- Email: [email protected]
Thrombin receptor function and cardiovascular disease
Tokuyasu K, Maher P: 1987. Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of a-actinin dots within titin spots at the time of the first myofibril formation. J Cell Biol 105:2795-2801. Toyofuku T, Doyle D, Zak R, Kordylewski L: 1993. Expression of phospholamban during early avian muscle morphogenesis is distinct from that of a-actin. Dev Dynam 196:103-l 13. Toyota N, Shimada Y: 1981. Differentiation of troponin in cardiac and skeletal muscles in chick embryos as studied by immunofluorescence microscopy. J Cell Biol9 1:497-504. Van Mierop L, Gessner I: 1970. The morphologic development of the sinoatrial node in the mouse. Am J Cardiol25:204-212.
Viragh S, Challice C: 1982. The development of the conduction system in the mouse embryo heart. IV. Differentiation of the atrioventricular conduction system. Dev Biol89:2540.
plasminogen
Vitadello M, Matteoli M, Gorza L: 1990. Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes. J Cell Sci 97: 1 l-2 1.
sel wall by an endothelial-dependent mechanism, probably by causing surface expression of GMP-140 on the endothelial surface (Hattori et al. 1989, Zimmerman et al. 1986) and directly activates neutrophils themselves (Cohen et al. 1991). These disparate functions of thrombin may be unified by viewing thrombin as an orchestrater of the response to vascular injury or wounding, mediating not only hemostatic but inflammatory and proliferative or reparative responses (Coughlin et al. 1992). Thrombin’s actions on cells also conjure hypotheses regarding possible roles in disease. Several pharmacologic studies demonstrate that thrombin activity is critical forplateletdependent arterial thrombosis in animal models (Eidt et al. 1989, Fitzgerald and Fitzgerald 1989, Hansen and Harker 1988, Heras et al. 1989, Jang et al. 1989). Moreover, the efficacy of antithrombin therapy for unstable angina (Theroux et al. 1989) suggests that thrombin activity is important in this disease. Beyond the platelet, thrombin’s proinflammatory actions on monocytes, neutrophils, and endothelial cells, and its proliferative effect on mesenchymal cells, suggest a possible role in pathologic responses of the vessel wall to injury. Candidate disorders include restenosis, glomerulosclerosis, and perhaps atherogenesis itself. While thrombin’s critical role in hemostasis and thrombosis is well established, the in vivo importance of its proliferative and inflammatory actions has not been defined. This introduction begs an understanding of the mechanisms by which thrombin activates cells. A recently cloned thrombin receptor has provided a framework for understanding how thrombin talks to cells (Vu et al. 1991a) and appears to account for many of the thrombin activities cited above. Structureactivity studies with this receptor have revealed a novel proteolytic mechanism of receptor activation. Moreover, this receptor has provided new tools for defining the role of thrombin-induced cell activation in vivo and may represent a new target for antithrombotic and other therapies (Coughlin et al. 1992). This review describes recent mechanistic
Woodcock-Mitchell J, Mitchell J, Low R, et al.: 1988. a-Smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation 39: 16 l166. Zeller R, Bloch K, Williams B, Arceci R, Seidman C: 1987. Localized expression of atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev 1:693-698. TCM
Thrombin Receptor Function and Cardiovascular Disease Shaun R. Coughlin
Thrombin, a multifunctional protease generated at sites of vascular injury, is a powerful agonist for a variety of cellular processes important in cardiovascular disease. A recently cloned thrombin receptor has provided a framework for understanding how thrombin, a protease rather than a classic ligand, activates cells. It has also yielded new tools for defining the role of thrombin and its receptor in cellular events. This review discusses a working model for how thrombin activates platelets and other cells, and possible roles for the thrombin receptor in thrombotic, proliferative, and inflammatory processes. (Trends Cardiovast bled 1994;4:77-83)
Thrombin
is a multifunctional
generated
at
sites
bin is the most potent activator of plate-
protease
of vascular
injury.
lets in vitro (Bemdt
While best known for its ability to cleave
Davey and Luscher
fibrinogen
also
thrombin variety
and trigger
fibrin formation,
is also a powerful agonist for a of cellular
responses
(Figure
1
chemotactic
and Phillips
1981,
1967). Thrombin for monocytes
Shavit et al. 1983) and is mitogenic lymphocytes,
fibroblasts,
and
is
(Barfor
vascular
[reviewed in Fenton (1988) and Coughlin
smooth muscle cells (Chen and Buchanan
et al. (1992)].
1975, Chen et al. 1976, McNamara
First and foremost,
throm-
1992). Shaun R. Coughlin is at the Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0524, USA.
