LOCALIZATION OF THE CROSS-LINKING SITES OF RGD AND KQAGDV PEPTIDES TO THE ISOLATED FIBRINOGEN RECEPTOR, THE HUMAN PLATELET INTEGRIN GLYCOPROTEIN-IIB/IIIA - INFLUENCE OF PEPTIDE LENGTH

The non-covalent and Ca2+-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha(IIb)beta-3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha-1), CGRGDF (alpha-2), CYHHLGGAKQAGDV (gamma-1) and CGAKQAGDV (gamma-2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha-2 and gamma-2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha-1 and gamma-1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha-1 at GPIIIa 63-87 and 303-350; gamma-1 at GPIIIa 9-37; alpha-2 at GPIIIa 151-191; and gamma-2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets. (b) The GPIIb regions which contribute to this ligand-binding domain are not so well defined, the greater molecular flexibility of GPIIb compared with that of GPIIIa being the most probable reason for the different pattern of peptide cross-linking to GPIIb/IIIa observed in solution and in activated platelets. (c) In 'activated' GPIIb/IIIa in solution the N-terminals of GPIIbH (42-73) and GPIIbL2 (30-75) and some segments of the GPIIIa 151-191 and 303-350 stretches are very close (less-than-or-equal-to 1 nm) not only to each other but also to the ligand binding site(s), in agreement with a recent rudimentary map of the compact domains and non-covalent associations in the heterodimer.