Thrombin is the most potent agonist of human platelets and its effects are primarily mediated
through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity
for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets.
Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in
selected patients. In contrast, despite the fact that several PAR-4 antagonists have been
developed, few of them have been tested in clinical trials.
The aim of the present study was to elucidate the molecular requirements involving the PAR-4
mechanism of activation by peptide analogues of its tethered-ligand.
Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for
their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to
describe the receptor–peptide interactions that are developed following PAR-4 exposure to the
above analogues. To provide a first structure-activity relationship rationale on the bioactivity
profiles recorded for the studied analogues, molecular docking was applied in a homology
model of PAR-4, derived using the crystal structure of PAR-1.
The following peptide analogues were synthesized: AYPGKF-NH2 (1), GYPGKF-NH2 (2), AcAYPGKF-NH2 (3), trans-cinnamoyl-AYPGKF-NH2 (4), YPGKF-NH2 (5), Ac-YPGKF-NH2 (6), transcinnamoyl-YPGKF-NH2 (7), and caffeoyl-YPGKF-NH2 (8). Peptide (1) is a selective PAR-4 agonist
inducing platelet aggregation with an IC50 value of 26.2 μM. Substitution of Ala-1 with Gly-1
resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25-
fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which
completely abolishes the agonistic activity of peptide (1) and renders it with a potent
antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides
tested were inactive. Tyr-2, residue, along with its neighboring environment was a key
determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the
transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as
YD-3, ML-354, and BMS-986120. π–π stacking interaction with Tyr-183 appears to be critical
and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKFNH2.
Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are
determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will
exert an agonistic or antagonistic activit
