title: Arginine-glycine-aspartic acid mimics can identify a transitional activation state of recombinant alphaIIb beta3 in human embryonic kidney 293 cells.
abstract: The platelet-specific integrin alphaIIb beta3 achieves a high affinity binding state in response to extracellular agonists such as thrombin, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different conformations of alphaIIb beta3, we expressed recombinant alphaIIb beta3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide recognition specificities of the full-length recombinant receptor resembled those of the native receptor in platelets. We used an array of peptidic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that specifically bind to human platelet alphaIIb beta3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated alphaIIb beta3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length alphaIIb beta3 is expressed in a "transitional" activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-alphaIIb beta3 antibody PAC1 to cells expressing the full-length recombinant alphaIIb beta3. Deletion of the entire cytoplasmic domain of the beta subunit was sufficient to convert the receptor in HEK 293 cells to a fully active form, as found in activated platelets. In addition, the full-length receptor was capable of mediating agonist-independent aggregation of cells in the presence of fibrinogen. Thus, by using RGD mimics, we have identified a functional transitional activation state of alphaIIb beta3 that is capable of mediating fibrinogen-dependent cell aggregation.
authors.affiliation: Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. dicky_abraham@merck.com
authors.forename: D G
authors.initials: DG
authors.lastname: Abraham
authors.forename: E M
authors.initials: EM
authors.lastname: Nutt
authors.forename: R A
authors.initials: RA
authors.lastname: Bednar
authors.forename: B
authors.initials: B
authors.lastname: Bednar
authors.forename: R J
authors.initials: RJ
authors.lastname: Gould
authors.forename: L T
authors.initials: LT
authors.lastname: Duong
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D001667:Binding, Competitive; D018821:CD18 Antigens / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002460:Cell Line; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D039421:Integrin alpha2; D009842:Oligopeptides / Q000737:chemistry; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D015703:Antigens, CD; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D005353:Fibronectins; D039421:Integrin alpha2; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid