Summary
We synthesized a series of hybrid peptides that correspond to the γ-chain dodecapeptide
(400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAK-QAGDV(G)
n
RGDS] to investigate the relationship of these receptor recognition domains of fibrinogen
to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide
and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 ± 1.6 μM, 15 ± 2.1 μM, and 87 ± 6.8 μM, respectively. The
inhibitory effect of hybrid peptides increased as the number of glycine residues increased,
plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 ± 0.14 μM. These hybrid peptides completely inhibited the binding of fibrinogen
to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 ± 0.6 μM. When the peptide Y-HHLGGAK-QAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min.
The binding sites of the hybrid peptide were 43,600 molecules/platelet (K
d = 3.1 ± 0.5 × 10-7 M) to stimulated platelets and 12,500 molecules/platelet (K
d = 1.4 ± 0.2 × 10-7 M) to nonstimulated platelets. The hybrid peptides had the same binding affinity
to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa
antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets.
These results indicate that in the native fibrinogen molecule the presence of both
RGD sequence or γ-chain domain at optimal distances increased the binding affinity
to GPIIb/IIIa. These domains may be the source of hybrid peptide, expanding a new
class of platelet inhibitors that act at membrane receptors for adhesive proteins.
