A MOLECULAR-BASIS FOR AFFINITY MODULATION OF FAB LIGAND-BINDING TO INTEGRIN ALPHA(IIB)BETA(3)

The Arg-Gly-Asp (RGD) sequence within the third complementarity-determ ining region (CDR3) of the heavy chain (H3) is responsible for the bin ding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the p latelet integrin alpha(IIb)beta(3). AP7 binding is minimally influence d by the conformational state of this receptor, whereas PAC1.1 binds p referentially to the activated state of the receptor induced by platel et agonists. To study the molecular basis for this functional differen ce, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of t he analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (V-H domain + C(gamma)1 domain) segments containing these H3 loop sequences were e xpressed as active Fab molecules by coinfection of Spo-doptera frugipe rda cell lines with recombinant baculoviruses containing Fd and AP7 ka ppa chain cDNA. Replacement of the entire AP7 H3 loop with that from P AC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified alpha(IIb) beta(3) in a manner identical to that of PAC1.1. Identical results wer e obtained when solely the sequences flanking the amino side of RGD wi thin the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibite d saturable but submaximal binding to activated gel-filtered platelets . Relative to AP7, the number of AP7.3 or PAC 1.1 Fab molecules bound per platelet was 17% in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40% i n the presence of 10 mu M 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 alpha(IIb)beta(3)( 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 ligand s to distinguish activated from resting conformations of the integrin alpha(IIb)beta(3) can be regulated by limited amino acid sequences imm ediately adjacent to the RGD tripeptide. Furthermore, those Fab molecu les that exhibit increased selectivity for the activated conformation of alpha(IIb)beta(3) bind to a subpopulation of this integrin on plate lets that is modulated by divalent cations.