TCM Vol.4,No. 2,1994
Thrombin
endothelium prostacyclin activating
01994,
acts
et al.
on the vascular
to stimulate
production
of
(Weksler et al. 1982), plateletfactor
(Prescott
et al. 1984),
ElsevierScience Inc., 1050-l738/94/$7.00
activator-inhibitor
(Camps
et al. 1993), and the potent smooth muscle cell mitogen tor (Daniel
platelet-derived
growth fac-
et al. 1986). Thrombin
induces neutrophil
adherence
also
to the ves-
77
Figure 1. Cellular actions of thrombin. Cellular actions of thrombin are illustrated in the context of a blood vessel. Thrombin is the most potent activator of blood platelets, an action probably critical for hemostasis and thrombosis. Thrombin is also chemotactic for monocytes, mitogenic for lymphocytes, activates neutrophils, and has a variety of effects on endothelial cells; these actions of thrombin may contribute to inflammatory responses to vascular injury. Thrombin is a potent mitogen for vascular smooth muscle cells and fibroblasts, actions that may contribute to proliferative and reparative responses to vascular injury or wounding. Reprinted with permission from J Clin Znvest (Coughlin et aI. 1992).
insights provided by the newly cloned thrombin receptor and its possible roles in cardiovascular disease.
l
ThromWm Receptor StructureActivityRelationships: How Does a Protease Talk to a Cell?
The thrombin receptor is a member of the seven-transmemb~e-domain receptor family, but is activated by a novel proteolytic mechanism (Vu et al. 1991 a). Thrombin binds to and cleaves its receptor’s extracellular amino terminal domain to unmask a new amino terminal. This new amino terminal then functions as a tethered peptide ligand, binding to as yet undefined sites within the body of the receptor to effect receptor activation (Figures 2 and 3). The studies that developed and extended this model are described below. Molecular Basis for Thrombin-Receptor Interaction Examination of the thrombin receptor’s amino acid sequence revealed a putative thrombin cleavage site resembling the known tbrombin cleavage site in protein C within the receptor’s amino terminal exodomain (Figure 2). Carboxyl to this 78
site was a sequence resembling the carboxy1 tail of hirudin, a structure known to interact with thrombin’s anionbinding exosite (Rydel et al. 1990). These analogies suggested that the receptor might interact with thrombin in the manner shown in Figure 2b and serve as a thrombin substrate (Vu et al. 199 la). This model has been supported by multiple studies: 1. Mutation of the thrombin receptor cleavage site to block cleavage rendered the receptor unactivatable by thrombin (Vu et al. 1991a). 2. Replacing the thrombin cleavage recognition sequence with that for enteropeptidase switched receptor specificity; cells (Hung et al. 1992d) or oocytes (Vu et al. 1991b) expressing this construct responded to enteropeptidase but not to thrombin. 3. Synthetic peptides mimicking the receptor’s cleavage site were cleaved by thrombin (Vu et al. 199 1b) and uncleavable “mutant“ peptides mimicking this region bound thrombin and inhibited its activity against synthetic substrates, fibrinogen, and its receptor (Hung et al. 1992b, Liu et al. 1991, Vu et al. 1991b). 4. Recent studies used an antibodybinding method to demonstrate receptor
01994, Else&r Science Inc., 1050-1738~4/$7.~
cleavage directly on intact cells and to show that receptor cleavage correlated with signaling [Ishii et al. (1993); see below]. 5. The bidentate interaction shown in Figure 2b is supported by the observations that receptor peptides require both the primary cleavage recognition sequence LDPR and the receptor’s hirudinlike domain sequence KYEPF for avid interaction with thrombin (Liu et al. 1991, Vu et al. 199ib). Moreover, alanine-scanning mutants of the KYEPF sequence suggest that this sequence bmds thrombin’s anionbinding exosite in a manner grossly analogous to the DFEEI sequence in hirudin’s carboxyl tail (Vu et al. 199lb). x-ray crystallographic studies of cocrystals of thrombin with receptor-based peptides are in progress and promise to reveal the details of this interaction. Several important questions regarding thrombin-receptor interaction remain to be answered. First, do other receptor domains beyond those described above participate in thrombin-receptor interaction? Second, do other thrombinbinding proteins on the surface of platelets and other cdls play an important role in presenting tbrombin to its receptor (either directly by formation of a ternary complex with thrombin and its receptor, or indirectly by protecting locally produced thrombin from inactivation)? Such a role has been suggested for the platelet surface glycoprotein GPIb (Okamura et al. 1978). Lastly, does the thrombin receptor cause a conformational change in thrombin to enable cleavage of the LDPR site? This question is raised by the identity of the human thrombin receptor’s thrombin cleavage site (LDPR) with that in bovine protein C (Vu et al. 1991a). When bound to thrombomodulin, thrombin gains the ability to cleave and activate the anticoagulant protein C. Thrombomodulin effects this change in thrombin substrate specificity in part by inducing a conformational change in thrombin such that it can accommodate the normally unfavorable LDPR cleavage recognition sequence (Ehrlich et al. 1990, Le Bonniec and Esmon 1991). Whether the thrombin receptor causes a similar conformational change in thrombin to accommodate the receptor’s own LDPR sequence and promote efficient receptor cleavage is unknown. TCM Vol. 4, No. .?z1994
\
HUMAN TR38-60 MOUSE TRB-62 a
A”IoN-8INDING BlH”ING DOM~I”
AGOVLST PEPriDE DaHarN
CLEavaGE SITE
I
Mechanism
ElOIiTE
Silent in the Vncleaved Receptor?
