Reversibility versus Persistence of GPIIb/IIIa Blocker-Induced Conformational Change of GPIIb/IIIa ( IIb 3, CD41/CD61)

Clinically used GPIIb/IIIa blockers are ligand mimetics, and thereby their binding can induce conformational changes of the platelet integrin GPIIb/IIIa. Since the reversibility of these conformational changes may be an important determinant of potential adverse effects of GPIIb/IIIa blockers, we produced a new monoclonal antibody (anti-LIBS-mAb), and by using its binding properties, we investigated the conformational changes of GPIIb/IIIa during the binding and especially the dissociation of GPIIb/IIIa blockers. Production of monoclonal antibody (mAb) clones was performed using purified GPIIb/IIIa in a high affinity conformation and using activated platelets. Clone anti-LIBS-145-mAb was chosen, since it allowed the sensitive probing of eptifibatide-induced conformational changes of GPIIb/IIIa. On resting and activated platelets and on GPIIb/IIIa-expressing Chinese hamster ovary cells, anti-LIBS145-mAb binding returned to background binding after dissociation of eptifibatide, indicating a complete reversibility of the eptifibatide-induced conformational change. Furthermore, with the mixing of eptifibatide-preincubated and nonincubated cells, a fast reversibility could be demonstrated. However, when fibrinogen was present in a physiological concentration, the GPIIb/IIIa blocker-induced conformation was partially retained after the dissociation of eptifibatide and to the same extent binding of fibrinogen and the activation-specific mAb Pac-1 was induced. In conclusion, a fast reversibility of the conformational change of GPIIb/IIIa after dissociation of GPIIb/IIIa blockers could be demonstrated as an intrinsic property of the GPIIb/IIIa receptor. This mechanism prevents general platelet aggregation after dissociation of ligand mimetic GPIIb/IIIa blockers. Nevertheless, in the presence of fibrinogen this reversibility is not complete, which may explain some of the side effects of GPIIb/IIIa blockers, especially those of the oral GPIIb/ IIIa blockers. Platelet activation results in a conformational change of the membrane spanning platelet integrin GPIIb/IIIa ( IIb 3, CD41/CD61) enabling the binding of the plasma protein fibrinogen. This binding is primarily reversible, but it enhances platelet activation by outside-in signaling causing receptor clustering, platelet secretion, and finally irreversible fibrinogen binding and platelet aggregation (Shattil et al., 1998). Similar conformational changes of GPIIb/IIIa are induced by several GPIIb/IIIa blockers, since these agents bind at or nearby the fibrinogen binding pocket of GPIIb/IIIa and thereby act as ligand mimetics (Gawaz et al., 1998; Jennings et al., 2000; Dickfeld et al., 2001). While the conformational change of GPIIb/IIIa after the binding of ligands or ligand mimetics has been investigated (Kamata and Takada, 2001; Hynes, 2002; Takagi et al., 2002), the conformational status of GPIIb/IIIa after dissociation of ligands or ligand mimetics has not been the focus of investigations yet. GPIIb/IIIa blockers have demonstrated limitations as intravenous drugs and have caused an increase in mortality in their use as oral drugs (Cox et al., 2000; Holmes et al., 2000; Chew et al., 2001; Quinn et al., 2002; Quinn et al., 2003; Topol et al., 2003). For several reasons, the reversibility of the GPIIb/IIIa blocker-induced conformational change after dissociation of the blocker might be essential for the understanding of the potential adverse effects associated with this class of anti-platelet drugs. GPIIb/IIIa blocker-associated Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.058883. ABBREVIATIONS: CD41, cluster of differentiation number for the GPIIb subunit; CD61, cluster of differentiation number for the GPIIIa subunit; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; GPIIb/IIIa, glycoprotein IIb/IIIa; LIBS, ligand-induced binding site; mAb, monoclonal antibody; PRP, platelet-rich plasma; RGD, aminoacid sequence on fibrinogen recognized by GPIIb/IIIa; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 0022-3565/04/3083-1002–1011$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 58883/1126565 JPET 308:1002–1011, 2004 Printed in U.S.A. 1002 at A PE T Jornals on A uust 1, 2017 jpet.asjournals.org D ow nladed from thrombocytopenia and platelet-activating effects of GPIIb/ IIIa blockers have been directly associated with the conformational changes of GPIIb/IIIa induced by the binding of ligand-mimetic blockers to the receptor (Peter et al., 1998; Madan and Berkowitz, 1999; Cox et al., 2000; Bougie et al., 2002; Bhatt and Topol, 2003; Quinn et al., 2003). Thus, the reversibility or persistence of these conformational changes can be expected to be determinants of the adverse effects of GPIIb/IIIa blockers. In the present study, we developed a new anti-ligandinduced binding site (LIBS) monoclonal antibody (mAb), which allows a sensitive probing of GPIIb/IIIa blocker-induced conformational receptor changes, and we set up experimental procedures using platelets as well as recombinant GPIIb/IIIa to study receptor conformation after dissociation of GPIIb/IIIa blockers. We could demonstrate that a fast reversibility of the GPIIb/IIIa blocker-induced conformational change is an intrinsic property of the receptor. However, in the presence of fibrinogen, reversibility is not complete. This finding is an important new aspect in the discussion of GPIIb/IIIa blocker-associated adverse effects. Materials and Methods Blood Preparation and Cells. Blood was collected by venipuncture with a 21-gauge butterfly needle from healthy volunteers and anticoagulated with citric acid. Platelet-rich plasma was obtained by centrifugation at 100g in plastic tubes at room temperature for 15 min in a laboratory centrifuge (Haereus Multifuge 3s). Platelets were counted using a Neubauer chamber and adjusted to 200,000/ l with