CHARACTERIZATION OF CATION-BINDING SEQUENCES IN THE PLATELET INTEGRINGPIIB-IIIA (ALPHA(IIIB)BETA(3)) BY TERBIUM LUMINESCENCE

The binding of cations to purified GPIIb-IIIa (alpha(IIb)beta(3)) and synthetic peptides corresponding to the potential cation-binding sites within this integrin has been assessed by terbium luminescence spectr oscopy. Tb3+ supported fibrinogen binding to purified GPIIb-IIIa, at l ower concentrations than Ca2+, consistent with its higher affinity for cation-binding motifs. Titration analyses indicated the presence of f ive Tb3+- binding sites of relatively high affinity in the receptor. T hese sites also could be filled by divalent cations. Six sequences wit hin GPIIb-IIIa have the appropriate spacing of five of the usual six c oordination sites for cations in functional Ca2+-binding EF-hand motif s. Peptides containing Tyr and/or Trp at selected positions as fluores cence energy donors were synthesized, and their Tb3+-binding capacity was assessed. The four potential Ca2+-binding sequences in the GPIIb s ubunit, GPIIb 242-255, 296-309, 364-377, and 425-438, were functional, despite lacking the usual Glu residue at the terminal coordination po sition. These peptides bound Tb3+ with the same affinity as typical Ca 2+-binding loop peptides and also bound Ca2+ and other divalent cation s without preference. Of the two candidate GPIIIa sequences, 118-131 a nd 208-221, the former bound Tb3+ and divalent cations with an affinit y similar to that of the GPIIb peptides, whereas the latter peptide wa s not functional. This functional difference, as well as data obtained with substituted peptides, emphasizes the importance of the first coo rdination position for interaction of synthetic peptide loops with cat ions. Together, these data identify the five cation-binding sites with in intact GPIIb-IIIa.