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J. Biol. Chem., Vol. 275, Issue 26, 19728-19734, June 30, 2000

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Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides

DETERMINANTS OF SPECIFICITY AND UTILITY IN ASSAYS OF RECEPTOR FUNCTION^*

Tatjana R. Faruqi^§, Ethan J. Weiss^§¶, Michael J. Shapiro^§**, Wei Huang^§, and Shaun R. Coughlin^§¶§§

From the  Cardiovascular Research Institute, the ^§ Daiichi Research Center, and the Departments of ^¶ Medicine and  Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0130

Received for publication, December 15, 1999, and in revised form, April 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structure-activity analysis of PAR4 peptides, AYPGKF was ~10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G_q and not G_i.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The coagulation protease thrombin elicits biological responses at least in part via G protein-coupled protease-activated receptors (PARs)¹ (1-5). PAR1, the prototype for this family, is activated when thrombin cleaves its amino-terminal exodomain at the R41/S42 peptide bond (1). This serves to unmask a new amino terminus beginning with the sequence SFLLRN, which serves as a tethered peptide agonist, binding intramolecularly to the body of the receptor to effect transmembrane signaling. PARs are thus in essence peptide receptors that carry their own ligands, which remain silent until unmasked by site-specific receptor cleavage. Synthetic peptides that mimic these tethered ligands function as PAR agonists and activate their receptors independent of protease and receptor cleavage (1, 4-6). Such peptides have been extremely useful for probing the roles of PARs in cells and tissues.

PAR4 is a recently characterized thrombin receptor that is expressed in human platelets along with PAR1 (4, 5, 7). Human PAR4 is activated when thrombin cleaves its amino-terminal exodomain at the Arg-47/Gly-48 peptide bond to unmask the tethered ligand GYPGQV (4, 5). The synthetic peptide GYPGQV does function as an agonist for PAR4, but a concentration of 200-500 µM is required for activity. This lack of potency severely limits the utility of this peptide for probing PAR4 function in culture systems and virtually precludes its use in vivo. Structure-function relationships for the PAR4 tethered ligand have not been explored. We here report such a study. In addition to identifying requirements for agonist activity and specificity, this study provides a new PAR4 specific agonist peptide, AYPGKF. This peptide was shown to be useful for probing PAR4 signaling in culture systems and in platelets.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- All chemicals, unless otherwise stated, were obtained from Sigma. myo-[³H]Inositol and [³H]adenine were obtained from Amersham Pharmacia Biotech. Fura-2/AM was obtained from Molecular Probes (Eugene, OR). Bordetella pertussis toxin islet-activating protein was obtained from List Biologicals (Campbell, CA). -Thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). The thromboxane receptor agonist, U46619, was obtained from Calbiochem-Novabiochem, Corp. (San Diego, CA). Peptides were synthesized at University of California San Francisco as carboxyl-terminal amides, purified by high-pressure liquid chromatography, and characterized by mass spectroscopy. Peptide solutions were made fresh from powder for most experiments.

Cells and Cell Lines-- Stable cell lines based on KOLFs, a cell line derived from lung fibroblasts from PAR1 knockout mice (8, 9), were generated by transfecting these cells with pBJ1-based mammalian expression vectors that directed hPAR1 or hPAR4 together with a hygromycin resistance vector (7, 10). The PAR1 and PAR4 cDNAs used in these studies encoded receptors bearing an amino-terminal FLAG epitope. KOLF clones resistant to hygromycin were screened for receptor expression by cell surface enzyme-linked immunosorbent assay for the FLAG epitope using M1 monoclonal antibody (10). KOLF lines were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum and 200 µg/ml hygromycin. Analogous Rat 1 cell lines were derived by transfecting the same expression vectors and a neomycin resistance vector and then screening G418-resistant clones for M1 antibody binding (10). COS7 cells were transiently transfected for 5 h with 2 µg/ml DNA (human PAR1 or PAR4 cDNA in pBJ1) using LipofectAMINE and Opti-MEM media (Life Technologies, Inc.) per the manufacturer's instructions. Following transfection, the cells were incubated in Dulbecco's modified Eagle's medium containing 10% calf serum overnight. The cultures were then split into 24-well plates for phosphoinositide hydrolysis assays. All cell culture was at 37 °C with 5% CO₂.

