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A peptide ligand of the human thrombin receptor antagonizes thrombin receptor activating peptide and α -thrombin-induced platelet aggregation
Fibrinolysis & Proteolysis (2000) 14 (1), 15–21 © Harcourt Publishers Ltd 2000 doi: 10.1054/ fipr.2000.0046, available online at http://www.idealibrary.com on
A peptide ligand of the human thrombin receptor antagonizes thrombin receptor activating peptide and α-thrombin-induced platelet aggregation R. Pakala,1 T. Chyou Liang,2 C. R. Benedict1 1
Department of Internal Medicine, Division of Cardiology, University of Texas Health Science Center – Medical School Houston, TX, USA Department of Biochemistry, University of Texas Health Center – Medical School, Houston, TX, USA
2
Summary Structure and function studies on thrombin receptor activating peptide have revealed that certain residues in this peptide could be replaced with alanine. Attempts to prepare antagonist peptides by single amino acid modification of thrombin receptor activating peptide have not resulted in potent antagonist peptide. In the present study, we report an antagonist peptide with multiple alanine substitutions in both critical and non-critical residues. At a concentration of 32 µM, this peptide could completely block agonist-induced platelet aggregation. The magnitude of the antagonist effect of this peptide depends on the concentration of the antagonist and preincubation time. This peptide blocked the platelet aggregation induced by the agonist peptide and also by α-thrombin, but did not have any effect on adenosine diphosphate or collagen-induced platelet aggregation indicating that the antagonist affects of this peptide may be pertained to thrombin receptor mediated events only. This peptide may be useful for blocking thrombinmediated events like thrombosis and restenosis or can be used as a template for developing more efficient thrombin receptor antagonists. © Harcourt Publishers Ltd 2000
INTRODUCTION Thrombin a serine protease is the central enzyme of the coagulation cascade. In addition to this pivotal role thrombin is also an essential component of proliferative and inflammatory responses to injury.1 Most if not all of these responses of thrombin are mediated by binding of thrombin to specific receptor.2,3 Since platelet aggregation is the main event in the thrombosis there has been an intense interest in elucidating the mechanism of thrombin action on its platelet receptors. The unique mechanism of action of thrombin receptor has made it possible to study some of the biological consequences specific for its activation.4–6 Thrombin receptor, a seven Received: 11 October 1999 Accepted after revision: 13 January 2000 Correspondence to: Rajbabu Pakala, Department of Internal Medicine, Division of Cardiology, University of Texas HSC- Medical School, 6431 Fannin, MSB 6.039, Houston, TX 77030, USA. Tel.: + 1 713 500 6622; fax: + 1 713 500 6625
transmembrane domain G-protein coupled receptor, is activated by a peptide that consists of the tethered ligand that is exposed after activation by thrombin cleavage.4–7 Receptor activation by this agonist peptide has revealed that many of the thrombin’s short-term effects can be attributed to the cloned receptor. In human platelets this includes activation of phospholipase C and phosphatidyl inostital 3- kinase,8,9 inhibition of adenylate cyclase,10 tyrosine phosphorylation of several proteins11 and full activation, secretion and aggregation.4,12,13 However, unlike thrombin, thrombin receptor activating peptide does not stimulate platelet procoagulent activity.14 The agonist peptide also has effects that parallel the thrombin-mediated short-term effects on fibroblasts,12,15 erythroleukemia cells,16 endothelial cells,17 glomerular mesangial cells18 and gastric smooth muscle cells.19 Furthermore, specific activation of the thrombin receptor by the agonist peptide induces growth of vascular smooth muscle cells20 and causes shape change in neuronal cells.21 15
16 Pakala et al.
Structure activity relationship between various synthetic peptides and the thrombin receptor have been studied in fibroblasts,22 platelets10,23,24 and cells expressing recombinant thrombin receptor.23 We describe here a peptide AFLARAA, that antagonizes thrombin and thrombin receptor activating peptide (SFLLRNA) induced platelet aggregation. Interestingly, the antagonist also induces platelet activation at very high concentrations (EC50 750–1000 µM). MATERIAL AND METHODS Materials Thrombin, Dicyclohexyl carboxiimide (DCC) were purchased from Sigma (St. Louis, MO). T-butyloxycorbonyl (BOC) protected amino acids and merrifield resins were obtained from advanced Chem Tech (Louisville, KY). Trifluroacetic (TFA) and trifluromethane sulfuric acids were from Lancaster synthesis (Windham, NH). Thioansisole, ethylenedithiol, N-hydroxyl succinimide, 1-hydroxybenzotriazole (HOBT), alumina (acid) and thiophenol were from Aldrich Chemical Co. (Milwaukee, WI) Dimethyl formamide was from Fisher Scientific and dried with acidic alumina and then stored over 4A molecule scives. Adenosine diphosphate (ADP) and collagen were obtained from Helena Laboratories (Beaumont, TX). Other reagent grade solvents and chemicals were obtained from commercial source and used without further purification. Peptide synthesis Peptides were synthesized using standard solid-phase peptide synthesis methodology using Boc amino acids and Merrified resins as described by Merrifield.25,26 The crude peptides were first precipitated from the cleavage solution by addition of ethyl ether and then purified on a LoBar C-18 reverse phase column (E. Merck; 2.5 × 30 cm). The pure fractions from these purification procedures were identified with analytical high performance liquid chromatography and combined. These combined fractions were first stripped of organic solvents on a rotary evaporator and then lyophilized. The identities of these peptides were confirmed by proton nuclear magnetic resonance spectroscopy and/or fast atom bombardment mass spectrometry. All the peptides were dissolved in normal saline. Preparation and aggregation of human platelets Nine volumes of blood were collected from healthy donors (no medication for at least 10 days before donation) in to 1 volume of citrate buffer (3.8% sodium
Fibrinolysis & Proteolysis (2000) 14(1), 15–21
citrate). The blood samples were centrifuged at 100 g for 15 min at room temperature and the supernatant platelet rich plasma (PRP) was carefully collected with a plastic pipette. Rest of the blood was further centrifuged at 2000 g for 15 min at room temperature to obtain platelet poor plasma (PPP). For preparation of washed platelets, PRP was centrifuged at 100 g for 10 min at room temperature and the resulting platelet pellet washed twice with tyrodes buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3 0.3 mM Na2HPO4, 2 mM CaCl2 5.5 mM glucose pH 7.35) containing 0.35% human albumin. Platelet counts in both PRP and washed platelets were adjusted to 3 × 105/ µl. Aggregation was measured at 37°C by a turbidimetric method in a biodata-4 aggregometer in a final volume of 400 µl with indicated additions and with continuous stirring at 1200 RPM. Before performing the assays the aggregometer was standardized with PPP for PRP and tyrodes buffer for washed platelets. In a typical aggregation assay, 350 µl of PRP or washed platelets were first equilibrated at 37°C for at least 10 min in siliconized glass tubes. To the stirred PRP, 50 µl aliquots of peptide stock solutions of appropriate concentration were added to achieve the desired final concentration. Progression of platelet aggregation was followed for 10 to 20 min. To study the inhibition of platelet activation, 25 µl aliquots of the antagonist peptide stock solution of appropriate concentrations were first added to the thermally equilibrated PRP. After specific time intervals, 25 µl aliquots of the agonist peptide stock solution of various concentrations were then added to start the aggregation. Washed platelets were used to study the effect of α-thrombin. To study the antagonist effects on other platelet receptors, 25 µl of antagonist peptide (final concentration 16 µM) or normal saline were added to 350 µl of thermally equilibrated PRP. After 5 min of incubation either ADP (25 µl from a 0.4 mM stock solution), collagen (25 µl from a 200 µg/ml stock solution) or agonist or antagonist peptide were added and platelet aggregation monitored for another 10 min. Results are representative of minimum three experiments and all the experiments were performed with plasma from different donors. RESULTS Effect of multiple alanine substitutions involving critical and non-critical residues in thrombin receptor activating peptide on platelet aggregation Earlier studies have indicated that the first five residues in the thrombin receptor related peptide are important for its platelet aggregating activity. Inclusion of sixth residue aspargine slightly improves the activity of this agonist peptide. However, addition of a seventh residue
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An antagonist peptide 17
platelet aggregation. Then, we synthesized peptides with multiple alanine substitutions which included one or two of the critical residues and tested them for their ability to reverse SFLLRNA-induced platelet aggregation. Results indicate (Table 1, Group II) that most of these peptides were not effective agonists or antagonists. One peptide, however, (peptide 14, AFLARAA) showed a weak agonist activity (EC50 700–100 µM) and a moderate antagonist activity (IC50 16–32 µM). The structural features important for the antagonist function of this peptide (peptide 14) were investigated by amino acid substitutions in this peptide. As shown in Table 1 (Group III), deletion of the n-terminal alanine resulted in a significant loss of antagonist potency (peptide 24). Another peptide (peptide 23) where N-terminal alanine was replaced with a propionyl group also had no significant antagonist activity. These results indicate that N-terminal alanine is important for the antagonist activity.
proline or residues further to the C-terminal end resulted in a slight loss of the activity. Decreased activities seen in longer peptides may be due to the presence of proline at the seventh position, and alanine substitution for this proline resulted in a slight improvement in the activity. Previous attempts to modify the agonist peptide by single amino acid substitutions did not result in an effective antagonist peptide. Therefore, using the hepta peptide (SFLLRNA) as a base sequence we synthesized peptides with multiple amino acid substitutions. Since it is known that side chains of second position phenylalanine, fourth position leucine and fifth position arginine are important for agonist binding, we synthesized peptides with double and triple alanine substitutions of non-critical residues, i.e. first position serine, third position leucine and sixth position asparagine. As shown in Table 1 (Group 1) these multiple alanine substituted peptides at reasonable concentrations did not antagonized the SFLLRNA-induced Table 1 No.
Peptide sequence
1. 2. 3. 4. 5. 6. 7. 8.
SFLLRNA Ac-FLLRNA Pr-FLLRNA SFLLRYA SFLYRNA SFYLRNA YFLLRNA SFLLLNA
EC50 (µM) as agonist*
IC50 (µM) as antagonist*
1.8–2 a a 9.6–12.8 32–50 16–32 a 100–125
– b b b b b b b
Group I: Multiple alanine substitutions involving noncritical residues 9. SFALRAA b 10. AFLLRAA b 11. AFALRNA b 12. AFALRAA b Group II: Multiple alanine substitutions involving critical residues 13. SFLARAA 125–250 14. AFLARAA 750–1000 15. AFAARNA b 16. AAALRNA b 17. AFALANA b 18. AALLRAA b 19. AFLLAAA b Group III: Analogues of the antagonist 20. AFYARAA 21. AYLARAA 22. YFLARAA 23. Pr-FLARA 24. FLARAA
b a a a a
b b b b a 16–33 b b b b b b* a** a** a** a**
*Concentrations are presented in ranges rather than specific numbers, since both the agonist and the antagonist effects are very sensitive to the changes in concentrations. The two numbers indicate the concentrations at which less than 20% or greater than 80% of the full responses were detected a: no effect at 500 µM; b: no effect at 250 µM. **Preincubated for 5 min. To determine the agonist activity of the peptide, 50 µl of peptide dissolved in normal saline was added to 350 µl of platelet rich plasma equilibrated at 37°C and platelet aggregation determined using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. For determing the antagonist activity of the peptide 25 µl of antagonist peptide to be tested and 25 µl of SFLLRNA were added to 350 µl of platelet rich plasma equilibrated at 37°C and platelet aggregation determined using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Different concentrations of stock solutions were used to achieve the desired concentrations. Results are representative of minimum three experiments performed with plasma from different donors.
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Fibrinolysis & Proteolysis (2000) 14(1), 15–21
18 Pakala et al.
Concentration and time-dependent action of the antagonist peptide In order to elucidate the mechanism of action of the antagonist peptide we examined the possible competition between the antagonist and agonist peptides. As shown in Figure 1, when platelets were incubated with 32 µM antagonist peptide and then challenged with 4 µM agonist peptide, antagonist peptide blocked agonist peptide-induced platelet aggregation by 50%, while the blocking effect was only 30% when platelet aggregation was induced with 8 µM agonist. These results suggest that the extent of antagonist effect of AFLARAA depends on the concentrations of both agonist and antagonist. To further elucidate the mechanism of action of the antagonist peptide we investigated the effect of preincubation of platelets with antagonist on agonist-induced platelet aggregation. Platelets in platelet-rich plasma were preincubated with 16 µM antagonist peptide for different time periods and at indicated time points were stimulated with the agonist peptide. As shown in Figure 2 the ability of antagonist peptide to block agonist-induced platelet aggregation increases with the length of preincubation. Since the sequence of antgonist peptide is based on the
Fig. 1 Effect of antagonist peptide concentration on agonist peptide-induced platelet aggregation. 25 µl aliquots of thrombin receptor activating peptide antagonist (TRAP) in normal saline and 25 µl of thrombin receptor activating peptide (TRAPA) in normal saline were added to 350 µl of platelet-rich plasma equilibrated at 37°C. Where TRAP alone was used 25µl of normal saline was added. Different concentrations of stock solutions were used to achieve the indicated concentrations. Platelet aggregation was measured using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors. ▲: 4 µM TRAP; ▲: 8 µM TRAP; •: 16 µM TRAPA + 4 µM TRAP; ●: 32 µM TRAPA + 8 µM TRAP; ■: 16 µM TRAPA + 4 µM TRAP; ■: 32 µM TRAPA + 8 µM TRAP
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agonist sequence we assessed the ability of this peptide to block α-thrombin induced platelet aggregation. Washed platelets were preincubated with carrier or antagonist peptide for 5 min and then stimulated with 22.5 nM (1 unit) of α-thrombin and the platelet aggregation monitored. The results indicate (Fig. 3) that the antagonist peptide blocked α-thrombin induced platelet aggregation. Effect of antagonist peptide on ADP and collageninduced platelet aggregation The antagonist peptide was synthesized based on the known thrombin receptor activating peptide sequence. Therefore, it is reasonable to presume that the antagonist peptide is specific to thrombin receptor mediated actions only and may not have any effect on ADP or collageninduced platelet aggregation. To demonstrate this platelets were first incubated with 16 µM antagonist peptide for 5 min and then stimulated with either ADP or collagen or agonist or antagonist peptide. Controls were incubated with normal saline for same time periods. The results (Fig. 4) indicate that antagonist peptide did not have any effect on ADP or collagen-induced platelet aggregation. Moreover, the results also indicate that incubation of platelets with antagonist peptide did not make them less responsive to ADP or collagen.
Fig. 2 Time-dependent inhibition of platelet aggregation by antagonist peptide. 25 µl of normal saline (control) or 25 µl of thrombin receptor activating peptide antagonist (TRAPA) in normal saline (final concentrations 16 µM) were added to 350 µl of plateletrich plasma equilibrated at 37°C. At indicated time points 25 µl of thrombin receptor activating peptide (TRAP) in normal saline was added (final concentration 4 µM) and platelet aggregation measured using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors. ●: control; ▼: 0 min preincubation; ■: 0.5 min preincubation; ▲: 1 min preincubation; ◆: 2 min preincubation; •: 5 min preincubation
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An antagonist peptide 19
Fig. 3 Effect of antagonist peptide on α-thrombin induced platelet aggregation. 350 µl of washed platelets in tyroed’s buffer and 350 µl of platelet rich plasma were incubated with either 25 µl of normal saline or 25 µl of thrombin receptor activating peptide antagonist (TRAPA) in normal saline (final concentration 16 µM). After 5 min of incubation 22.5 nM (1 unit) of thrombin was added to washed platelets incubated with saline (■) and TRAPA (■ ■). Similarly, 4 µM thrombin receptor activating peptide was added to platelet-rich plasma incubated with saline (•) and TRAPA (● ●). Platelet aggregation was measured using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors.
Fig. 4 Effect of antagonist peptide on ADP and collagen-induced platelet aggregation. 25 µl of normal saline or 25 µl of thrombin receptor activating peptide antagonist (TRAPA, final concentration 16 µM) were added to 350 µl of platelet-rich plasma equilibrated at 37°C. After 5 min of preincubation saline-incubated platelets were stimulated with 25 µl of saline (◆ ◆) or 25 µl of thrombin receptor activating peptide (TRAP, final concentration 4 µM, ●) or 25 µl of ADP (final concentration 25 µM, ▲) or 25 µl of collagen (final concentration 5 µg, ■). Similarly, TRAPA-incubated platelets were also stimulated with saline (◆), TRAP (•), ADP (▲) or collagen (■) and platelet aggregation measured using a Biodata-4 aggregometer. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors.
DISCUSSION In the present study by multiple alanine substitutions in the thrombin receptor activating peptide, we have shown that non-critical residues first position serine third position leucine and sixth position aspergine may also contribute to the activity of the peptide. Although, single alanine substitutions of these residues were reported to have minimal effects on the agonist activity of the resultant peptides, simultaneously replacing two or three of these residues with alanine resulted in a marked reduction in the agonist activity. The magnitude of this reduced activity was significantly greater than the combined effect resulted from two individual substitutions indicating that when more than one non-critical residue is replaced with alanine they act synergistically. One of the peptides with multiple substitutions at first position serine, fourth position leucine and sixth position aspergine (both critical and non-critical residues, AFLARAA) acted as an antagonist (IC50 16–32 µM). Giesberts et al.9 have reported that 3-mercaptapropionic acid-Phecyclohexylalanine-cyclohexylalanine-Arg-LysPro-Asn-Asp-Lys-NH2, inhibited ATP secreation induced by α-thrombin (IC50 5 µM). Similarly, Scarborough et al.27, 28 have demonstrated that 3-mercaptopropionylPhe- cyclohexylanine- cyclohexylalanine-Arg- LysPro-Asn-Asp-Lys-Amide prevented α-thrombin and © Harcourt Publishers Ltd 2000
TRAP-induced platelet aggregation with little effect on collagen-induced platelet aggregation. They have also shown that the same peptide prevented α-thrombin and TRAP-induced calcium mobilization with little effect on calcium mobilization in response to thromboxane receptor antagonist. Like most of the antagonists developed based on the thrombin receptor activating peptide, ALFARAA also is a week agonist. However, the concentrations of ALFARAA required for platelet activation are much more higher (EC50 750–1000 µM) as compared to Phe-Leu-Leu-Arg-Asn-Pro (EC50 200 µM, Vassallo et al. 1992)10 and Tyr-Phe-Leu- Leu-Arg-Asn-Pro (EC50 100 µM, Rasmussen et al. 1993).29 Unlike the dipeptide antagonists which were not based on thrombin receptor activating peptide structure30,31 AFLARAA had no effect on adenosite diphosphate or collagen-induced platelet aggregation indicating that AFLARAA is a specific thrombin receptor inhibitor. The observation that the inhibitory effect of the antagonist peptide could be reversed by the agonist in a concentration-dependent manner suggests that the antagonist may simply compete with the agonist for the same binding site on the receptor. However, the following observations imply that this may not be true: (1) the time-dependent nature of the inhibition; (2) antagonist Fibrinolysis & Proteolysis (2000) 14(1), 15–21
20 Pakala et al.
behaving like an agonist at higher concentrations; (3) reduction in antagonistic property with a simultaneous in the agonist property upon replacing first alanine with serine. This result is in contrast with the observation that peptides with either alanine or serine as the first residue have similar agonist properties.24 Based on these observations it is likely that the antagonist peptide reported here may bind to thrombin receptor at a site different from that of the agonist. Alternatively, if the agonist and antagonist do bind at the same site, the specific interactions (Eg H-brand, hydrophobic and ion interactions) involved for the two effects may be different. Receptor phosphorylation has been shown to be involved in the regulation of many G-protein coupled receptors.32 It is also thrown that most of the G-protein coupled receptors could be desensitized if they are preincubated with subthreshold levels of agonist.32,33 For thrombin receptor such homodesensitization was shown to involve receptor phosphorylation.38 In the present study also binding of antagonist to the receptor (either at the same site as the agonist binding site, but with a different set of interactions or at a different site) may lead to phosphorylation of the thrombin receptor making the thrombin receptor desensitized and unavailable to the agonist. In summary, we have shown that the peptide AFLARAA is an antagonist to α-thrombin and SFLLRNA-induced human platelet aggregation. Thus, AFLARAA may be a useful tool for differentiating between several possible activation states of the human thrombin receptor, or it may provide a structural template for developing more efficient antagonists targeted to the thrombin receptor. ACKNOWLEDGEMENTS The authors would like to thank Shirley Mcwhorter for her help in typing this manuscript. REFERENCES 1. Fenton JW. II Regulation of thrombin generation and functions. Sem in Thromb and Hemost 1988; 14: 234–240. 2. Coughlin SR. Thrombin receptor function and cardiovascular disease. Trends Cardiovasc Med 1994; 4: 77–83. 3. Ogletree ML, S Natarajan, SM Seiler. Thrombin receptors as drug discovery targets. Perspect Drug Discov Design 1994; 1: 527–536. 4. Vu T-K-H, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–1068. 5. Rasmussen UB, Vouret-Craviari V, Jallat S et al. cDNA cloning and expression of a hamster-thrombin receptor coupled to Ca2+ mobilization. FEBS 1991; 288: 123–128. 6. Zhong C, Hayzer DJ, Carson MA, Runge MS. Molecular cloning at the rat vascular smooth muscle thrombin receptor: evidence for in vitro regulation by basic fibroblast growth factor. J Biol Chem 1992; 267: 16975–16979.
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7. Vu T-KH, Wheton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674–677. 8. Hung R-S, Sorisky A, Church WR, Simons ER, Rittenhouse SE. Thrombin receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3phosphorylated phosphoinositides. J Biol Chem 1991; 266: 18435–18438. 9. Giesberts AN, van Willigen G, Lapetina EG, Akkerman JW. Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor. Biochem J 1995; 309: 613–620. 10. Seiler SM, Michel IM, Fenton II JW. Involvement of the tethered ligand receptor in thrombin inhibition of platelet adenylate cylase. Biochem Biophys Res Comm 1992; 182: 1296–1302. 11. Vassallo JR RR, Kieber-Emmons T, Chichowski K, Brass LF. Structure-function relationship in the activation of platelet thrombin receptors by receptor derived peptides. J Biol Chem 1992; 267: 6081–6085. 12. Vouret-Craviari V, Van Obberghen-Schilling E, Rasmussen UB, Pavirani A, Lecocq J-P, Pouyssegur J. Synthetic-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol of Cell 1992; 3: 95–102. 13. Hung DT, Vu T-KH, Wheaten VI, Coughlin SR. Clones platelet receptor in necessary for thrombin-induced platelet activation. J Clin Invest 1992; 89: 1350–1353. 14. Goodwin CA, Wheeler-Jones CP, Kakkar VV, Deadman JJ, Authi KS, Scully MF. Thrombin receptor activating peptide does not stimulate platelet procoagulant activity. Biochem Biophys Res Commun 1994; 202: 321–327. 15. Vouret-Craviari V, Van Obberghen-Schilling E, Scimeca JC, Van Obberghen E, Pouyssegun J. Differential activation of p44mapk (ERK1) by α-thrombin and thrombin-receptor peptide agonist. Biochem J 1993; 289: 209–214. 16. Brass LF, Vassallo JR RR, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J Biol Chem 1992; 267: 13795–13798. 17. Ngaiza JR, Jaffe EA. A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells. Biochem Biophys Res Commun 1991; 179: 1656–1661. 18. Troyer D, Padilla R, Smith T, Kreisberg J, Glass WI. Stimulation of the thrombin receptor of human glomerular mesangial cells by Ser-Phe-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Pro-Phe peptide. J Biol Chem 1992; 267: 20126–20131. 19. Hollenberg MD, Yang S-G, Laniyonu AA, Moore GJ, Saifeddine M. Action of a thrombin receptor polypeptide in gastric smooth muscle: identification of a core pentapeptide retaining full thrombin-mimetic activity. Mol Pharmacol 1992; 42: 186–191. 20. McNamara CA, Sarembock IJ, Gimple LW, Fenton II JW, Coughlin SR, Owen GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94–98. 21. Suidan H, Stone SR, Hemmings BA, Monard D. Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors. Neuron 1992; 8: 363–375. 22. Van Obberghen-Schilling E, Pouyssegur J. Signalling pathways of the thrombin receptor. Thromb Haemostas 1993; 70: 163–167. 23. Scarborough RM, Naughton MA, Teng W et al. Thethered ligand agonist peptides: structural requirements for thrombin
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receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem 1992; 267: 13146–13149. Chao BH, Kalkunte S, Marganore JM, Stone SR. Essential groups in synthetic agonist peptide for activation of the platelet thrombin receptor. Biochem 1992; 31: 6175–6178. Merrifield B. Solid phase synthesis. Science 1986; 232: 341–347. Merrifield B. Concept and early development of solid-phase peptide synthesis. Methods Enzymol 1997; 289: 3–13. Scarborough R, Teng W, Naughton MA, Rose JW, Alves V, Arfsten A. A thrombin receptor antagonist designed from tethered ligand agonist peptides. Circulation 1992; 86: 1–151. Scarborough RM, Teng W, Naughton MA et al. Thrombin receptor antagonists derived from “thethered ligand” agonist peptides. 13th American Peptide Symposium. Edmonton, Canada: Abst. 1993; P632. Rasmussen UB, Gachet C, Schlesinger Y et al. A peptide ligand of the human thrombin receptor antagonizes alpha-thrombin and patially activates platelets. J Biol Chem 1993; 268: 14322–14328. Ruda EM, Petty A, Scrutton MC, Tuffin DP, Manley PW. Identification of small peptide analogues having agonist and antagonist activity at the platelet thrombin receptor. Biochem Pharmacol 1988; 37: 2417–2426. Ruda EM, Scrutton MC, Manley PW, Tuffin DP. Thrombin receptor antagonist: Structure-activity relationships for the
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A peptide ligand of the human thrombin receptor antagonizes thrombin receptor activating peptide and α-thrombin-induced platelet aggregation R. Pakala,1 T. Chyou Liang,2 C. R. Benedict1 1
Department of Internal Medicine, Division of Cardiology, University of Texas Health Science Center – Medical School Houston, TX, USA Department of Biochemistry, University of Texas Health Center – Medical School, Houston, TX, USA
2
Summary Structure and function studies on thrombin receptor activating peptide have revealed that certain residues in this peptide could be replaced with alanine. Attempts to prepare antagonist peptides by single amino acid modification of thrombin receptor activating peptide have not resulted in potent antagonist peptide. In the present study, we report an antagonist peptide with multiple alanine substitutions in both critical and non-critical residues. At a concentration of 32 µM, this peptide could completely block agonist-induced platelet aggregation. The magnitude of the antagonist effect of this peptide depends on the concentration of the antagonist and preincubation time. This peptide blocked the platelet aggregation induced by the agonist peptide and also by α-thrombin, but did not have any effect on adenosine diphosphate or collagen-induced platelet aggregation indicating that the antagonist affects of this peptide may be pertained to thrombin receptor mediated events only. This peptide may be useful for blocking thrombinmediated events like thrombosis and restenosis or can be used as a template for developing more efficient thrombin receptor antagonists. © Harcourt Publishers Ltd 2000
INTRODUCTION Thrombin a serine protease is the central enzyme of the coagulation cascade. In addition to this pivotal role thrombin is also an essential component of proliferative and inflammatory responses to injury.1 Most if not all of these responses of thrombin are mediated by binding of thrombin to specific receptor.2,3 Since platelet aggregation is the main event in the thrombosis there has been an intense interest in elucidating the mechanism of thrombin action on its platelet receptors. The unique mechanism of action of thrombin receptor has made it possible to study some of the biological consequences specific for its activation.4–6 Thrombin receptor, a seven Received: 11 October 1999 Accepted after revision: 13 January 2000 Correspondence to: Rajbabu Pakala, Department of Internal Medicine, Division of Cardiology, University of Texas HSC- Medical School, 6431 Fannin, MSB 6.039, Houston, TX 77030, USA. Tel.: + 1 713 500 6622; fax: + 1 713 500 6625
transmembrane domain G-protein coupled receptor, is activated by a peptide that consists of the tethered ligand that is exposed after activation by thrombin cleavage.4–7 Receptor activation by this agonist peptide has revealed that many of the thrombin’s short-term effects can be attributed to the cloned receptor. In human platelets this includes activation of phospholipase C and phosphatidyl inostital 3- kinase,8,9 inhibition of adenylate cyclase,10 tyrosine phosphorylation of several proteins11 and full activation, secretion and aggregation.4,12,13 However, unlike thrombin, thrombin receptor activating peptide does not stimulate platelet procoagulent activity.14 The agonist peptide also has effects that parallel the thrombin-mediated short-term effects on fibroblasts,12,15 erythroleukemia cells,16 endothelial cells,17 glomerular mesangial cells18 and gastric smooth muscle cells.19 Furthermore, specific activation of the thrombin receptor by the agonist peptide induces growth of vascular smooth muscle cells20 and causes shape change in neuronal cells.21 15
16 Pakala et al.
Structure activity relationship between various synthetic peptides and the thrombin receptor have been studied in fibroblasts,22 platelets10,23,24 and cells expressing recombinant thrombin receptor.23 We describe here a peptide AFLARAA, that antagonizes thrombin and thrombin receptor activating peptide (SFLLRNA) induced platelet aggregation. Interestingly, the antagonist also induces platelet activation at very high concentrations (EC50 750–1000 µM). MATERIAL AND METHODS Materials Thrombin, Dicyclohexyl carboxiimide (DCC) were purchased from Sigma (St. Louis, MO). T-butyloxycorbonyl (BOC) protected amino acids and merrifield resins were obtained from advanced Chem Tech (Louisville, KY). Trifluroacetic (TFA) and trifluromethane sulfuric acids were from Lancaster synthesis (Windham, NH). Thioansisole, ethylenedithiol, N-hydroxyl succinimide, 1-hydroxybenzotriazole (HOBT), alumina (acid) and thiophenol were from Aldrich Chemical Co. (Milwaukee, WI) Dimethyl formamide was from Fisher Scientific and dried with acidic alumina and then stored over 4A molecule scives. Adenosine diphosphate (ADP) and collagen were obtained from Helena Laboratories (Beaumont, TX). Other reagent grade solvents and chemicals were obtained from commercial source and used without further purification. Peptide synthesis Peptides were synthesized using standard solid-phase peptide synthesis methodology using Boc amino acids and Merrified resins as described by Merrifield.25,26 The crude peptides were first precipitated from the cleavage solution by addition of ethyl ether and then purified on a LoBar C-18 reverse phase column (E. Merck; 2.5 × 30 cm). The pure fractions from these purification procedures were identified with analytical high performance liquid chromatography and combined. These combined fractions were first stripped of organic solvents on a rotary evaporator and then lyophilized. The identities of these peptides were confirmed by proton nuclear magnetic resonance spectroscopy and/or fast atom bombardment mass spectrometry. All the peptides were dissolved in normal saline. Preparation and aggregation of human platelets Nine volumes of blood were collected from healthy donors (no medication for at least 10 days before donation) in to 1 volume of citrate buffer (3.8% sodium
Fibrinolysis & Proteolysis (2000) 14(1), 15–21
citrate). The blood samples were centrifuged at 100 g for 15 min at room temperature and the supernatant platelet rich plasma (PRP) was carefully collected with a plastic pipette. Rest of the blood was further centrifuged at 2000 g for 15 min at room temperature to obtain platelet poor plasma (PPP). For preparation of washed platelets, PRP was centrifuged at 100 g for 10 min at room temperature and the resulting platelet pellet washed twice with tyrodes buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3 0.3 mM Na2HPO4, 2 mM CaCl2 5.5 mM glucose pH 7.35) containing 0.35% human albumin. Platelet counts in both PRP and washed platelets were adjusted to 3 × 105/ µl. Aggregation was measured at 37°C by a turbidimetric method in a biodata-4 aggregometer in a final volume of 400 µl with indicated additions and with continuous stirring at 1200 RPM. Before performing the assays the aggregometer was standardized with PPP for PRP and tyrodes buffer for washed platelets. In a typical aggregation assay, 350 µl of PRP or washed platelets were first equilibrated at 37°C for at least 10 min in siliconized glass tubes. To the stirred PRP, 50 µl aliquots of peptide stock solutions of appropriate concentration were added to achieve the desired final concentration. Progression of platelet aggregation was followed for 10 to 20 min. To study the inhibition of platelet activation, 25 µl aliquots of the antagonist peptide stock solution of appropriate concentrations were first added to the thermally equilibrated PRP. After specific time intervals, 25 µl aliquots of the agonist peptide stock solution of various concentrations were then added to start the aggregation. Washed platelets were used to study the effect of α-thrombin. To study the antagonist effects on other platelet receptors, 25 µl of antagonist peptide (final concentration 16 µM) or normal saline were added to 350 µl of thermally equilibrated PRP. After 5 min of incubation either ADP (25 µl from a 0.4 mM stock solution), collagen (25 µl from a 200 µg/ml stock solution) or agonist or antagonist peptide were added and platelet aggregation monitored for another 10 min. Results are representative of minimum three experiments and all the experiments were performed with plasma from different donors. RESULTS Effect of multiple alanine substitutions involving critical and non-critical residues in thrombin receptor activating peptide on platelet aggregation Earlier studies have indicated that the first five residues in the thrombin receptor related peptide are important for its platelet aggregating activity. Inclusion of sixth residue aspargine slightly improves the activity of this agonist peptide. However, addition of a seventh residue
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An antagonist peptide 17
platelet aggregation. Then, we synthesized peptides with multiple alanine substitutions which included one or two of the critical residues and tested them for their ability to reverse SFLLRNA-induced platelet aggregation. Results indicate (Table 1, Group II) that most of these peptides were not effective agonists or antagonists. One peptide, however, (peptide 14, AFLARAA) showed a weak agonist activity (EC50 700–100 µM) and a moderate antagonist activity (IC50 16–32 µM). The structural features important for the antagonist function of this peptide (peptide 14) were investigated by amino acid substitutions in this peptide. As shown in Table 1 (Group III), deletion of the n-terminal alanine resulted in a significant loss of antagonist potency (peptide 24). Another peptide (peptide 23) where N-terminal alanine was replaced with a propionyl group also had no significant antagonist activity. These results indicate that N-terminal alanine is important for the antagonist activity.
proline or residues further to the C-terminal end resulted in a slight loss of the activity. Decreased activities seen in longer peptides may be due to the presence of proline at the seventh position, and alanine substitution for this proline resulted in a slight improvement in the activity. Previous attempts to modify the agonist peptide by single amino acid substitutions did not result in an effective antagonist peptide. Therefore, using the hepta peptide (SFLLRNA) as a base sequence we synthesized peptides with multiple amino acid substitutions. Since it is known that side chains of second position phenylalanine, fourth position leucine and fifth position arginine are important for agonist binding, we synthesized peptides with double and triple alanine substitutions of non-critical residues, i.e. first position serine, third position leucine and sixth position asparagine. As shown in Table 1 (Group 1) these multiple alanine substituted peptides at reasonable concentrations did not antagonized the SFLLRNA-induced Table 1 No.
Peptide sequence
1. 2. 3. 4. 5. 6. 7. 8.
SFLLRNA Ac-FLLRNA Pr-FLLRNA SFLLRYA SFLYRNA SFYLRNA YFLLRNA SFLLLNA
EC50 (µM) as agonist*
IC50 (µM) as antagonist*
1.8–2 a a 9.6–12.8 32–50 16–32 a 100–125
– b b b b b b b
Group I: Multiple alanine substitutions involving noncritical residues 9. SFALRAA b 10. AFLLRAA b 11. AFALRNA b 12. AFALRAA b Group II: Multiple alanine substitutions involving critical residues 13. SFLARAA 125–250 14. AFLARAA 750–1000 15. AFAARNA b 16. AAALRNA b 17. AFALANA b 18. AALLRAA b 19. AFLLAAA b Group III: Analogues of the antagonist 20. AFYARAA 21. AYLARAA 22. YFLARAA 23. Pr-FLARA 24. FLARAA
b a a a a
b b b b a 16–33 b b b b b b* a** a** a** a**
*Concentrations are presented in ranges rather than specific numbers, since both the agonist and the antagonist effects are very sensitive to the changes in concentrations. The two numbers indicate the concentrations at which less than 20% or greater than 80% of the full responses were detected a: no effect at 500 µM; b: no effect at 250 µM. **Preincubated for 5 min. To determine the agonist activity of the peptide, 50 µl of peptide dissolved in normal saline was added to 350 µl of platelet rich plasma equilibrated at 37°C and platelet aggregation determined using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. For determing the antagonist activity of the peptide 25 µl of antagonist peptide to be tested and 25 µl of SFLLRNA were added to 350 µl of platelet rich plasma equilibrated at 37°C and platelet aggregation determined using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Different concentrations of stock solutions were used to achieve the desired concentrations. Results are representative of minimum three experiments performed with plasma from different donors.
