title: 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, alphaIIbbeta3. In unstimulated platelets, alphaIIbbeta3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables alphaIIbbeta3 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 alphaIIbbeta3 or alphaIIbbeta3 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 alphaIIbbeta3 molecules, indicating that there are only two states of alphaIIbbeta3 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 activation/inactivation state.
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA.
authors.forename: Rustem I
authors.initials: RI
authors.lastname: Litvinov
authors.forename: Henry
authors.initials: H
authors.lastname: Shuman
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bennett
authors.forename: John W
authors.initials: JW
authors.lastname: Weisel
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000502:physiology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000502:physiology*; D005340:Fibrinogen / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D007834:Lasers; D008854:Microscopy, Electron; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D011336:Probability; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000737:chemistry / Q000502:physiology
chemical_list: D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014841:von Willebrand Factor; D005340:Fibrinogen