Phosphoinositide Hydrolysis-- Cells cultured in 24-well plates were labeled overnight with myo-[³H]inositol (2 µCi/ml) in serum-free Dulbecco's modified Eagle's medium containing 1 mg/ml bovine serum albumin, 20 mM HEPES, and penicillin/streptomycin. Following labeling, the cells were incubated in the absence or presence of pertussis toxin (0.1 -100 ng/ml) for 5 h at 37 °C. Agonists were then added together with 20 mM LiCl in serum-free media and incubated at 37 °C for 20-120 min. Cells were extracted, and total [³H]inositol phosphates were quantitated as described (11).

Intracellular Ca²⁺ Mobilization-- Intracellular calcium mobilization was measured fluorometrically using fura-2 as described previously (12, 13).

Adenylyl Cyclase Activity-- The accumulation of [³H]cAMP in [³H]adenine-labeled cells was measured as described previously (14). Briefly, cells were grown in 24-well plates and labeled with [³H]adenine (2 µCi/ml) over night at 37 °C in 1 ml of HEPES-buffered serum-free Dulbecco's modified Eagle's medium + 0.1% bovine serum albumin. Cells were treated in the presence or absence of pertussis toxin (0.1-100 ng/ml) for 5 h at 37 °C and then incubated with forskolin and 3-isobutyl-1-methylxanthine (1 mM) for 30 min at 37 °C in the presence or absence of -thrombin (30 nM) or agonist peptides. Cells were then extracted, and [³H]cAMP and total [³H]adenine nucleotides were assayed as described previously (14).

Platelet Function-- Platelet-rich plasma and washed human or mouse platelets were prepared, and platelet ATP secretion and aggregation in response to various agonists was measured by standard lumiaggregometry (Chrono-log Corp.) as described previously (7, 12).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To define the structure-function requirements for PAR4-activating peptides, we tested a series of hexapeptides based on the sequence GYPGKF. GYPGKF represents the first six amino acids of the new amino terminus unmasked when thrombin cleaves mouse PAR4 and was chosen as the starting sequence because it was more potent than the cognate human sequence GYPGQV on both the mouse and human receptors. PAR1-deficient mouse lung fibroblasts (KOLFs) (8, 9) that had been stably transfected with cDNAs encoding human PAR4 (KOLF-PAR4) or PAR1 (KOLF-PAR1) (7, 8) were used for functional assays of receptor activation and agonist specificity. Agonist-triggered phosphoinositide hydrolysis was used as an end point. Untransfected KOLFs showed no responses to thrombin or to the peptides tested (Refs. 8 and 9 and data not shown); thus, responses in KOLF-PAR1 and KOLF-PAR4 cells were dependent upon PAR1 and PAR4, respectively.

We first compared the native PAR4 and PAR1 peptides GYPGKF and SFLLRN. GYPGKF elicited only 55% of the maximal response triggered by thrombin in PAR4-expressing cells, even when added at 500 µM. By contrast, in PAR1-expressing cells, the maximal responses to SFLLRN and thrombin were of similar magnitude. Both peptides were specific for their respective receptors in this cell system (Table I).

View this table: [in this window] [in a new window]   Table I Synthetic peptides screened for PAR1 and PAR4 receptor activation Peptide-triggered release of [3H]inositol phosphates from KOLF stable cell lines expressing human PAR4 or PAR1 was measured as an index of agonist-induced phosphoinositide hydrolysis. Untransfected KOLFs exhibited no response to peptides or -thrombin. Results were expressed as a percentage of the response to 30 nM -thrombin determined in each experiment. Over the many experiments represented here, 30 nM -thrombin triggered a 7-16-fold increase in phosphoinositide hydrolysis in PAR1-expressing cells and a 10-36-fold increase in PAR4-expressing cells. Peptides were used at 500 µM. Each experiment was done in triplicate and repeated the indicated number of times. Results shown are means ± S.E. of the results of repeated experiments. Important results were confirmed using Rat1 cell lines expressing human PAR4 or PAR1 (see text).