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Fibrinolysis & Proteolysis (2000) 14(1), 15–21
18 Pakala et al.
Concentration and time-dependent action of the antagonist peptide In order to elucidate the mechanism of action of the antagonist peptide we examined the possible competition between the antagonist and agonist peptides. As shown in Figure 1, when platelets were incubated with 32 µM antagonist peptide and then challenged with 4 µM agonist peptide, antagonist peptide blocked agonist peptide-induced platelet aggregation by 50%, while the blocking effect was only 30% when platelet aggregation was induced with 8 µM agonist. These results suggest that the extent of antagonist effect of AFLARAA depends on the concentrations of both agonist and antagonist. To further elucidate the mechanism of action of the antagonist peptide we investigated the effect of preincubation of platelets with antagonist on agonist-induced platelet aggregation. Platelets in platelet-rich plasma were preincubated with 16 µM antagonist peptide for different time periods and at indicated time points were stimulated with the agonist peptide. As shown in Figure 2 the ability of antagonist peptide to block agonist-induced platelet aggregation increases with the length of preincubation. Since the sequence of antgonist peptide is based on the
Fig. 1 Effect of antagonist peptide concentration on agonist peptide-induced platelet aggregation. 25 µl aliquots of thrombin receptor activating peptide antagonist (TRAP) in normal saline and 25 µl of thrombin receptor activating peptide (TRAPA) in normal saline were added to 350 µl of platelet-rich plasma equilibrated at 37°C. Where TRAP alone was used 25µl of normal saline was added. Different concentrations of stock solutions were used to achieve the indicated concentrations. Platelet aggregation was measured using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors. ▲: 4 µM TRAP; ▲: 8 µM TRAP; •: 16 µM TRAPA + 4 µM TRAP; ●: 32 µM TRAPA + 8 µM TRAP; ■: 16 µM TRAPA + 4 µM TRAP; ■: 32 µM TRAPA + 8 µM TRAP
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agonist sequence we assessed the ability of this peptide to block α-thrombin induced platelet aggregation. Washed platelets were preincubated with carrier or antagonist peptide for 5 min and then stimulated with 22.5 nM (1 unit) of α-thrombin and the platelet aggregation monitored. The results indicate (Fig. 3) that the antagonist peptide blocked α-thrombin induced platelet aggregation. Effect of antagonist peptide on ADP and collageninduced platelet aggregation The antagonist peptide was synthesized based on the known thrombin receptor activating peptide sequence. Therefore, it is reasonable to presume that the antagonist peptide is specific to thrombin receptor mediated actions only and may not have any effect on ADP or collageninduced platelet aggregation. To demonstrate this platelets were first incubated with 16 µM antagonist peptide for 5 min and then stimulated with either ADP or collagen or agonist or antagonist peptide. Controls were incubated with normal saline for same time periods. The results (Fig. 4) indicate that antagonist peptide did not have any effect on ADP or collagen-induced platelet aggregation. Moreover, the results also indicate that incubation of platelets with antagonist peptide did not make them less responsive to ADP or collagen.
Fig. 2 Time-dependent inhibition of platelet aggregation by antagonist peptide. 25 µl of normal saline (control) or 25 µl of thrombin receptor activating peptide antagonist (TRAPA) in normal saline (final concentrations 16 µM) were added to 350 µl of plateletrich plasma equilibrated at 37°C. At indicated time points 25 µl of thrombin receptor activating peptide (TRAP) in normal saline was added (final concentration 4 µM) and platelet aggregation measured using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors. ●: control; ▼: 0 min preincubation; ■: 0.5 min preincubation; ▲: 1 min preincubation; ◆: 2 min preincubation; •: 5 min preincubation
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An antagonist peptide 19
Fig. 3 Effect of antagonist peptide on α-thrombin induced platelet aggregation. 350 µl of washed platelets in tyroed’s buffer and 350 µl of platelet rich plasma were incubated with either 25 µl of normal saline or 25 µl of thrombin receptor activating peptide antagonist (TRAPA) in normal saline (final concentration 16 µM). After 5 min of incubation 22.5 nM (1 unit) of thrombin was added to washed platelets incubated with saline (■) and TRAPA (■ ■). Similarly, 4 µM thrombin receptor activating peptide was added to platelet-rich plasma incubated with saline (•) and TRAPA (● ●). Platelet aggregation was measured using a Biodata-4 aggregometer with continuous stirring at 1200 RPM. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors.
Fig. 4 Effect of antagonist peptide on ADP and collagen-induced platelet aggregation. 25 µl of normal saline or 25 µl of thrombin receptor activating peptide antagonist (TRAPA, final concentration 16 µM) were added to 350 µl of platelet-rich plasma equilibrated at 37°C. After 5 min of preincubation saline-incubated platelets were stimulated with 25 µl of saline (◆ ◆) or 25 µl of thrombin receptor activating peptide (TRAP, final concentration 4 µM, ●) or 25 µl of ADP (final concentration 25 µM, ▲) or 25 µl of collagen (final concentration 5 µg, ■). Similarly, TRAPA-incubated platelets were also stimulated with saline (◆), TRAP (•), ADP (▲) or collagen (■) and platelet aggregation measured using a Biodata-4 aggregometer. Results are representative of minimum three experiments, and all experiments were performed with plasma from different donors.
DISCUSSION In the present study by multiple alanine substitutions in the thrombin receptor activating peptide, we have shown that non-critical residues first position serine third position leucine and sixth position aspergine may also contribute to the activity of the peptide. Although, single alanine substitutions of these residues were reported to have minimal effects on the agonist activity of the resultant peptides, simultaneously replacing two or three of these residues with alanine resulted in a marked reduction in the agonist activity. The magnitude of this reduced activity was significantly greater than the combined effect resulted from two individual substitutions indicating that when more than one non-critical residue is replaced with alanine they act synergistically. One of the peptides with multiple substitutions at first position serine, fourth position leucine and sixth position aspergine (both critical and non-critical residues, AFLARAA) acted as an antagonist (IC50 16–32 µM). Giesberts et al.9 have reported that 3-mercaptapropionic acid-Phecyclohexylalanine-cyclohexylalanine-Arg-LysPro-Asn-Asp-Lys-NH2, inhibited ATP secreation induced by α-thrombin (IC50 5 µM). Similarly, Scarborough et al.27, 28 have demonstrated that 3-mercaptopropionylPhe- cyclohexylanine- cyclohexylalanine-Arg- LysPro-Asn-Asp-Lys-Amide prevented α-thrombin and © Harcourt Publishers Ltd 2000
TRAP-induced platelet aggregation with little effect on collagen-induced platelet aggregation. They have also shown that the same peptide prevented α-thrombin and TRAP-induced calcium mobilization with little effect on calcium mobilization in response to thromboxane receptor antagonist. Like most of the antagonists developed based on the thrombin receptor activating peptide, ALFARAA also is a week agonist. However, the concentrations of ALFARAA required for platelet activation are much more higher (EC50 750–1000 µM) as compared to Phe-Leu-Leu-Arg-Asn-Pro (EC50 200 µM, Vassallo et al. 1992)10 and Tyr-Phe-Leu- Leu-Arg-Asn-Pro (EC50 100 µM, Rasmussen et al. 1993).29 Unlike the dipeptide antagonists which were not based on thrombin receptor activating peptide structure30,31 AFLARAA had no effect on adenosite diphosphate or collagen-induced platelet aggregation indicating that AFLARAA is a specific thrombin receptor inhibitor. The observation that the inhibitory effect of the antagonist peptide could be reversed by the agonist in a concentration-dependent manner suggests that the antagonist may simply compete with the agonist for the same binding site on the receptor. However, the following observations imply that this may not be true: (1) the time-dependent nature of the inhibition; (2) antagonist Fibrinolysis & Proteolysis (2000) 14(1), 15–21
