Abstract
The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.
Highlights
From the $Hematology-OncologySection and Department of Medicine, University of Pennsylvania School of Medicine, Philadelwhia.Pennsvluania. 19104 and the Dewartmentsof Whemistry and IIBiology, Memphis State University, Memphk, Tennessee38152
Gly-Asp-Ser (RGDS), and the y chain peptide, Leu- of this family is its interaction with proteins and peptides
To differentiate between these possibilities, we compared the abilityof RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and tides containing this sequence inhibit the binding of these proteins to activated platelets [4, 8,9,10]
Summary
DIFFERENTIALEFFECTS OF O( AND y CHAIN FIBRINOGEN PEPTIDES ON THE GLYCOPROTEIN IIb-IIIa COMPLEX*. Exclusive binding suggests that either the a and y chain peptides bindto identical or overlapping siteosn the GPIIb-IIIacomplex or thaot ne peptide induces a change in the complex that excludes the other To differentiate between these possibilities, we compared the abilityof RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and tides containing this sequence inhibit the binding of these proteins to activated platelets [4, 8,9,10]. Hotoaffinity labeling experiments using modified Arg-Gly-Aspand y chain peptides suggest that the peptides interact with GPIIb-IIIa at separate sites[15].To resolve this antibody A2A9, LGGAKQAGDV was a partial inhibi- question, we compared the ability of each type of peptide to tor of A2A9 binding to activated platelets.
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