We next performed an alanine scan and a series of other substitutions designed to identify sites important for activity and specificity at PAR4 versus PAR1. Surprisingly, alanine or serine substitution at position one of GYPGKF yielded a gain of function for PAR4 activation (Table I). Threonine substitution caused a loss of function. These data suggest that small aliphatic side chains are preferred at position 1. Substitution of a mercaptoproprionic acid for serine in SFLLRN nearly abolished agonist activity for PAR1 (15, 16). The cognate substitution of mercaptoproprionic acid for glycine in GYPGKF also resulted in loss of agonist function. Mercaptoproprionic acid-YPGKF and SYPGKF differ at only two positions. The mercaptoproprionic acid-peptide lacks an amino-terminal protonated amino group and has a sulfur atom at the position corresponding to oxygen in the serine side in SYPGKF. The amino-terminal protonated amino group of SFLLRN is critical for agonist function at PAR1; thus, it is likely that the protonated amino group in GYPGKF is critical for agonist activity at PAR4. This is consistent with the notion that the proteolytic switch in PARs involves both removal of sequence amino to the cleavage site and creation of a new protonated amino group that functions in the context of the tethered ligand (17-19).

In contrast to the gain of function seen with alanine substitution at position 1, substitution of alanine for the tyrosine at position 2 in GYPGKF resulted in a loss of agonist activity (not shown). Thus, as in the PAR1 agonist SFLLRN (17, 20), the aromatic side chain at position 2 is critical for activity. Interestingly, substitution of phenylalanine or para-fluorophenylalanine for tyrosine did not decrease agonist activity for PAR4 but did cause a remarkable gain of function for PAR1 (Table I). This result suggests that the presence of a tyrosine at position two is an important determinant of the activity of GYPGKF and of its specificity for PAR4 over PAR1. More importantly, this result shows that is it possible to develop agonists and, by extension, perhaps antagonists, that act on both PAR1 and PAR4.

Substitution of alanine for proline at position three or for glycine at position four in GYPGKF resulted in substantial loss of agonist function for PAR4 (Table I). This loss of function was also noted in the context of the SYPGKF better-than-native peptide. Proline and glycine are often found in -turns; thus, proline 3 and glycine 4 may be important for a conformation necessary for agonist function.

Substitution of leucine, cyclohexylalanine, or isoleucine for glycine at position 4 all caused gain of function for PAR1, consistent with published results (17, 21), but also caused loss of function at PAR4. Substitutions at positions 5 and 6 that might be expected to yield gain of function at PAR1 failed to do so (Table I). Conversely, the better-than-native PAR1 agonists S(F)(Cha)(Cha)RK and S(F)(Cha)(Cha)(homoR)K (where Cha indicates cyclohexylalanine) had no activity at PAR4 (Table I).

The results in Table I were confirmed in two ways. First, all of the phosphoinositide hydrolysis studies were verified at a semiquantitative level using peptide-triggered increases in cytoplasmic calcium, measured fluorometrically, as an end point (see below). Second, important experiments were replicated using Rat 1 cell lines stably transfected with hPAR1 or hPAR4. These studies yielded results very similar to those obtained with the KOLF-based cell lines shown in Table I. AYPGKF was again shown to be a more potent agonist than GYPGKF. In PAR4-expressing Rat 1 cells, the maximum response to thrombin (30 nM) was an ~18-fold increase in phosphoinositide hydrolysis (mean of triplicate determinations; standard deviations were ~5% of means). Untransfected cells showed no significant response. Responses to 30, 100, and 500 µM AYPGKF were 72, 101, and 116% of the maximal thrombin response to thrombin, respectively. The corresponding responses to GYPGKF were 4, 30, and 92%, respectively.

The relative specificity of AYPGKF for PAR4 and SFLLRN for PAR1 was also confirmed. In Rat 1 cells expressing PAR1 or PAR4, the maximum response to thrombin (30 nM) was an 11- or 19-fold increase in phosphoinositide hydrolysis, respectively, and untransfected cells showed no significant response. In cells expressing PAR1 or PAR4, the response to 500 µM SFLLRN was 130 or 3% of the maximal response to thrombin, respectively. The cognate responses to 500 µM AYPGKF were 4 or 97% of the maximal response to thrombin, respectively. Thus, even at 500 µM, SFLLRN and AYPGKF were relatively specific for their respective receptors. By contrast, the peptides GFPGKF and G(F)PGKF, which demonstrated an ability to activate both PAR1- and PAR4-expressing KOLFs, showed similar activity in the Rat1 system. The response to 500 µM GFPGKF in Rat1 cells expressing PAR1 or PAR4 was 72 or 81% of the maximal thrombin response, respectively. The cognate responses to G(F)PGKF were 131 or 76%, respectively.

