TETHERED LIGAND DERIVED PENTAPEPTIDE AGONISTS OF THROMBIN RECEPTOR - A STUDY OF SIDE-CHAIN REQUIREMENTS FOR HUMAN PLATELET ACTIVATION AND GTPASE STIMULATION

Proteolytic action of alpha-thrombin on human thrombin receptor results in cleavage of a portion of the N-terminus, thereby generating a 'tethered ligand' at the newly exposed N-terminus, which then activates the receptor in an intramolecular fashion. Agonist peptides incorporating the amino acid sequence of the newly exposed N-terminal portion of the cleaved receptor cause receptor activation without requiring prior cleavage of the receptor by thrombin. The pentapeptide amide Ser-Phe-Leu-Leu-Arg-NH2, which retains the N-terminal sequence of the 'tethered ligand' of the receptor, has been shown to be the minimum sequence to cause receptor activation. To understand the importance of the side chains of various residues within the pentapeptide amide, we carried out an extensive structure-activity study of the ability of peptides to stimulate gel-filtered platelet aggregation. In this study 106 pentapeptide amides were synthesized, utilizing naturally occurring L-amino acids, unnatural amino acids, D-amino acids and N-methyl amino acids for replacements. At position-1, charged residues (acidic or basic) were not tolerated, and the size and shape of the residue were important. Position-2 tolerated only aromatic residues. Position-3 accommodated various residues. A significant finding of this study was that two very different residues, [3-(2-naphthyl)]-L-alanine and L-arginine, when substituted for leucine residue at position-3, resulted in more active agonists. At position-4 aromatic and aliphatic residues were well tolerated, whereas basic and acidic residues were less tolerated. Position-5 mimicked position-3 in its ability to tolerate a wide range of residues. In general, an acidic residue was not preferred in any position. In all positions a -Phe-residue was tolerated. Positions 1 and 3 tolerated -Pro-residue, whereas positions 2, 4 and 5 did not. D-Amino acid substitution was not tolerated in any position. While N-methylation of residues at positions-3 and -4 led to poor agonists, at position-2 it resulted in an inactive analog. These studies define the side chain requirements for the 'tethered ligand' derived pentapeptide agonists of thrombin receptor for human platelet activation. Selected peptides were tested for ability to stimulate platelet membrane GTPase. A good correlation was observed between peptides that stimulate GTPase and those that stimulate platelet aggregation, with the general finding that 3-10-fold higher concentrations were required to half maximally activate GTPase when compared with the concentrations needed for activation of platelet aggregation. (C) Munksgaard 1995.