20 Pakala et al.
behaving like an agonist at higher concentrations; (3) reduction in antagonistic property with a simultaneous in the agonist property upon replacing first alanine with serine. This result is in contrast with the observation that peptides with either alanine or serine as the first residue have similar agonist properties.24 Based on these observations it is likely that the antagonist peptide reported here may bind to thrombin receptor at a site different from that of the agonist. Alternatively, if the agonist and antagonist do bind at the same site, the specific interactions (Eg H-brand, hydrophobic and ion interactions) involved for the two effects may be different. Receptor phosphorylation has been shown to be involved in the regulation of many G-protein coupled receptors.32 It is also thrown that most of the G-protein coupled receptors could be desensitized if they are preincubated with subthreshold levels of agonist.32,33 For thrombin receptor such homodesensitization was shown to involve receptor phosphorylation.38 In the present study also binding of antagonist to the receptor (either at the same site as the agonist binding site, but with a different set of interactions or at a different site) may lead to phosphorylation of the thrombin receptor making the thrombin receptor desensitized and unavailable to the agonist. In summary, we have shown that the peptide AFLARAA is an antagonist to α-thrombin and SFLLRNA-induced human platelet aggregation. Thus, AFLARAA may be a useful tool for differentiating between several possible activation states of the human thrombin receptor, or it may provide a structural template for developing more efficient antagonists targeted to the thrombin receptor. ACKNOWLEDGEMENTS The authors would like to thank Shirley Mcwhorter for her help in typing this manuscript. REFERENCES 1. Fenton JW. II Regulation of thrombin generation and functions. Sem in Thromb and Hemost 1988; 14: 234–240. 2. Coughlin SR. Thrombin receptor function and cardiovascular disease. Trends Cardiovasc Med 1994; 4: 77–83. 3. Ogletree ML, S Natarajan, SM Seiler. Thrombin receptors as drug discovery targets. Perspect Drug Discov Design 1994; 1: 527–536. 4. Vu T-K-H, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–1068. 5. Rasmussen UB, Vouret-Craviari V, Jallat S et al. cDNA cloning and expression of a hamster-thrombin receptor coupled to Ca2+ mobilization. FEBS 1991; 288: 123–128. 6. Zhong C, Hayzer DJ, Carson MA, Runge MS. Molecular cloning at the rat vascular smooth muscle thrombin receptor: evidence for in vitro regulation by basic fibroblast growth factor. J Biol Chem 1992; 267: 16975–16979.
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7. Vu T-KH, Wheton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674–677. 8. Hung R-S, Sorisky A, Church WR, Simons ER, Rittenhouse SE. Thrombin receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3phosphorylated phosphoinositides. J Biol Chem 1991; 266: 18435–18438. 9. Giesberts AN, van Willigen G, Lapetina EG, Akkerman JW. Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor. Biochem J 1995; 309: 613–620. 10. Seiler SM, Michel IM, Fenton II JW. Involvement of the tethered ligand receptor in thrombin inhibition of platelet adenylate cylase. Biochem Biophys Res Comm 1992; 182: 1296–1302. 11. Vassallo JR RR, Kieber-Emmons T, Chichowski K, Brass LF. Structure-function relationship in the activation of platelet thrombin receptors by receptor derived peptides. J Biol Chem 1992; 267: 6081–6085. 12. Vouret-Craviari V, Van Obberghen-Schilling E, Rasmussen UB, Pavirani A, Lecocq J-P, Pouyssegur J. Synthetic-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol of Cell 1992; 3: 95–102. 13. Hung DT, Vu T-KH, Wheaten VI, Coughlin SR. Clones platelet receptor in necessary for thrombin-induced platelet activation. J Clin Invest 1992; 89: 1350–1353. 14. Goodwin CA, Wheeler-Jones CP, Kakkar VV, Deadman JJ, Authi KS, Scully MF. Thrombin receptor activating peptide does not stimulate platelet procoagulant activity. Biochem Biophys Res Commun 1994; 202: 321–327. 15. Vouret-Craviari V, Van Obberghen-Schilling E, Scimeca JC, Van Obberghen E, Pouyssegun J. Differential activation of p44mapk (ERK1) by α-thrombin and thrombin-receptor peptide agonist. Biochem J 1993; 289: 209–214. 16. Brass LF, Vassallo JR RR, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J Biol Chem 1992; 267: 13795–13798. 17. Ngaiza JR, Jaffe EA. A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells. Biochem Biophys Res Commun 1991; 179: 1656–1661. 18. Troyer D, Padilla R, Smith T, Kreisberg J, Glass WI. Stimulation of the thrombin receptor of human glomerular mesangial cells by Ser-Phe-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Pro-Phe peptide. J Biol Chem 1992; 267: 20126–20131. 19. Hollenberg MD, Yang S-G, Laniyonu AA, Moore GJ, Saifeddine M. Action of a thrombin receptor polypeptide in gastric smooth muscle: identification of a core pentapeptide retaining full thrombin-mimetic activity. Mol Pharmacol 1992; 42: 186–191. 20. McNamara CA, Sarembock IJ, Gimple LW, Fenton II JW, Coughlin SR, Owen GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94–98. 21. Suidan H, Stone SR, Hemmings BA, Monard D. Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors. Neuron 1992; 8: 363–375. 22. Van Obberghen-Schilling E, Pouyssegur J. Signalling pathways of the thrombin receptor. Thromb Haemostas 1993; 70: 163–167. 23. Scarborough RM, Naughton MA, Teng W et al. Thethered ligand agonist peptides: structural requirements for thrombin
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