The structure-activity scan in Table I yielded two peptides, SYPGKF and AYPGKF, that were more potent than the native GYPGKF sequence at stimulating PAR4-mediated phosphoinositide hydrolysis. Because cells were exposed to agonist for 60 min in the phosphoinositide hydrolysis assay, it was possible that increased activity of a given peptide might be due to better stability in cell culture. To test this possibility and to further probe the specificity of these peptides, we examined their ability to trigger immediate increases in cytoplasmic calcium in KOLF-PAR4 and KOLF-PAR1 cell lines (Fig. 1). AYPGKF and SYPGKF triggered increases in cytoplasmic calcium concentration in KOLF-PAR4; over several experiments, the peak calcium response elicited by AYPGKF was similar to that elicited by thrombin in these cells. AYPGKF and SYPGKF triggered little response in KOLF-PAR1. Conversely, SFLLRN induced calcium mobilization in KOLF-PAR1 but not in KOLF-PAR4 cells. We next compared the relative potencies of AYPGKF, SYPGKF, and GYPGKF in the phosphoinositide hydrolysis and cytoplasmic calcium assays (Fig. 2 and data not shown) in KOLF-PAR4 cells. GYPGKF appeared to be a partial agonist for PAR4-triggered phosphoinositide hydrolysis; even at 500 µM, GYPGKF elicited only approximately 50% of the maximal response to thrombin (Fig. 2A). By contrast, both AYPGKF and SYPGKF were full agonists for PAR4 activation and showed EC₅₀ values of 20 and 50 µM, respectively, in this assay. Similarly, AYPGKF stimulated calcium mobilization in KOLF-PAR4 cells with an EC₅₀ of approximately 25 µM (Fig. 2B), whereas more than 200 µM GYPGKF was required to elicit similar responses in this assay (data not shown). Thus in assays in which cellular effects are measured over either seconds or hours, AYPGKF was substantially more potent than GYPGKF. These data militate against peptide stability as being responsible for the greater activity of AYPGKF and instead suggest that AYPGKF is intrinsically more active than GYPGKF at PAR4.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Intracellular calcium mobilization in response to synthetic agonist peptides. KOLF-PAR1 and KOLF-PAR4 cells were loaded with fura-2/AM, and increases in cytoplasmic calcium in response to -thrombin (30 nM), AYPGKF peptide (500 µM), SYPGKF peptide (500 µM), and SFLLRN (100 µM) were measured fluorometrically. These data are representative of three replicate experiments done in duplicate.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Phosphoinositide hydrolysis and intracellular calcium mobilization in response to PAR4 activation. A, KOLF-PAR4 cells were treated with either -thrombin (30 nM) (filled square) or the indicated concentrations of GYPGKF (open squares), AYPGKF (open triangles), or SYPGKF (open circles) in the presence of LiCl (20 mM) for 1 h at 37 °C. Total [3H]inositol phosphates were collected and quantitated as described under "Experimental Procedures." Values from untreated cells (ranging from 500 to 1200 cpm in different experiments) were subtracted as background. Data are the means ± S.E.; n = 3. Similar results were obtained in three separate experiments. B, KOLF-PAR4 cells were loaded with fura-2/AM and assayed for intracellular calcium mobilization in response to increased concentrations of AYPGKF. Concentrations were (bottom curve to top curve) 1, 5, 10, 100, and 500 µM. Data are representative of two similar experiments.

