Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Abstract

Integrin activation states determine the ability of these receptors to mediate cell-matrix and cell-cell interactions. The prototypic example of this phenomenon is the platelet integrin, ␣IIb␤3. In unstimulated platelets, ␣IIb␤3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables ␣IIb␤3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified ␣IIb␤3 or ␣IIb␤3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80 -100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single ␣IIb␤3 molecules, indicating that there are only two states of ␣IIb␤3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation͞inactivation state.T he detailed biochemistry of receptor-ligand interactions can be determined from solution and͞or surface studies, but these results do not take into account the response of receptormediated cell functions to externally applied forces encountered in vivo. This consideration is particularly relevant for integrins because of the cellular capacity to regulate the state of integrin activation. The extent to which cells regulate integrin function is highly variable. In some cellular environments, the ability of a specific integrin to support cellular adhesion may be constitutive, whereas in others, the integrin may be inactive or only partially active (1). Thus, it has been concluded that integrins exist in a variety of activation states, although the basis for this conclusion has generally been indirect, based on whole-cell adhesion assays and the interaction of integrins with specific monoclonal antibodies. Accordingly, direct studies of the activation states of individual receptors are important. In addition, such studies can reveal the relative contribution to integrin regulation of changes in receptor conformation (affinity modulation) vs. receptor clustering (avidity; ref. 2).The platelet integrin ␣IIb␤3 (GPIIb͞IIIa), which is inactive on resting platelets but is activated by agonists such as ADP and thrombin, is the prototypic example of adhesion receptor modulation. This tight regulation of ␣IIb␤3 activity is imperative to prevent the spontaneous formation of thrombi. In this paper, we demonstrate a model employing laser tweezers to determine the force between single ligand-receptor bonds eithe...

Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening

“…Techniques such as atomic force microscopy, micropipette aspiration, or optical traps have been traditionally used for the study of single integrin-ligand molecular adhesion forces in the range of 30-150 pN (41)(42)(43)(44)(45). By contrast, our method can apply much higher force levels (0-1.5 nN), well suited to study the concerted action of many adhesion receptors simultaneously.…”

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

“…This type of assay is typified by the measurement of the force and displacement of optically trapped kinesin coated beads moving along fixed microtubules, pioneered by Block and coworkers 20,21,57 . In a similar assay, the binding probability and unbinding force was measured for virus coated beads brought into contact with erythrocytes 58 , and the binding strength and activation state of single fibrinogenintegrin pairs was measured on living cells 16 . Recently, Kessemaker and colleagues directly observed individual incorporation and shrinkage events at the dynamic end of a microtubule 59 .…”

“…The power and breadth of these techniques is highlighted by the wide variety of measurements and systems investigated. Single cells can be manipulated to probe the strength and location of receptor binding 16 and adhesion or to measure traction and adhesion forces 17 . Viscoelastic properties can be measured on short length-scales and in small volumes 18 , such as within cells 19 .…”

The Platelet Integrin αIIbβ3 Differentially Interacts with Fibrin Versus Fibrinogen

“…When the immobilized fibrinogen or fibrin monomer on the bead interacted with ␣IIb␤3 on the pedestal, tension was produced when the bead was displaced from the laser focus until the ␣IIb␤3-fibrinogen or ␣IIb␤3-fibrin bond ruptured. The applied force was then displayed as a signal proportional to the strength of ␣IIb␤3-fibrinogen or ␣IIb␤3-fibrin binding (29,31). Such signals were on the order of piconewtons and quantitatively characterize the interactions of ␣IIb␤3 with fibrin and fibrinogen at the nanomechanical molecular level.…”