Yoshimi Sato

Discovery and structure--activity relationship studies of a novel and specific peptide motif, Pro-X-X-X-Asp-X, as a platelet fibrinogen receptor antagonist.

A novel hexapeptide, H-Pro-Ser-Nva-Gly-Asp-Trp-OH 6, a specific antagonist of platelet fibrinogen receptor (GpIIb/IIIa), was discovered in a structure-activity relationship (SAR) study where the role of the N-terminal Pro moiety of an RGD-containing peptide, H-Pro-Ser-Arg-Gly-Asp-Trp-OH 1, which is a potent but not specific antagonist toward GpIIb/IIIa integrin, was investigated. This novel peptide 6 exhibits very high activity as a human platelet aggregation inhibitor (IC50 0.59 microM, human PRP/collagen) as well as marked specificity for GpIIb/IIIa. A series of substitutions at the third position (Nva residue) in this hexapeptide, focused on the conformational rigidity, led to compounds which are superior to the original novel peptide 6 with regard to anti-platelet activity. The peptides, H-Pro-Ser-Hyp-Gly-Asp-Trp-OH 17 and H-Pro-Ser-delta Pro-Gly-Asp-Trp-OH 18 with the 5-membered ring structure, which restricted the conformation of the peptide backbone at the third position, inhibited the aggregation of human platelets at submicromolar concentrations (IC50 0.39 and 0.30 microM, respectively). Further structure-activity relationship studies at each position of the peptide sequence suggest a novel motif sequence, Pro-X1-X2-X3-Asp-X4, for specific GpIIb/IIIa integrin recognition, in which the N-terminal free Pro residue and the Asp residue at the fifth position are essential to the activity. This motif sequence is summarized as follows: (1) a small amino acid such as Ser, Ala or Gly is preferable at X1 position; (2) X2 may be any amino acid, preferably a bulky amino acid such as Tle or a cyclic amino acid such as Pro; (3) X3 must be a small amino acid such as Gly; and (4) X4 is preferably an amino acid with an aromatic side chain.

A Novel Peptide Motif for Platelet Fibrinogen Receptor Recognition

To develop a specific antagonist of platelet αIIbβ3 using small linear peptides, we synthesized a series of hexapeptides that did not have an Arg-Gly-Asp (RGD) sequence and examined their anti-platelet activity and their specificity for αIIbβ3. We found a novel motif sequence, Pro-X1-X2-X3-Asp-X4, where X1 to X4 were all L-form α-amino acids, which specifically inhibited aggregation of human platelets at submicromolar concentrations. The Pro residue at the N terminus was essential to the anti-platelet activity, and the acetylation of the imino group of this residue also resulted in the complete loss of the activity. The results of the binding assay using purified human platelet αIIbβ3 and placental αvβ3 and those of the cell adhesion assay suggest that this motif peptide is highly specific for platelet αIIbβ3 among other integrins. Flow cytometric studies using an fluorescein isothiocyanate-labeled RGD peptide showed that this motif peptide inhibited the binding of an RGD peptide to activated platelets, suggesting that it has the same inhibitory mode as RGD peptides. Conformational analysis of this motif peptide and an RGD-containing peptide suggests that the imino group of the Pro residue may substitute for the role of the guanidino group of the Arg residue of the RGD sequence.

In vitro and in vivo properties of NSL-9511, a novel anti-platelet hexapeptide without an RGD sequence

Integrins are heterodimeric cell surface receptor molecules and are thought to be particularly important mediators of cell adhesion to extracellular matrix proteins, cell migration, cell-to-cell contact, etc. Many of them, such as platelet GpIIb/IIIa (fibrinogen receptor), vitronectin receptor and fibronectin receptor recognize the Arg-GlyAsp sequence (RGD) as a common recognition motif within their putative ligands [1,2]. Because the RGD sequence has been found in many types of proteins, there are apparent redundancies in integrin-ligand interactions. These redundancies are interesting aspects of integrin receptors and may be beneficial to cell functions. We are developing small peptides which are specific for each integrin as a tool for investigating ligand-integrin interactions and as agents for therapeutic uses. In this study, we have found a novel motif sequence which exhibited potent and highly specific GpIIb/IIIa antagonistic activity. In vivo and in vitro properties of one of these motif peptides are examined and discussed.