the analysis and interpretation of the forces reflecting specific knob-hole binding.…”

Activation of Platelet αIIbβ3 by an Exogenous Peptide Corresponding to the Transmembrane Domain of αIIb

“…Rupture Force Spectroscopy (Laser Tweezers)-A custombuilt laser tweezers setup was used to measure the strength of fibrinogen and osteopontin binding to CHO cells or platelets (21,22). Purified human fibrinogen and recombinant osteopontin (OPN) were covalently bound to 1.87-m carboxylate-modified latex beads.…”