Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRPs)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC) 1.
Using endothelium-denuded and intact rat aortic rings, the contractile and relaxant structure-activity profile for a series of TRPs were determined, based on the human and rat receptor sequences, SFLLR (P5), SFLLR-NH2 (P5-NH2) SFFLR (Rat P5), SFFLR-NH2 (Rat P5-NH2), SFLLRNP (P7), SFLLRNP-NH2 (P7-NH2), SFFLRNP (Rat P7), SFFLRNP-NH2 (Rat P7-NH2), and SFLLRNPNDKYEPF (P14) 2.
In 1992, many groups like Chao et al., Hui et al., Sabo et al., Vassallo et al, observed that peptides derived from the TRPs, beginning with serine and ranging from five (P5) to fourteen (P14) amino acids, mimiced the actions of thrombin in a variety of target tissues ranging from platelets to vascular and gastric smooth muscle 2.
Based on the minimal peptide sequence (Phe-Leu-Leu-Arg) that has been found to exhibit biological activity in a gastric smooth muscle contractile assay for thrombin receptor-activating peptides, the cyclic peptide analogues cyclo(Phe-Leu-Leu-Arg-Acp) (1), cyclo(Phe-Leu-Leu-Arg-eLys) (2), and cyclo(Phe-Leu-Leu-Arg-Gly) (3) have been synthesized by the solid-phase method using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoroborate or 2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate as cyclization reagents. The contractile activities of compounds 1-3 have been compared with that of the linear TRP Ser-Phe-Leu-Leu-Arg-NH2 (compound 4) using a gastric smooth muscle strip assay. Compound 2, wherein the e-amino group of lysine was coupled to the a-carboxyl of arginine, exhibited a contractile activity comparable to that of the linear TRP, compound 4. However compound 1, wherein the aminocaproic linker group yielded a ring size the same as for compound 2 but without a primary amino group, exhibited a contractile activity 600-1000-fold lower than compounds 2 and 4. Compound 3, which exhibited partial agonist activity, was about 100-fold less potent than either compound 2 or 4. NMR spectroscopy of compound 2 revealed a proximity of the Phe and Arg side chains, leading to a molecular model generated by distance geometry and molecular dynamics, wherein the Phe and Arg residues are shown in proximity on the same side of the peptide ring. It has been concluded that the Phe and Arg side chains along with the primary amino group form an active recognition motif that is augmented by the presence of a primary amino group in the cyclic peptide 3. It has been suggested that a comparable cyclic conformation may be responsible for the interaction of linear TRAPs with the thrombin receptor. The validity of this proposition was tested by the synthesis of four active nonpeptide thrombin receptor mimetics. Substance (S)-N-(6-guanidohexanoyl)-N'-(2-amino-3-phenylpropionyl)piperazine (3), in which the pharmacophoric phenyl, guanidino, and amino groups were incorporated onto a piperazine template, was found to be the most active compared to the other synthesized compounds which lack the amino pharmacophoric group 4.
Mode of Action
Thrombin receptor activation, by thrombin or SFLLR-containing peptides, stimulates GTPase activity in platelet and CHRF-288 membranes. Polyclonal antibodies to peptides derived from the thrombin receptor (anti-TR52-69 and anti-TR36-49), which block many of thrombin's actions on platelets and endothelial cells, also block thrombin activation of membrane GTPase (as does thrombin active site and anion-binding exosite inhibitors). Most of the receptor-activated GTPase, stimulated by both thrombin and SFLLRNP in platelet membranes, was inhibited by prior treatment with pertussis toxin or N-ethylmaleimide, suggesting that under these conditions much of the thrombin receptor-stimulated GTPase in platelet membranes is a member of the pertussis toxin-sensitive G alpha i family. In platelet membrane preparations, the peptide agonists stimulated approximately twice as much GTPase activity as stimulated by alpha-thrombin. In contrast, the membranes prepared from CHRF-288 cells showed similar maximal SFLLRNP- and alpha-thrombin-stimulated GTPase activity. Stimulation of the platelet membrane GTPase by a variety of different peptide agonists correlated with their ability to stimulate platelet aggregation. Studies show that the tethered- ligand receptor mediates the GTPase activation by thrombin in platelet and CHRF-288 cell membranes, and this provides a specific, reliable, and convenient cell-free assay system with which one can evaluate agonists and partial agonists 5.
Thrombin, apart from its widely recognized role as a coagulation factor, via the proteolytic cleavage of fibrinogen and the activation of platelets, has also been known for some time to regulate vascular contractility. Depending on the vascular preparation, thrombin can cause either an endothelium-dependent relaxation or an endothelium-independent contraction. The proteolytic mechanism whereby thrombin activates target tissues such as the platelet has been elucidated by Coughlin and colleagues, who in parallel with others, cloned the G-protein-coupled receptor for thrombin and demonstrated that proteolytic activation of the receptor involves the exposure of an amino terminal sequence (beginning with serine-42 in the human receptor), that acts as an 'anchored' or 'tethered' receptor activating moiety. Remarkably, peptides derived from the TRPs, beginning with serine and ranging from five (P5) to fourteen (P14) amino acids, have been found to mimic the actions of thrombin in a variety of target tissues ranging from platelets to vascular and gastric smooth muscle 2.
1. Shankar R, de la Motte CA, Poptic EJ, DiCorleto PE (1994). Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells. The Journal of Biological Chemistry, 269:13936-13941.
2. Laniyonu AA, Hollenberg MD (1995). Vascular actions of thrombin receptor-derived polypeptides: structure-activity profiles for contractile and relaxant effects in rat aorta. Br J Pharmacol., 114(8):1680–1686.
3. Matsoukas JM, Panagiotopoulos D, Keramida M, Mavromoustakos T, Yamdagni R, Wu Q, Moore GJ, Saifeddine M, Hollenberg MD (1996). Synthesis and contractile activities of cyclic thrombin receptor-derived peptide analogues with a Phe-Leu-Arg motif : Importance of the Phe/Arg relative conformation and the primary amino group for activity. Journal of medicinal chemistry, 39(18):3585-3591.
4. Alexopoulos K, Panagiotopoulos D, Mavromoustakos T, Fatseas P, Paredes-Carbajal MC, Mascher D, Mihailescu S, Matsoukas J. 2001. Design, synthesis, and modeling of novel cyclic thrombin receptor-derived peptide analogues of the Ser42-Phe-Leu-Leu-Arg46 motif sequence with fixed conformations of pharmacophoric groups: importance tg rg yhy a Phe/Arg/NH2 cluster for receptor activation and implications in the design of nonpeptide thrombin receptor mimetics. J Med Chem., 44(3):328-339.
5. Seiler SM, Peluso M, Tuttle JG, Pryor K, Klimas C, Matsueda GR, Bernatowicz MS (1996). Thrombin receptor activation by thrombin and receptor-derived peptides in platelet and CHRF-288 cell membranes: receptor-stimulated GTPase and evaluation of agonists and partial agonists. Mol Pharmacol., 49:190-197.
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