Abstract
The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.
Highlights
Conforformational changes in GPIIb-IIIaas evidenced by the mational changes in bound ligands may be anticipated; induction of ligand-induced binding sites which are and, a corollary to the LIBS hypothesis can be recognized by antibodies that rseealectctively with the forwarded to predict that “receptor-induced binding sites”
Formational changesis demonstrated by the iden-An anti-RIBS monoclonal antibody would react with the tification and characterization of a receptor-induced bound but not the free ligand
Expression of Fg-RIBS-I by Platelet-bound Fibrinogen-In a previous study,we haddemonstrated that the 2G5 monoclonal antibody, designated anti-Fg-RIBS-I in this study, reacted with fibrinogen immobilized onto plastic microtiter plates or filter paper but did not react with soluble fibrinogen
Summary
Proteins and Their Fragments-The monoclonal antibody, designated anti-Fg-RIBS-I, is an IgG of the y1subclass with a kappa light chain. An occupied receptor may express a new enzymatic activity (l),become a substrate for a cellular enzyme [2], or bind to cytoplasmic or skeletal elements of the cell [3]. Such conformational changes may elicit new sites elsewhere [11]. Fab fragments of the antibody were prepared by papain digestion (Sigma) within the occupied receptor which can serve as epitopes for at anenzyme to substrate ratio of 1:lOO (w/w) for 6 h at 37 “C [14]. These sites, which we have termed Undigested antibody and Fc fragments were removed by chromatography on protein A-Sepharose (Pharmacia, AB, Uppsala, Sweden)
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