I. Introduction

Top Previous Next References

Seven transmembrane G-protein-coupled receptors comprise the biggest group of receptors in mammalian systems, and a large number of cognate receptors and associated ligands have been identified. A recently described novel subset of this group, the proteinase-activated receptors (PARs), has been shown to have unique mechanism of activation. Rather than being activated by simple ligand occupancy, based on the law of mass action, they are activated enzymatically through proteolysis of the receptor. This proteolytic cleavage is specifically mediated by a well characterized family of enzymes that require serine within the active site, the serine proteases. Classically, serine proteases have been shown to play important roles in diverse biological functions, particularly in relation to clot formation and wound healing. However, proteolytic cleavage of PARs as a mode of receptor activation now represents an increasingly important feature of this family of enzymes. In this review, we will outline the pharmacological characteristics of the four members of the PAR family, PAR-1 through -4, their mechanism of activation by serine proteases, coupling to intracellular signaling pathways, and their potential physiological and pathophysiological roles. Such is the rapidly expansive nature of the field; the reader is directed to a number of excellent recent shorter reviews that will complement this current work (Grand et al., 1996; Hollenberg, 1996; Dery et al., 1998; Cocks and Moffatt, 2000; Coughlin, 2000).

	II. Historical PerspectivesCellular Effects of Thrombin and the Cloning of the Thrombin Receptor, Proteinase-Activated Receptor-1

Top Previous Next References

The role of thrombin as a key intermediate in the coagulation process has been established for a number of decades. Thrombin was originally identified as a trypsin-like serine protease, produced from prothrombin by the action of factor Xa, which mediated the formation of fibrin, the fibrous matrix of blood clots, from fibrinogen (Davie et al., 1991; Stubbs and Bode, 1993). Since the initiating factor in the cascade, tissue factor, was also found primarily on cells that under normal conditions do not access the bloodstream, thrombin was seen primarily as a component of a coagulation process linking tissue damage to wound repair. However, thrombin in the absence of other products of the coagulation cascade, was also found to be a strong activator of platelet aggregation, suggesting the potential of cellular effects in addition to a role in clot formation (Davey and Luscher, 1967). In addition, several studies established thrombin to have direct effects upon a number of other cell types, including monocytes, smooth muscle cells, endothelial cells, and lymphocytes, among others (Chen and Buchanan, 1975; Chen et al., 1976; Bar-Shavit et al., 1983; Bizios et al., 1986; Daniel et al., 1986; Hattori et al., 1989) (see Section IV.). Significantly, several of these studies used serine protease inhibitors to confirm that the protease activity of thrombin was essential for these cellular effects (Shuman, 1986). Although classical radioligand binding studies with modified thrombin had identified several candidate thrombin-binding proteins (Okamura et al., 1978; Gronke et al., 1987), up until 1990 a functional thrombin receptor had not been identified.

In 1991, Coughlin and colleagues (Vu et al., 1991a) used a dilution cloning approach in an attempt to isolate the cDNA encoding the thrombin receptor. Initially, mRNA, from cells highly responsive to thrombin, was injected into ⁴⁵Ca²⁺-labeled Xenopus oocytes and thrombin-stimulated ⁴⁵Ca²⁺ release assayed. The mRNA transcript encoding the receptor was fractionated and used to construct a size-specific cDNA library, which was then plated into 50 pools of estimated 20,000 clones each. By injecting in vitro transcribed cRNA from each pool into Xenopus oocytes, and functionally assaying each pool for thrombin-stimulated ⁴⁵Ca²⁺ release, positives were identified and could then be progressively subdivided and plated into fractions containing fewer cDNAs. Eventually, a single cDNA species was isolated which, when assayed in oocytes, displayed 100-fold higher calcium release and chloride entry when compared with oocytes injected with a similar quantity of nonpurified Dami cell mRNA. In oocytes expressing the receptor, thrombin was found to be an extremely potent agonist (EC₅₀ = 50 pM), whereas trypsin, although effective, was considerably less potent and efficacious. Furthermore, responses to thrombin were found to be blocked by the thrombin antagonists hirudin and hirugen, a hirudin-derived peptide (Vu et al., 1991a) indicative of the thrombin-specific nature of the cloned receptor.

Sequencing of the functional clone revealed a 3.5-kb insert, containing an open reading frame encoding a 425 amino acid protein. Hydropathy analysis of the sequence revealed the protein to be a member of the seven transmembrane domain receptor superfamily, being most closely related to the peptide (e.g., substance P) and glycoprotein hormone receptor subfamilies. The protein was found to contain an N-terminal hydrophobic signal sequence with potential cleavage sites at Thr²⁴ and Ala²⁶. The remainder of the 75 amino acid N terminus is extracellularly disposed and contains several asparagine-linked glycosylation sites. Crucially, a putative thrombin cleavage site (LDPR/S), similar to the activation cleavage site in the zymogen protein C, was identified in the amino terminus, suggesting that receptor activation involves proteolytic cleavage. Mutation of this residue, Arg⁴¹ to Ala, rendered the receptor insensitive to stimulation when expressed in oocytes (Vu et al., 1991a), whereas a peptide mimicking the new amino terminus created by cleavage at Arg⁴¹, SFLLRNPNDKYEPF (TRAP-14), was able to activate both wild-type and mutant receptors. In addition, Northern blotting of mRNA revealed high levels of receptor in HEL and DAMI cells, both known to be highly responsive to thrombin, and also in platelets and endothelial cells by RT-PCR.

In the same year, Rasmussen et al. (1991) cloned the hamster thrombin receptor from CCL39 hamster lung fibroblasts. Functional expression in Xenopus oocytes indicated a functional thrombin receptor, and the deduced amino acid sequence revealed a thrombin consensus cleavage site in the extracellular N terminus, followed by a negatively charged cluster of residues comprising a binding region for the anion exosite found in thrombin (see below). Cellular and tissue-specific expression was also consistent with that observed for the human receptor. Other thrombin receptors cloned to date include those from rat (Zhong et al., 1992), mouse (Tanaka et al., 1993), Xenopus laevis (Gerszten et al., 1994), and bovine (Ma et al., 1996) sources. Taken together, these findings confirmed the identification of PAR-1 and a novel, proteolytic, mechanism of activation (see Figs. 1 and 2).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Protease activation of PARs. Proteolytic cleavage the N terminus generates a new tethered ligand designated by the filled section that interacts with the extracellular loop-2 of the receptor. A peptide sequence derived from the N terminus is able to activate the receptor in the absence of protease-mediated cleavage of the N terminus.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Structural features of PARs 1-4. The key areas of PAR receptor activation are highlighted. The N terminus cleavage domain and the hirudin-like binding domain, the ECL-2 where tethered ligand/receptor interactions occur, and the C-terminal tail that is involved in desensitization and some aspects of intracellular signaling.

Several further studies identified additional features regarding the mechanism of the interaction between thrombin and the receptor. Initially, the crucial role of the N terminus was confirmed. A mutant receptor with the LDPR/S site replaced by an enterokinase site was fully responsive to enterokinase, suggesting no requirement for an additional mechanism of activation other than that initially proposed (Vu et al., 1991b). Subsequently, a mutant lacking the N terminus was found to be both inactive and unresponsive to thrombin (Chen et al., 1994a). The findings of this study not only confirmed the crucial role for this region of the receptor, but also provided an argument against the idea that the role of the N terminus was to prevent receptor activation, and that cleavage left the receptor free to form an active conformation. Additionally, the differences in the potency between enterokinase and thrombin in their ability to activate wild-type or mutant receptors suggested additional binding sites for thrombin within the N terminus. Mutation of the N terminus identified the presence of a hirudin-like domain within region 51-63 that was essential for high affinity binding and the potent effects of thrombin (Vu et al., 1991b). Peptides not susceptible to thrombin cleavage but which encompass this region, or other exosite ligands, such as thrombomodulin and fibrinogen, blocked the actions of thrombin in whole cells or thrombin-stimulated cleavage of a GST/N-terminal receptor fusion protein expressed in Escherichia coli (Bouton et al., 1995). Furthermore, -thrombin, which lacks the anion exosite, was found to be considerably less (100-fold) potent than thrombin in activating the receptor (Bouton et al., 1995; Seiler et al., 1995). Subsequent studies have confirmed the importance of the N-terminal DKYEPF hirudin-like domain in reducing the kinetic barrier to thrombin/receptor complex formation. These studies have also provided evidence to suggest that thrombin binding at this site initiates a conformational change in the active center of the enzyme that accommodates the LDPR cleavage sequence and facilitates binding (Ishii et al., 1995).

The ability of thrombin receptor activating peptide (TRAP) to activate a thrombin receptor lacking the amino terminal exodomain implicated a site, or sites, of interaction with the other extracellular loops. Experiments utilizing chimeras generated from human and Xenopus receptors or antibodies directed against different segments of the thrombin receptor (Bahou et al., 1994) showed that both the N-terminal exodomain and the second extracellular loop determine SFLLRN binding to the receptor. Subsequent studies using PAR-1/PAR-2 chimeras (see below) confirmed the role of ECL-2 in determining the specificity of this interaction (Lerner et al., 1996). Similar studies also generated detailed information regarding the molecular basis of thrombin/receptor interactions. The N terminus and the ECL-2 regions of the receptor were shown to dictate the selectivity of either Xenopus or human thrombin receptor for stimulation by human and Xenopus TRAPs. Point mutation at only two residues within the Xenopus receptor, Phe for Asn⁸⁷ in the N-terminal exodomain and Glu for Leu²⁶⁰ in the second extracellular domain, conferred human receptor-like specificity (Nanevicz et al., 1995). Additional experiments using substituted TRAPs showed that Arg⁵ of the peptide was involved in binding to Glu²⁶⁰ since a human receptor with Glu²⁶⁰ mutated to arginine lost the ability to signal to SFLLRN. This mutation was also complementary for activation in response to SFLLEN, normally inactive at the wild-type receptor, indicating the importance of Arg⁵-Glu²⁶⁰ in defining the specificity for thrombin activating peptides for human PAR-1. This interaction is also likely to be important in initiating a conformational change in the receptor and subsequent intracellular signaling since a human receptor containing a Xenopus ECL-2 domain encompassing region 259-268 is constitutively active (Nanevicz et al., 1996). However, no subsequent studies utilizing only the human thrombin receptor have confirmed this hypothesis.

	III. Pharmacology of Proteinase-Activated Receptor-1

Top Previous Next References

A series of studies utilizing substituted TRAP analogs representing the cleaved N terminus were undertaken to derive information regarding the structure-function relationship for activation of the thrombin receptor (see Table 1). Initial studies using a number of functional assays, in particular platelet aggregation and [³H]IP accumulation, showed that the pentapeptide SFLLR-NH₂ was a minimum requirement for full agonist activity, although the hexapeptide SFLLRN was 2- to 3-fold more potent, suggesting it to be the preferred functional sequence. Peptides truncated from the amino terminus displayed substantially reduced potency, whereas a series of peptides with extended C termini showed similar or reduced potency to the hexapeptide (Chao et al., 1992; Sabo et al., 1992; Scarborough et al., 1992a; Vassallo et al., 1992). A series of single amino acid substitutions indicated that, although Ser¹ was essential for binding, changes could be tolerated as long as the free amino group was maintained (Scarborough et al., 1992a; Sakaguchi et al., 1994; Shimamoto et al., 1995). Removal or acetylation of the amino group at Ser¹ reduced potency considerably (Sakaguchi et al., 1994). Phe² was found to be essential for agonist activity and tolerated substitution poorly, displaying complete loss of activity with alanine replacement (Scarborough et al., 1992a), but allowed substitution with tyrosine (Nose et al., 1993; Natarajan et al., 1995). Leu³ was noted to be relatively unimportant in that it could be substituted with many different residues. However, some loss in potency was recorded following alanine substitution at Leu⁴ and, in particular, Arg⁵ (Chao et al., 1992; Scarborough et al., 1992a; Vassallo et al., 1992; Natarajan et al., 1995). A bulky aliphatic residue at position 4 and either a basic or aromatic residue at position 5 are moderately important for activity. Positions 1 and 3 tolerate proline substitution, while scanning through positions 1-5 with D- or N-Me amino acids has been shown to cause a major loss of agonist potency (Feng et al., 1995; Natarajan et al., 1995). More recently, reduced amide (CH₂N) and ester (COO) scans have revealed the importance of the amide nitrogen between residues 1 and 2 for agonist recognition and the potential involvement of carbonyl groups along the backbone in hydrogen bonding with the receptor (Shimamoto et al., 1995; Ceruso et al., 1999). From these and other studies, a consensus peptide structure has been developed that has provided a template from which additional compounds have been synthesized.

Additional consideration has also been given to the favored bioactive conformations of TRAP-5. Information derived from NMR and other modeling techniques has suggested an extended structure for the active form of the peptide. These studies also suggest a limited conformation for Phe², a  torsional angle similar to Pro, a  torsional angle close to that of a -sheet for Leu³, and a trans configuration for the amide bonds of S-F and F-L (Shimamoto et al., 1995). Furthermore, despite the finding that Leu³ can tolerate a wide variety of substitutions, the peptide bond itself is sensitive to conformational changes possibly due to a hydrophobic contact between Phe² and Leu⁴ side chains (Ceruso et al., 1999). Thus, this region may play a crucial role in changes in conformation during interaction with the receptor.

Although the aforementioned studies have indicated an extended structure for the peptide, another group has proposed a curved cyclic backbone structure for the active form of TRAP-5. This hypothesis is based on the potential of weak contacts between the Arg⁵ side chain and the Ser¹ and Phe² residues (Matsoukas et al., 1997). Consistent with this is the finding that a 19-membered-ring macrocyclic SFLLR, linked from the P1 side chain to the C terminus, is nearly equipotent with SFLLR in induction of gastric smooth muscle contraction (Matsoukas et al., 1996). However, it has been subsequently shown that these compounds are generally less potent than SFLLRN in platelet aggregation assays (McComsey et al., 1999). The contradictions between studies may be further exacerbated by the fact that the respective conclusions, despite utilizing sophisticated modeling techniques, are based substantially on extrapolation of data derived from experiments using the untethered ligand rather than the tethered bioactive form. Future development of studies using X-ray crystallography allowing direct examination of peptide/receptor interactions will represent a vital step forward in this area.

Recent studies have further refined the structure activity relationships for PAR-1 and its ligand, leading to the synthesis of a number of penta- and tetrapeptides with enhanced agonist potency (Table 1). Substitution of Phe² with p-fluorophenylalanine, but not other larger halogen derivatives, increases agonist potency by approximately 5-fold (Nose et al., 1993), possibly by enhancing the - bonding between the ligand and the receptor (Nose et al., 1998). Replacement of Leu³ with residues containing either neutral or basic side chains, such as (2-napthyl) alanine (Natarajan et al., 1995; Seiler et al., 1996) p-guanidinophenylalanine (Bernatowicz et al., 1996) or arginine (Feng et al., 1995; Natarajan et al., 1995), also results in enhanced agonist potency. Introducing a hydrophobic cyclohexylalanine in place of Leu⁴ increases potency a further 2-fold (Feng et al., 1995). One synthetic peptide combining some of these modifications with an additional tyrosine substitution in position 6, H-Ala-(pF-)Phe-Arg-Cha-hArg-Tyr-NH₂, has been shown to give an EC₅₀ value of 10 nM in platelet aggregation assays and a K_d of 15 nM when a tritium-labeled form is used in radioligand binding assays (Feng et al., 1995; Ahn et al., 1997). Despite these findings, peptides such as these show only moderate selectivity (100-fold) over the recently described PAR-2 in both activation and desensitization assays (Kawabata et al., 1999b), and more selective PAR-1 agonists, such as Ala-(pF)Phe-Arg-Cha-Cit-Tyr-NH₂ (Kawabata et al., 1999b) with increased potency, are still required.

Rational drug design methodology has also been utilized to generate a series of substituted peptides displaying partial agonist and antagonist properties. The peptide 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys amide (C186-65), initially designed from agonist peptides (Scarborough et al., 1992b), was found to inhibit both SFLLR and thrombin-stimulated platelet Ca²⁺ mobilization and aggregation, but not the similar responses produced by collagen or TXA₂, suggesting some specificity for thrombin receptors (Seiler et al., 1995). However, the potency of C186-65 was relatively low, and partial agonist activity at PAR-1 has been recorded in some cell types. Indeed, recently, this peptide has been found to also have PAR-2 agonist activity in HEK cells (Kawabata et al., 1999b). Nevertheless, using this strategy, a potent antagonist, N-trans-cinnamoyl-p-fluoro-Phe-p-guanidino-Phe-Leu-Arg-NH₂ (BMS-197525), was synthesized and found to have an IC₅₀ value of approximately 10 nM in radioligand binding assays and 0.2 µM in platelet aggregation studies (Bernatowicz et al., 1996). Furthermore, addition of a single arginine residue at the C terminus further enhanced antagonist potency in functional assays by 5- to 10-fold, whereas further substitution of arginine for ornithine at position 6 generated a peptide suitable for radiolabeling and use in binding studies (Elliott et al., 1999). The relative potencies of these and other analogs were also tested in GTPase assays and Ca²⁺ mobilization experiments, the results from which agreed well with the initial values obtained in platelets. Similar approaches have generated a number of peptide antagonists with variable potency (Hoekstra et al., 1998; Fujita et al., 1999) (see Table 1).

Despite apparent advances in the development of PAR-1 antagonist peptides, a number of problems remain. Not only do the compounds have only moderate potency for PAR-1, the recent isolation of other PARs has brought into question the relative selectivity of the these compounds and the apparent lack of potency in particular preparations. For example, it has recently been shown that a derivative of BMS-197525 has partial activity at PAR-2, as well as PAR-1 in HEK cells (Kawabata et al., 1999b). Furthermore, although substituted peptide compounds have been shown to inhibit TRAP stimulation in general, they have been shown to be much less effective against thrombin stimulation. Although this may again be due to the use of preparations, where other PARs exist, another likely possibility is the disparity between the conformations achieved by the N-terminal tethered ligand in interaction with the receptor and by receptor-activating peptides in free solution. Considering the spatial constraints of groups in the SFLLRN agonist and the need for a rigid molecular structure, Andrade-Gordon and coworkers (1999) recently synthesized a peptide mimetic PAR-1-selective antagonist RWJ-56110, based on an indole template (Fig. 3). This compound demonstrated consistent, relatively potent (approximately 1 to 300 µM), inhibitory actions against both thrombin- and SFLLRN-stimulated responses, including platelet aggregation and smooth muscle Ca²⁺ mobilization. Other nonpeptide PAR-1 antagonists, including FR171113 (Kato et al., 1999) and SCH 79797, one of a pyrroloquinazoline class of molecules (Ahn et al., 1999, 2000), have recently been identified (Fig. 3). Both compounds strongly inhibited SFLLRN- and thrombin-stimulated platelet aggregation, whereas the latter was also demonstrated to be selective for PAR-1 over PARs 2-4 (see Section VIII.B.). Taken together, these compounds represent good potential lead candidates for the future development of orally active PAR-1 antagonist drugs.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   PAR-1 antagonists developed from thrombin receptor-activating peptides. Structural comparison of the thrombin receptor-activating peptide, SFLLR-NH2, and synthetic antagonists. RWJ5610, undeclared; FR171113 = 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one; SCH79797= N-3-cyclopropyl-7-{[4-(1-methyl-ethyl)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1, 3-diamine.

It should be noted that absolute potency estimations between synthetic agonists generated within different laboratories is difficult due to differences in assay systems used and initial estimates for the EC₅₀ values of TRAPs that vary between studies. This also includes the potential for peptide degradation, since in one study substituting isoserine for Ser¹ of SFLLRN was shown to confer resistance to cleavage by aminopeptidase M (Coller et al., 1993). This may generate artifactual differences in potency estimations, depending on the assay system involved. Second, many of these values have been generated using human platelet aggregation and other systems, which may be altered by the presence of PAR-4.

	IV. Functional Responses to Proteinase-Activated Receptor-1 Activation

Top Previous Next References

Many of the cellular effects of thrombin are consistent with a primary role in vessel wound healing and revascularization (Carney et al., 1992). This not only includes clot formation, but also effects upon a multitude of cell types known to play a role in the systemic response to vascular damage. Target cells for the effects of thrombin include not only platelets, endothelial, and smooth muscle cells, but also cell types such as neutrophils, leukocytes, neurons, and glial cells. Activation of a wide range of cell types therefore facilitates a co-ordinated response to vessel damage, including platelet aggregation, leukocyte extravasation, angiogenesis, nerve regeneration, and even initiation of a controlled immune response. Since thrombin-generating systems are primarily restricted to blood, few extravascular effects have been reported that cannot be either directly or indirectly extrapolated to vessel damage and repair. However, recent studies have indicated the presence of a thrombin generating system in brain, suggesting potential extravascular sites of thrombin production (Gingrich and Traynelis, 2000). It is therefore clear that thrombin, acting through PAR-1, is capable of affecting a wide range of physiological systems (see Table 2).

A very large number of studies have now confirmed that thrombin is a major stimulus for platelets, initiating a series of co-ordinated events that result in platelet aggregation in vitro or in vivo (Eidt et al., 1988). Early attempts to characterize the action of thrombin upon platelet aggregation, prior to the cloning of PAR-1, demonstrated that aggregation was not due to generation of an active molecule from the clotting process, but rather involves a direct effect of the enzyme and requires protease activity (Davey and Luscher, 1967; Martin et al., 1975; Tam et al., 1980). Thrombin mediates shape change and stimulates the release of 5-HT (Harmon and Jamieson, 1986b), adenosine triphosphate (Detwiler and Feinman, 1973), thromboxane A₂, and other granule contents. It also activates the plasma membrane localization of integrin IIb/3, which results in the binding of fibrinogen and von Willebrand factor and platelet aggregation (McGregor et al., 1989; Watts et al., 1989). In addition, thrombin also mediates the translocation of P-selectin and CD40 ligand to the plasma membrane, which facilitate the binding of platelets to endothelial cells (Stenberg et al., 1985; Henn et al., 1998). Other factors such as VEGF may also be released, which may promote endothelial cell growth as an initial step in angiogenesis (Mohle et al., 1997). Numerous studies have confirmed that these responses can be mimicked by PAR-1 activating peptides (Section III.) and involve a number of intracellular signaling events that regulate cytoskeletal reorganisation associated with the aggregation process (Section V.).

As part of the early attempts to characterize the action of thrombin on platelets, several groups demonstrated saturable ¹²⁵I radiolabeled thrombin binding to platelet membranes (Ganguly, 1974; Harmon and Jamieson, 1986b; Greco and Jamieson, 1991). However, extended analysis of radioligand binding data in platelets indicated the presence of three affinity binding states for thrombin with K_d values of 0.3, 10, and 3 mM, respectively (Harmon and Jamieson, 1986b; Greco and Jamieson, 1991). Whereas the moderate and low affinity site are related to PAR-1 and possibly PAR-4 interactions, the high affinity thrombin binding site is likely to be associated with an interaction between the anion binding exosite of thrombin with the platelet membrane glycoprotein complexes GP1-IX-V complex (Berndt et al., 1986). High affinity thrombin binding is lost in platelets derived from patients with Bernard-Soulier syndrome (Demarco et al., 1991), a condition in which GP1 is not expressed, or following preincubation with either monoclonal antibodies directed against GP1 (Greco et al., 1996b) or the metalloprotease Serratia marcesens (Greco et al., 1996a), which removes 70-90% of GP1 from the platelet surface. In these conditions, thrombin-induced platelet aggregation is either delayed or requires higher concentrations to be maximally effective (Demarco et al., 1991; Greco et al., 1996a,b), suggesting that binding to this site, although nonfunctional, nevertheless enhances thrombin function. Thrombin binding to GP1 is believed to be within a specific "hirudin-like" extracellular cytoplasmic domain, spanning residues 271-284, within which lies a cluster of negatively charged amino acids that are common to other thrombin binding molecules (Demarco et al., 1994).

Although more recent studies have shown that ¹²⁵I- thrombin binds strongly to GP1 rich fractions from solubilized platelets (Harmon and Jamieson, 1986a) or to cell lines expressing recombinant GP1 IX-V functional complexes (Dong et al., 1997), at least one study has identified an additional thrombin binding site on platelets distinct from the GP1-IX-V complex. Inhibition of binding to this site by binding of a mutant thrombin, Quick II, enhances rather than reduces thrombin-stimulated platelet activation (Leong et al., 1992), suggesting that occupation of this site results in a negative regulation of platelet responsiveness. Furthermore, an antibody raised against the C terminus of hirudin has recently been shown to bind directly to a site on platelets distinct from GP1 and PAR-1, despite the presence of a hirudin-like domain within these proteins (Hayes and Tracy, 1999).

Irrespective of the identity of the high-affinity binding site for thrombin in platelets, it is likely to have a functional significance. Clearly, such a site may either positively or negatively regulate the threshold concentration of thrombin required to initiate platelet aggregation. Another possibility may be that the high-affinity site acts to promote chemotaxis, binding low concentrations of thrombin, and targeting platelets to an area of higher thrombin concentration. Such a system would allow platelets to be attracted to specific sites where the formation of a thrombus was necessary, and the higher concentration of thrombin present could cleave PAR-1 and induce platelet aggregation. These possibilities await examination.

A key component of the clotting and wound healing process is the activation of endothelial cells. Thrombin released from platelets stimulates the release of von Willebrand factor, cell surface redistribution of P-selectin, and increased expression of tissue factor and adhesion molecules, ICAM-1, VCAM-1, and E-selectin (Hattori et al., 1989; Bartha et al., 1993; Henn et al., 1998). These actions not only further promote the coagulation process and the binding and aggregation of platelets, but also facilitate the rapid adherence of neutrophils, monocytes, and later lymphocytes to the endothelial cell layer (Malik et al., 1986; Sugama and Malik, 1992; Zimmerman et al., 1994). Thrombin also stimulates endothelial cell contraction and increased permeability (Garcia et al., 1986; Malik et al., 1986; Lum and Malik, 1996) partly through the regulation of cell-to-cell junction organization (Rabiet et al., 1996). These events, along with increased adhesion molecule expression, facilitate rolling and transmigration of neutrophils and other cells to the site of vessel damage.

Concomitant with these effects, thrombin also stimulates aggregation of neutrophils and chemotaxis of neutrophils and monocytes (Bizios et al., 1986). However, several studies have demonstrated that the chemotactic response to thrombin is unrelated to the proteolytic properties of the enzyme, but rather the hirudin binding site appears to be the important feature of the protein (Bizios et al., 1986). The lack of PAR-1 expression on neutrophils (Jenkins et al., 1995), coupled with the noncatalytic nature of the interaction between thrombin and neutrophils, strongly indicates the presence of another binding site for thrombin on these cells. Such a binding site may be similar to that defined in platelets or to the nonproteolytically activated receptor found in other related cell types (Naldini et al., 1998). Further studies are required to confirm the existence and function of such a site on neutrophils; however, it is possible that all cells of megakaryocyte origin may possess a high thrombin affinity site to aid in cell movement.

Although thrombin is unable to induce Ca²⁺ mobilization in neutrophils, consistent with a lack of PAR-1 expression, the PAR-1 agonist peptide SFLLRNPND has been shown to raise intracellular Ca²⁺ levels (Jenkins et al., 1995). Subsequent studies indicate that PAR-2 is expressed on neutrophils (Howells et al., 1997) and that TRAPs, in addition to activating PAR-1, are capable of activating this receptor (Lerner et al., 1996) (see Section X.). It is therefore likely that other actions of TRAP on cells unresponsive to thrombin are due to PAR agonist cross-reactivity.

Thrombin, released from platelets, is a potent mitogen for cells of mesenchymal origin. In fibroblasts and vascular smooth muscle and endothelial cells, thrombin stimulates increases in DNA synthesis and promotes cellular proliferation with an efficacy comparable with serum (Chen and Buchanan, 1975; Carney et al., 1978; McNamara et al., 1993). These effects require thrombin serine-protease activity and are mimicked to some extent by SFLLLRNPNDKY-EPF (McNamara et al., 1993; Herbert et al., 1994), consistent with the effect being mediated by PAR-1. At lower concentrations, thrombin can act as a co-mitogen, suggesting roles as both a competence and progression factor. Thrombin is also mitogenic for selected cells of myeloid origin, such as lymphocytes, splenocytes (Chen and Buchanan, 1975), and other cells types, such as oesteoblasts (Abraham and Mackie, 1999). In addition to direct effects upon cell growth, thrombin also facilitates the production and release of promitogenic factors, such as PDGF and ET-1 through induction of PDGF and ET-1 prepro mRNA (Daniel et al., 1986; Yanagisawa et al., 1988; Garcia et al., 1993; Golden et al., 1998) and also regulates the subsequent release of these factors, in particular ET-1 (Kohno et al., 1992). Other similar actions of thrombin include the induction of receptors for VEGF, KDR and Flt (Maragoudakis et al., 2000) and the induction of TGF- (Bachhuber et al., 1997). These effects provide a basis for synergy between thrombin, or other mitogens and/or the potential for thrombin to mediate mitogenesis indirectly through release of other factors.

Activation of PAR-1 also results in marked effects on the synthesis of extracellular factors that are now known to be important in the normal wound healing process and in the development of vascular disorders (see Section XV.). Thrombin stimulates procollagen synthesis in smooth muscle cells and lung fibroblasts (Chambers et al., 1998; Dabbagh et al., 1998), and the expression of Cy61 and connective tissue growth factor (Pendurthi et al., 2000). Thrombin also regulates the induction and release of matrix metalloproteinases (MMPs), including progelatinase A (Zucker et al., 1995; Nguyen et al., 1999a) and MMPs 1, 2, and 3. These are key enzymes involved in degradation of the underlying basement membranes which, along with endothelial cell migration and proliferation, is an important first step in the initiation of angiogenesis. Consistent with these findings, thrombin has been shown to stimulate endothelial tube formation in matrigel and to stimulate angiogenesis in the chick chorioallantoic membrane system and in vivo (Tsopanoglou et al., 1993; Haralabopoulos et al., 1997). Thrombin also promotes MMP-2 release in vascular smooth muscle (Fernandez-Patron et al., 1999), suggesting that these events are common to many cells of the vasculature and are likely to participate in a co-ordinated wound healing process.

The effects of thrombin upon cell growth and division is not restricted to peripheral tissues. Both PAR-1 and prothrombin mRNA are expressed in a number of regions within the brain, such as the thalamus, hypothalamus, cortex, and cerebellum (Weinstein et al., 1995), indicating the presence of a functional thrombin effector system in the brain. Indeed, in neuronal cells, thrombin or TRAPs mediate neurite retraction and reversal of astrocyte stellation (Gurwitz and Cunningham, 1988; Grand et al., 1989; Cavanaugh et al., 1990; Beecher et al., 1994; Suidan et al., 1996), stimulate astrocyte proliferation (Grabham and Cunningham, 1995), and can protect against neuronal cell death induced by -amyloid, oxidative stress, or hypoglycemia (Vaughan et al., 1995; Pike et al., 1996). Furthermore, biochemical studies show increased synthesis of nerve growth factor and ET-1 in response to thrombin (Ehrenreich et al., 1993; Neveu et al., 1993), and a decrease in the expression of some subtypes of the metabotropic glutamate receptor (Miller et al., 1996). Taken together, these findings support a role for thrombin in mediating neuronal cell survival at least in response to some environmental insults.

At higher concentrations, thrombin per se causes death of hippocampal neurones (Pike et al., 1996) and in some studies can, at lower concentrations, potentiate -amyloid-induced cell death (Smith-Swintosky et al., 1995, 1997). These contradictory results suggest that thrombin, as well as aiding neuronal cell survival, may also function as a mediator of some disease states. For example, in Alzheimer's, the levels of an endogenous inhibitor of thrombin, protease nexin-1, have been shown to be reduced (Vaughan et al., 1994, 1995). This might lead to neuronal damage due to the presence of higher effective concentrations of thrombin. It has also been postulated that higher levels of systemic thrombin perhaps entering the brain following damage to the blood-brain barrier may act as a neurodegenerative agent.

Thrombin and TRAPs mediate a substantial endothelial-dependent relaxation of aortic and coronary blood vessels from species such as rat, guinea pig, and dog in vitro (Muramatsu et al., 1992; Tesfamariam, 1994a,b; Zaleski and Ku, 1993; Ku and Dai, 1997). This is likely to be mediated by both release of cyclooxygenase products, including possibly prostaglandin I₂ and by nitric oxide (NO), because many of the responses can be reversed by indomethacin and L-NAME or related analogs (Zaleski and Ku, 1993; Ku and Dai, 1997). Following removal of the endothelium thrombin, in some preparations, generates strong contractile responses (Zaleski and Ku, 1993; Ku and Dai, 1997) consistent with expression of PAR-1-linked Ca²⁺ influx in the underlying smooth muscle (Deblois et al., 1992; Antonaccio et al., 1993; Antonaccio and Normandin, 1994). In other vessels, for example, human umbilical and placental arteries, contractile responses can prevail even in endothelium intact vessels (Tay-Uyboco et al., 1995), indicating differences in the relative expression and function of PAR-1 on endothelial and smooth muscle cells in different vessels. These differences are reflected in whole organ responses to PAR-1 activation: administration of TRAPs causes vasodilation in perfused piglet lung, but vasoconstriction in the guinea pig (Pinheiro et al., 1993; Lum et al., 1994). In coronary vessels in vivo, TRAP generates a transient increase in blood flow followed by a sustained decrease (Damiano et al., 1996a). Indeed, the contractile effects of PAR-1 activation in coronary vessels can also mediate secondary changes in heart function, such as decreases in cardiac output and mean arterial pressure (Damiano et al., 1996a,b), despite the fact that thrombin can directly stimulate both via a positive ionotrophic effect through increased intracellular Ca²⁺ (Steinberg et al., 1991).

Administration of TRAP to mice in vivo causes a rapid hypotension followed by a sustained moderated hypotension (Darrow et al., 1996; Cheung et al., 1998). However, when NO release is prevented following pretreatment with L-NAME, a rebound hypertension is revealed reflecting the expression of PAR-1 on vascular smooth muscle. Despite these findings, a physiological role for thrombin in the regulation of cardiovascular function is not overwhelming, since in mice deficient in PAR-1, parameters of cardiac function and blood pressure are not different from normal mice (Darrow et al., 1996). It is, therefore, more likely that thrombin plays a role in the control of local blood flow following tissue damage.

	V. Proteinase-Activated Receptor-1-Mediated Cellular Signaling

Top Previous Next References

In common with several other helipthical receptors, PAR-1 has been shown to couple to multiple heterotrimeric G-proteins (Fig. 4). In a number of early studies, two main signaling events were characterized that were assumed to involve receptor G-protein coupling. The first event involves the inhibition of cAMP through interactions with inhibitory G-protein of the G_i class (Hung et al., 1992; Kanthou et al., 1996). The second event is stimulation of phospholipase C (PLC)-catalyzed hydrolysis of polyphosphoinositides, resulting in the formation of InsP₃, mobilization of intracellular Ca²⁺, and generation of diacylglycerol, the endogenous activator of protein kinase C (PKC) (Babich et al., 1990; Hung et al., 1992). Thrombin also stimulates the rapid hydrolysis of other phospholipids, implying roles for PLD, PLA₂ and phosphatidylcholine-specific PLC in the initial generation of lipid activators of protein kinase C isoforms (McNicol and Robson, 1997; Cheng et al., 1999).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   G-protein-dependent signaling pathways regulated through activation of PAR-1. The major signaling events regulated by PAR-1. Dashed lines represent putative pathways where the intermediates have not been identified or fully demonstrated for PAR-1, but are a feature of other G-protein-coupled receptors. Other signaling pathways implicated in PAR-1 activation are outlined in Table 3. Some well identified intermediates and precursors have been omitted for sake of clarity. DAG, diacylglycerol; SRC, pp60src and related kinases; MAPK, p42/44 mitogen-activated protein kinase; PKB, protein kinase B; GER, Rho GTP exchange factor; PIP3, phosphatidylinositol 3,4,5-trisphosphate.

The recent identification of multiple G-protein subunits and their corresponding effector enzymes allowed examination of these transduction mechanisms. Microinjection of antibodies directed against G_q/11 into CCL-39 cells inhibited PAR-1-mediated Ca²⁺ mobilization (Baffy et al., 1994), whereas the same antibodies abrogated GTPase activity in thrombin-stimulated platelet membranes (Benka et al., 1995). Furthermore, in platelets derived from transgenic mice lacking G_q, thrombin-stimulated phosphoinositide hydrolysis was abrogated (Offermanns et al., 1997).

A direct interaction between PAR-1 and G_q/11 and G_i2 has been recently demonstrated by immunoprecipitation of PAR-1 with G_i2 and G_q/11 in thrombin-stimulated human neuroblastoma SH-EP cells (Ogino et al., 1996), clearly indicating interaction of PAR-1 with these two G-protein subunits. In a number of cell systems, pertussis toxin (PTX)-mediated ADP ribosylation of G_i/G_o -subunits also reduced thrombin-stimulated InsP₃ formation and Ca²⁺, indicating the potential for coupling of PAR-1 to G_i/G_o subunits (Babich et al., 1990; Brass et al., 1991). Antibodies to G_o also reduced PAR-1-mediated responses (Baffy et al., 1994), suggesting that this subunit contributes to PLC activation. However, G_o expression is cell-specific, and it is likely that another pertussis sensitive G-protein, possibly G_i2, may also be involved. At present, it remains unclear for thrombin receptor systems whether subunits derived from G_i2 or G_o are able to activate other isoforms of PLC-, such as PLC-2 or PLC-3.

PAR-1 is also linked to other second messenger systems via pertussis-sensitive G-proteins. Thrombin-mediated inhibition of adenylyl cyclase has been demonstrated to involve a direct interaction of the receptor with G_i2 (Hung et al., 1992; Kanthou et al., 1996; Magnaldo et al., 1988; Swift et al., 2000). Stimulation of other phospholipase activity, such as PLD and PLA₂ has also been shown to be sensitive to PTX in some cell types (Banga et al., 1988; Suzuki et al., 1996). However, evidence supporting a direct interaction between the receptor and a G-protein -subunit in a manner analogous to G_q/11/PLC-1 is minimal. One study has shown that expression of a mutant G_i2 protein can specifically inhibit arachidonic acid release in response to thrombin (Winitz et al., 1994) through a mechanism that does not involve intermediates known to regulate PLA₂ activity. In general, regulation of these phospholipases following PAR-1 activation is likely to be downstream of initial activation of PLC- isoforms and, indeed, in cells where PLC-1 is poorly expressed, thrombin stimulation of PLD and cPLA₂ is diminished (Fee et al., 1994).

Recent studies have also demonstrated that PAR-1 also transduces important cell signals via G₁₂ and G₁₃. In platelet membranes, thrombin stimulates the incorporation of the photoreactive GTP analog [-³²P]GTP azidoanilide into G₁₂ and G₁₃ as assessed by immunoprecipitation studies (Offermanns et al., 1994), suggesting a direct interaction of both G-protein -subunits with PAR-1. Furthermore, injection of antibodies directed against G₁₂ prevents thrombin-mediated gene transcription and DNA synthesis (see below) strongly implicating a PTX-insensitive, and thus G_i/G_o-independent mechanism, as being responsible for many of the cellular effects of PAR-1.

Although thrombin was able to activate PKC isoforms in several cell types, principally by hydrolysis of multiple phospholipids (Baron et al., 1993; Godin et al., 1995), other pathways were implicated in the pro-mitogenic effects of PAR-1 activation. This was based on several key observations. Firstly, thrombin was found to be a potent mitogen relative to other G-protein coupled receptor agonists, despite generating comparable phospholipid and Ca²⁺ signals (Seuwen et al., 1990). Second, PAR-1-activating peptides stimulated inositol phosphate formation to a level comparable with thrombin itself but were unable to stimulate mitogenesis (Vouret-Craviari et al., 1992) and lastly, in a number of cell types thrombin-stimulated mitogenesis was PTX-sensitive while phospholipid hydrolysis was PTX-independent (Babich et al., 1990).

Since the identification of the mitogen-activated protein (MAP) kinases, key signaling events central to the action of thrombin have been identified (see Fig. 4). Multiple signaling paradigms have since been established for PAR-1, including activation of PI-3 kinase (Mitchell et al., 1990; Walker et al., 1998), Src family tyrosine kinases (Cichowski et al., 1992; Rao et al., 1995), stress-activated protein (SAP) kinases (Mitsui et al., 1997, 1998; Malcolm et al., 2000;), Rho kinase (ROK) (Seasholtz et al., 1999; Carbajal et al., 2000), Janus activated kinase-2 (JAK-2) (Rodriguez-Linares and Watson, 1994; Huang et al., 2000), focal adhesion kinase, pp125^fak (Negrescu et al., 1995; Choudhury et al., 1996), and proline-rich tyrosine kinase 2 (Pyk-2) (Ohmori et al., 2000) (see Table 3).

A paradigm for the activation of p42/44 MAP kinase or extracellular-regulated kinases (ERKs) has now been established for tyrosine kinase-linked receptors (Malarkey et al., 1995). Phosphotyrosine residues within the intracellular domain of an activated receptor interact with the adaptor protein SHC that in turn recruits GRB-mSos resulting in increased rate guanine-nucleotide exchange by the monomeric G-protein p21^ras. This initiates binding of Raf-1 isoforms to the plasma membrane for activation by Ras and some other factor, and downstream activation of MEK-1, the direct activator of MAP kinase. Multiple variations of this model can be applied for a number of growth factors and G-protein coupled receptors, and depending on cell type, PAR-1 incorporates many components of such a paradigm (Fig. 4).

Early studies demonstrated that thrombin stimulated p42/44 MAP kinase activation was essential for initiation of DNA synthesis (Pages et al., 1993). However, in contrast with agonists for other G-protein-coupled receptors, thrombin was also found to stimulate a biphasic activation of p42/44 MAP kinase, the sustained phase of which was essentially PTX-sensitive (Kahan et al., 1992). Furthermore, thrombin was found to stimulate GTP/GDP exchange on p21^ras indicating the potential for a `growth factor-like' MAP kinase cascade to also be activated via PAR-1 and other G-protein-coupled receptors, such as the receptor(s) for lysophosphatidic acid (Van Corven et al., 1993). In this instance, activation of p21^ras was inhibited by both PTX pretreatment and genestein, a nonselective tyrosine kinase inhibitor, suggesting the involvement of both a G_i protein and tyrosine kinase in mediating the activation of p42/44 MAP kinase by PAR-1. Several recent studies have shown for other G-protein-coupled receptors, although not for PAR-1, a role for -subunits in the activation of Src, tyrosine phosphorylation of p52^SHC, and formation of SHC-GRB-2 complexes as a mechanism by which G_i-dependent activation of p42/44 MAP kinase could be achieved. Thrombin-mediated stimulation of pp60^src and phosphorylation of SHC has been demonstrated in a number of cell types (Chen et al., 1994b, 1996b; Rao et al., 1995), consistent with this model of ERK activation. However, these events are not in all instances PTX-sensitive and indicate the potential for G_i-independent pathways to regulate early events in the MAP kinase signaling cascade (Chen et al., 1996b). Recently in some cell types, G protein-coupled receptor agonists such as lysophosphatidic acid and thrombin have also been found to stimulate the tyrosine phosphorylation of growth factor receptors such as the basic fibroblast growth factor (Weiss and Maduri, 1993) and insulin-like growth factor-1 receptors (Delafontaine et al., 1996) resulting in the recruitment to the receptor of SHC and other intermediate proteins, and the subsequent activation of the MAP kinase signaling cascade, a phenomenon known as transactivation. This is likely to involve pp60^src or a similar tyrosine kinase; however, the events that regulate these events have not been elucidated.

A similar mode of activation of other signaling pathways may also be a feature of PAR-1. PI-3 kinase plays important roles in thrombin-mediated regulation of cytoskeletal structure, cell motility, cell survival, and mitogenesis and, also in some cell types, functions as an intermediate in activation of ERKs (Malarkey et al., 1995; Touhara et al., 1995). Thrombin stimulates the accumulation of PtdIns(3,4,5)P₃ in platelets, neutrophils, human and bovine airway smooth muscle cells, and others through activation of multiple PI-3 kinase isoforms, including a novel 110-kDa isoform that can be directly activated by G-protein -subunits, rather than through binding of the tyrosine kinase receptor-associated protein p85 PI-3 kinase. In platelets, thrombin-stimulated PI-3 kinase activity involves the small molecular weight G-protein Rho (Zhang et al., 1995). In addition, activity can also be regulated by sequestration of G-protein -subunits, consistent with a role for -p110 and thus a G_i/G_o-dependent pathway. This latter model of activation is likely to be restricted to certain cell types, where PAR-1 mediated second messenger formation is largely PTX-sensitive, although it is likely that even in a single cell type multiple pathways for activating PI-3 kinase isoforms exists. In platelets, the activity of other small molecular weight G-proteins, such as Rac and Cdc42, may also be regulated through PAR-1 activation, although their inter-relationship with PI-3 kinase signaling and aggregation remains unclear (Azim et al., 2000).

In both human and bovine airway smooth muscle cells and in pulmonary artery fibroblasts, PI-3 kinase is implicated in PAR-1-mediated activation of p70^s6k (Belham et al., 1997; Walker et al., 1998; Johanson et al., 1999; Krymskaya et al., 1999), and protein kinase B (Walker et al., 1998), two important regulators of cell survival and mitogenesis. Although thrombin-stimulated p70^s6k is partially PTX-sensitive in pulmonary artery fibroblasts, suggestive of the involvement of a -regulated form of PI-3 kinase, in human and bovine airway smooth muscle cells, thrombin has been shown to stimulate the tyrosine phosphorylation of the classical growth factor receptor-associated p85/110 isoform (Walker et al., 1998; Krymskaya et al., 1999). Thus, as with activation of p42/44 MAP kinase cascade, intermediate stimulation of pp60^src and/or transactivation of a growth factor receptor is also likely to be involved in the activation of this pathway.

In some cell types where PTX-independent cellular responses to PAR-1 have been recorded, G₁₂/G₁₃ signaling pathways have been implicated. Injection of antibodies directed against G₁₂ abolished thrombin-stimulated DNA synthesis in 1321N1 astrocytoma cells (Aragay et al., 1995), whereas reconstitution of PAR-1 with G₁₂ in COS-7 cells gives rise to substantial AP-1-mediated gene expression in response to thrombin (Post et al., 1996). This is in turn likely to be mediated via Ras- or Rac-dependent activation of JNK, as expression of a dominant negative mutant of MEKK-1, an upstream regulator of JNK, or mutant forms of Ras and Rac inhibit thrombin-stimulated AP-1 gene expression in NIH3T3 cells (Collins et al., 1996). Recent evidence also suggests that G₁₂ is essential for thrombin-stimulated tyrosine phosphorylation of SHC and AP-1 reporter activity (Collins et al., 1997), whereas Src has been implicated in JNK activation mediated by G₁₂ (Nagao et al., 1998). Thus, it is possible that PAR-1 may utilize G₁₂ to activate Src, resulting in the phosphorylation of SHC, activation of JNK and regulation of AP-1 activity. Further studies are required, however, to confirm whether this pathway mediates JNK activation in other PAR-1 expressing cell systems. Thrombin has also been shown to activate both JNK and p38 MAP kinase in other cell types, and both PTX-sensitive G-proteins and PKC have been implicated, suggesting additional roles for G_i- and G_q-dependent pathways (Mitsui et al., 1997, 1998; Malcolm et al., 2000).

In other cell types however, G₁₂ has also been implicated in the regulation of Rho-dependent events initiated via PAR-1. As well as being implicated in the regulation of PI-3 kinase, JNK and others, Rho plays an intimate role in the regulation of cellular responses to thrombin through activation of a number of target kinases, including in particular ROK. PAR-1-mediated responses in which Rho or ROK have been implicated include: the activation of cell rounding and apoptosis in cultured neurones and astrocytes (Donovan et al., 1997; Majumdar et al., 1998), stimulation of smooth muscle DNA synthesis and cell migration (Seasholtz et al., 1999), stress fiber formation (Crouch, 1997), platelet aggregation (Zhang et al., 1995), endothelial cell and smooth muscle contraction (Essler et al., 1998), and endothelial cell barrier dysfunction (Vouret-Craviari et al., 1998; Carbajal et al., 2000). Many of these events are also activated by G₁₂- or G₁₃-dependent mechanisms, and recent studies have provided evidence for the direct coupling of G₁₂ to Rho via a group of Rho-specific guanine nucleotide exchange factors (Majumdar et al., 1998; Fukuhara et al., 1999). Taken together, these studies suggest that the G₁₂/Rho/Rho kinase axis may represent a new and important pathway in mediating PAR-1 response in a variety of cell types.

	VI. Desensitization of Proteinase-Activated Receptor-1

Top Previous Next References

The intramolecular basis for PAR-1 activation through the generation of a tethered N terminus ligand has important implications for the magnitude and kinetics of thrombin responses. Firstly, a single thrombin molecule may proteolytically cleave multiple thrombin receptors and secondly, cleavage could result in sustained activation of each receptor. This does not seem to be the case, however, because the extent of phosphoinositide hydrolysis is directly proportional to the concentration of thrombin (Ishii et al., 1993). This implies the generation of a fixed "quanta" of second messenger followed by a rapid termination mechanism. Thus, PAR-1 desensitization has been examined in considerable detail and closely compared with that observed in other G-protein-coupled receptors activated through a normal ligand/receptor mechanism (Hein et al., 1994; Vouret-Craviari et al., 1995). For such receptors, desensitization essentially entails uncoupling of the receptor from the G-protein, followed by subsequent internalization (Bohm et al., 1997) (Fig. 5). Desensitization also includes the potential for the long-term down-regulation of mRNA expression (Weinstein et al., 1998); however, relatively few studies of this type have been performed regarding PAR-1.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Modes of PAR-1 desensitization. C-terminal phosphorylation at distinct residues can mediate rapid receptor shutoff (A), followed by further phosphor