AYPGKF appeared to be potentially useful as a probe of PAR4 function. To further assess specificity, we examined the ability of this peptide to activate calcium mobilization in Xenopus oocytes (1, 22) expressing human PAR1, PAR2, PAR3, or PAR4. AYPGKF (500 µM) elicited ~10-fold increases in ⁴⁵Ca release from hPAR4-expressing oocytes, but only ~1.5-fold increases in hPAR1 expressing oocytes and no responses in oocytes expressing hPAR2 or hPAR23 (data not shown). Oocytes expressing hPAR1, hPAR2, and hPAR3 did respond robustly to SFLLRN, SLIGRL, and thrombin, respectively. Thus, even in our most sensitive assay system, AYPGKF was relatively specific for PAR4 versus the other known PARs.

PAR1 has been shown to couple to G proteins of the G_i, G_q, and G_12/13 subfamilies (14, 23-25). We first determined whether PAR4 might exhibit a different coupling pattern by measuring phosphoinositide hydrolysis and inhibition of adenylyl cyclase in the presence or absence of pertussis toxin and then compared signaling in response to thrombin versus AYPGKF to determine whether such agonists recapitulated the signaling pattern elicited by thrombin. Thrombin or SFLLRN inhibited forskolin-stimulated adenylyl cyclase activity in a pertussis toxin-sensitive manner in PAR1-KOLF cells (Fig. 3 and data not shown), consistent with the known coupling of PAR1 to G_i (14). Moreover, phosphoinositide hydrolysis in response to thrombin or SFLLRN was slightly inhibited by pertussis toxin in KOLF-PAR1 cells and was more substantially inhibited by pertussis toxin in COS7 cells transiently transfected with PAR1 (Fig. 4 and data not shown). This is consistent with a G_i-mediated contribution to phospholipase C activation (26). By contrast, neither thrombin, GYPGKF, nor AYPGKF inhibited adenylyl cyclase in PAR4-expressing KOLFs or COS7 cells (Fig. 3 and data not shown). Moreover, PAR4-mediated phosphoinositide hydrolysis was pertussis toxin-insensitive. Under the conditions used in these experiments, treatment with 100 ng/ml pertussis toxin ADP-ribosylates all detectable pertussis substrate in cell membranes as assayed by subsequent ADP-ribosylation of membrane preparations in vitro using activated pertussis toxin and ³²P-NAD (11). Thus these data suggest that, unlike PAR1, PAR4 does not couple to G_i. Moreover, the observation that the responses to thrombin and the various agonist peptides were concordant, at least in terms of which G proteins were activated, suggests that these peptides are useful probes of PAR4 signaling in mammalian cells. The observation that the response of PAR4-expressing cells to GYPGKF, even at 500 µM, was less than 60% of the maximum response to thrombin again emphasizes the utility of better than native PAR4 agonists.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The effect of thrombin on forskolin-stimulated adenylyl cyclase activity. KOLF-PAR1 (A) or KOLF-PAR4 (B) cells were loaded with [3H]adenine as described under "Experimental Procedures" and treated with 50 µM forskolin in the presence of the indicated concentrations of -thrombin for 30 min at 37 °C. Prior to agonist treatment, cells were either left untreated (closed circles) or treated (open circles) with pertussis toxin (100 ng/ml) for 5 h at 37 °C. Data presented are [3H]cAMP as a fraction of [3H]cAMP + [3H]ATP (mean ± S.E.; n = 3). Similar results were obtained in three separate experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Phosphoinositide hydrolysis in response to -thrombin or agonist peptides in the presence of increasing concentrations of pertussis toxin. KOLF-PAR4 or KOLF-PAR1 or transfected COS7 cells were incubated with the indicated concentration of pertussis toxin for 5 h at 37 °C and then either left untreated (triangles), treated with -thrombin (30 nM) (circles), or treated with the receptor agonist peptides (squares): GYPGKF (500 µM) for PAR4-expressing cells or SFLLRN (100 µM) for PAR1-expressing cells. Agonist treatment was for 1 h at 37 °C in the presence of LiCl (20 mM). Total [3H]inositol phosphates released (mean ± S.E.; n = 3) were quantitated as described under "Experimental Procedures." Similar results were obtained in three separate experiments for each cell type. Note pertussis sensitivity of PAR1 but not PAR4 response in COS7 cells.

Having identified a relatively potent and specific PAR4 agonist peptide, we investigated its utility for probing PAR4 function in a differentiated cell that naturally expresses PAR4, the human platelet. In platelet-rich plasma, AYPGKF caused platelet shape change at 20 µM (Fig. 5, top left; shape change is indicated by the dip in the solid aggregation curve). At 100 µM and above, AYPGKF reliably stimulated platelet ATP secretion and aggregation (Fig. 5). By comparison, under the conditions used in these experiments, GYPGKF elicited only shape change even at 500 µM. The PAR1 agonist SFLLRN was considerably more potent than AYPGKF; the threshold for SFLLRN for shape change and secretion was ~4 µM, and substantial responses occurred at 20 µM. However, maximum responses to AYPGKF were at least as great at those to SFLLRN (Fig. 5). AYPGKF also elicited robust increases in platelet cytoplasmic calcium concentration (Fig. 6). Responses to AYPGKF were not inhibited by preincubating platelets with the PAR1 antagonist BMS200261 (100 µM) (27); by contrast, SFLLRN responses were blocked by this reagent (data not shown). Thus, platelet responses to AYPGKF are not attributable to agonist activity at PAR1. These results are consistent with expression of both PAR1 and PAR4 on human platelets (4, 5, 7).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Aggregation and ATP secretion in human platelets in response to PAR1 and PAR4 agonist peptides. AYPGKF, GYPGKF, and SFLLRN were added to human platelet-rich plasma at the indicated concentrations (µM) and aggregation (left) and secretion (right) followed in a lumiaggregometer. Tracings representative of at least four determinations are shown.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Calcium mobilization in human platelets following PAR4 desensitization. Washed human platelets were loaded with fura-2/AM, and agonist-triggered increases in cytosolic calcium were measured fluorometrically. Platelets were either left untreated prior to measurements (gray bars) or were pretreated with the PAR1-antagonist peptide BMS200261 (100 µM) for 5 min and AYPGKF (500 µM) for 30 min at room temperature (white bars). These platelets were then stimulated with a saturating concentration of agonist as indicated: -thrombin (30 nM), SFLLRN (100 µM), AYPGKF (500 µM), or U46619 (10 µM). Data shown are means ± S.E. (n = 2). This experiment was replicated twice.

Desensitization with PAR-specific agonist peptides has been useful for probing the role of specific receptors in differentiated cells, and AYPGKF indeed proved useful in this regard. Increases in cytoplasmic calcium were measured fluorometrically as an index of PAR signaling. In platelets in which PAR1 was blocked by the antagonist BMS200261 (27), pretreatment with AYPGKF caused platelets to become refractory to both AYPGKF and to thrombin (Fig. 6). Only limited heterologous desensitization to the thromboxane receptor agonist U46619 was noted. These results indicate that AYPGKF effectively desensitized PAR4-mediated signaling in human platelets. Moreover, they confirm and extend previous studies that suggest PAR1 and PAR4 account for most if not all thrombin signaling in human platelets (7).

Genetically manipulated mice are increasingly utilized to identify the molecules and mechanisms that regulate platelet function. In our hands, thrombin is the most effective agonist for activation of mouse platelets, with ADP, collagen, epinephrine, and U46619 being considerably less potent. However, because thrombin clots fibrinogen, thrombin cannot be used to study platelet function in platelet-rich plasma; washed platelets must be used, and preparation of washed platelets entails substantial additional ex vivo manipulation. In the human system, the PAR1 agonist SFLLRN has proven useful as a strong agonist that can activate platelets in platelet-rich plasma. However, mouse platelets utilize PAR3 and PAR4 for thrombin signalingPAR1 does not contribute. No peptide agonist capable of activating mouse PAR3 has been described, and recent evidence suggests that mPAR3 functions as a cofactor for thrombin cleavage of PAR4 rather than as a bona fide receptor (28). AYPGKF proved to be a potent activator of mouse platelets in platelet-rich plasma, unlike the wild-type GYPGKF peptide (Fig. 7). AYPGKF will therefore be useful for exploring platelet function in genetically manipulated mice. Indeed, AYPGKF caused shape change but not secretion or aggregation in platelets from G_q-deficient mice (data not shown), suggesting that PAR4 mediates shape change through a G protein other than G_q, probably G_12/13 (29).

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Mouse platelet secretion and aggregation in platelet-rich plasma: effectiveness of AYPGKF. Platelet-rich plasma was prepared from wild-type C57BL6 mice, and agonist-triggered ATP secretion (right) and aggregation (left) were measured as a function of time in response to the indicated concentrations of AYPGKF, GYPGKF, or the thromboxane receptor agonist U46619. Concentrations are in µM. Tracings representative of at least four determinations are shown. Note that the native PAR4 agonist GYPGKF triggered minimal aggregation (bottom left) and no detectable secretion (not shown) even at 500 µM.

In summary, this exploration of structure-activity relationships for PAR4 tethered ligand peptides has provided useful structure-function lessons, a new PAR4 agonist, and new information regarding PAR4 coupling to G proteins. Several structure-function lessons were learned. First, the amino-terminal protonated amino group of the PAR4 tethered ligand appears to be important for agonist activity at PAR4. This is consistent with the notion that the proteolytic switch that activates the tethered ligand involves not only removal of sequence amino to the scissile bond between Arg-47 and Gly-48 but also generation of a new protonated amino group at the nitrogen that was part of that peptide bond. This is analogous to previous findings with PAR1 (17, 18) and, not surprisingly, suggests that the proteolytic switch in PARs utilizes a common mechanism (19). Second, the basis for agonist specificity for PAR4 versus PAR1 was in part attributable to the tyrosine at position 2 in GYPGKF and AYPGKF. Several peptides in which this tyrosine was substituted by phenylalanine or p-fluorophenylalanine were capable of acting at both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to block activation of both receptors to prevent or treat thrombosis. The observation that it is possible to generate an agonist capable of activating both receptors raises the possibility that it might also be possible to generate an antagonist capable of blocking both receptors.

These studies also generated a new PAR4 agonist, AYPGKF, which was used to provide new information about PAR4 signaling and function in platelets and other cells. AYPGKF, unlike GYPGKF or GYPGQV (peptides based on native tethered ligand sequence), was a full agonist for PAR4 and ~10-fold more potent than the native agonist GYPGKF. AYPGKF was also relatively specific for PAR4 versus the other known PARs. The increased potency of AYPGKF made it a useful tool for investigating PAR4 signaling in various settings. In the course of characterizing thrombin and peptide signaling in PAR-transfected cells, we noted that whereas PAR1 coupled to G_q and G_i, PAR4 coupled to G_q but not G_i. Elevated cAMP levels inhibit platelet activation; hence, the ability of PAR1 to activate G_i and inhibit adenylyl cyclase (in addition to activating G_q) may contribute to its effectiveness in activating human platelets (14, 30). Conversely, the lack of ability to activate G_i of PAR4 may be a factor in its apparent lesser role in thrombin-triggered platelet activation (7). AYPGKF also proved useful for directly probing PAR4 function in platelets (Figs. 5 and 6). When combined with PAR1 inhibition, desensitization of PAR4 signaling by prolonged incubation with AYPGKF effectively blocked platelet activation by thrombin. This observation supports the model that PAR1 and PAR4 together account for most if not all platelet activation by thrombin (7). One specialized use of AYPGKF will be probing mouse platelet function, as it elicits robust platelet activation in platelet-rich plasma. Reliable activators of mouse platelets are needed given the increasing use of genetically modified mice to investigate platelet function (4, 8, 31-33). Collectively, these results further our understanding of platelet activation and thrombin signaling and provide a valuable new tool for future studies. We expect that the availability of the AYPGKF peptide will promote study of PAR4 function, much as the availability of SFLLRN promoted study of PAR1.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL44907 and HL59202 and by the Daiichi Research Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by National Institutes of Health Institutional National Research Service Award HL07731.

^** Supported by the American Heart Association.

^§§ To whom correspondence should be addressed: University of California San Francisco, HSE 1355, Box 0130, 513 Parnassus Ave., San Francisco, CA 94143. Tel.: 415-476-6174; Fax: 415-476-8173; E-mail: coughlin@cvrimail.ucsf.edu.

Published, JBC Papers in Press, April 21, 2000, DOI 10.1074/jbc.M909960199

	ABBREVIATIONS

The abbreviation used is: PAR, protease-activated receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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