title: A molecular basis for affinity modulation of Fab ligand binding to integrin alphaIIb beta3.
abstract: The Arg-Gly-Asp (RGD) sequence within the third complementarity-determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin alphaIIbbeta3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (VH domain + Cgamma1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 kappa chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified alphaIIbbeta3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1. 1 Fab molecules bound per platelet was 17% in the presence of 1 m Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 microM Mn2+. The ratio of Fab molecules bound after versus before activation (mean =/- S.D.; n = 3) was: for AP7.3, 9.8 =/- 0.6; for PAC1.1, 8.8 +/- 0.3; and for AP7, 1.4 =/- 0.2. In addition, AP7 bound to the stably expressed integrin mutant alphaIIbbeta3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin alphaIIbbeta3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of alphaIIbbeta3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.
authors.affiliation: Roon Research Center for Arteriosclerosis and Thrombosis, Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Kunicki
authors.forename: D S
authors.initials: DS
authors.lastname: Annis
authors.forename: Y J
authors.initials: YJ
authors.lastname: Deng
authors.forename: J C
authors.initials: JC
authors.lastname: Loftus
authors.forename: S J
authors.initials: SJ
authors.lastname: Shattil
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D016466:CHO Cells; D002413:Cations, Divalent; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D039421:Integrin alpha2; D039482:Integrin alpha5; D039661:Integrin beta3; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D015703:Antigens, CD; D002413:Cations, Divalent; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D039421:Integrin alpha2; D039482:Integrin alpha5; D039661:Integrin beta3; D008024:Ligands; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid