Binding of RGD-peptide Mimics to intact Human Platelets: an NMR Study

The interactions of small peptides with biological membranes is central to a number of biological processes. Interference with these recognition events could be used to modulate or alter signal transmission or to prevent the onset of deseaes. The biophysical environment of a membrane is considerably different from the isotropic extracellular medium. It is therefore desirable to investigate membrane proteins and their binding specificity directly in living cells. We are investigating these problems using as model the integrin IIb3 which is the most abundant platelet cell surface glycoprotein. This integrin plays a key role in adhesion of platelet to protein-coated surfaces and in platelet/platelet aggregation. Therefore, IIb3 receptor is an excellent target for drug design. New cyclic pentapeptide mimics (1) were designed incorporating the tripeptide sequence Arg-Gly-Asp (RGD)[1], a common amino acid motif found in a number of adhesive proteins. In order to study the binding processes between the platelet IIb3 integrin and the RGD ligands, at molecular level, we used transfer-NOE and STD (Saturated Transfer Difference) experiments directly on whole human platelets.[2] NMR studies were performed to investigate the conformation of the peptidomimetics, first in solution and then upon binding to platelets by transferred nuclear Overhauser effect (TR-NOE) measurements. STD experiments were also performed in order to define the portions of the ligands which are in closest contact with the protein. Analysis of the NMR data allowed to correlate the biological results and to derive workable models of the ligand:protein complexes. Utilizing structural information by NMR experiments and docking studies, we have discovered a new high affinity IIb3 ligand, which is able to inhibit the platelet aggregation and the adhesion of fibrinogen to platelets