I
LOPR/SFLLRN/PND-K/YEPFWE-DEE.. VNPR/SFFLRN/PSENT/::L'IPLGDEE..
HIRUDIN 55-65
The model just described casts the throm-
. ..DFEEIPEEY'LO-Coo
Figure 2. (a) Functional domains within the thrombin receptor’s amino terminal extension. The cleavage recognition sequence (LDPR), thrombin cleavage site, agonist peptide domain, and anion-binding exosite binding domain as defined by structure-activity studies with the human receptor are shown. These are aligned with the murine thrombin receptor sequence and the anion-binding exosite-binding sequence of the leech anticoagulant hirudin. (b)A model for interaction of these domains with thrombin. Thrombin has an extended substrate-binding surface (represented by the canyon running laterally) that recognizes residues both amino and carboxyl to its substrate’s cleavage site. Structure-function studies suggest that the receptor’s hirudinlike domain (KYEPF) interacts with thrombin’s anion-binding exosite, while its cleavage site (LDPRIS) interacts with thrombin’s Sl-S4 subsites. This model has important implications for the development of blocking antibodies and receptor peptide-based thrombin inhibitors. Reprinted with permission from Nature (Vu et al. 1991b).
Proteolytic Unmasking of a Tethered Peptide Ligand: A Novel Mechanism of Receptor Activation How might proteolysis within the thrombin receptor’s amino terminal extension activate the receptor? There are several precedents for a protease activating a target protein by unmasking a new amino terminus within that protein. In particular, proteolytic activation of the zymogen trypsinogen occurs when enteropeptidase cleaves it to unmask a new amino terminus that then binds intramolecularly to effect a conformational change and create an active trypsin molecule. A grossly analogous mechanism exists for the thrombin receptor (Figure 3) (Vu et al. 1991a). Synthetic peptides that mimic the new amino
TCM Vol. 4, No. 2, 1994
terminus
created
when
cleaves its receptor receptor
thrombin
are full agonists
activation,
quirement
and bypass
for receptor
for
the re-
proteolysis
(Fig-
ures 2 and 3) (Vu et al. 1991a).
This
observation
suggests
the model
shown
in Figure 3. Thrombin
cleaves its recep-
tor’s amino
extension
terminal
mask a new amino amino
terminus
terminus. then
to un-
This new
functions
as a
tethered peptide ligand, binding to an as yet undefined receptor
site within the body of the
to effect
receptor
activation
(Vu et al. 1991a).
As discussed
synthetic
mimicking
peptides
onist peptide
domain” receptor
below, this “ag-
(Figure
vide a new tool for defining the thrombin
2) pro-
the role of
in various
cellu-
lar events.
01994.
of the “Proteolytic Switch’:
How Does the Tethered Ligand Remain
ElsevierScienceInc., lOSO-1738/94/$7.00
bin receptor as a peptide receptor that contains its own ligand. This ligand remains cryptic until unmasked when thrombin cleaves the receptor. How might this “proteolytic switch” work? Recent structure-function studies of the receptor’s agonist peptide domain suggest a possible answer. The first five amino acids of the receptor’s agonist peptide domain (SPLLR in the single-letter code) are sufficient to specify agonist activity (Scarborough et al. 1992, Vassallo et al. 1992, Vouret-Craviari et al. 1992). The protonated amino group of Serl and the Phe2 side chain are vital for agonist function; the Leu4 and At-g5 side chains play less important roles (Coller et al. 1992, Scarborough et al. 1992, Vassal10 et al. 1992). The importance of Serl’s protonated amino group is particularly appealing, as this group is created by receptor cleavage. This may explain in part how the agonist peptide domain’s activity is masked when the receptor is in the uncleaved state (Scarborough et al. 1992). Steric and structural contributions to maintaining the agonist peptide silent in the uncleaved receptor remain to be defined. Kinetics of Thrombin Receptor Cleavage and Relation to Signaling: How Does a Protease Elicit Concentration-Dependent Responses? Like other important signaling molecules, thrombin effects concentration-dependent and graded responses in its target cells (Berndt and Phillips 1981, Detwiler and Feinman 1973, Martin et al. 1975 and 1976, Paris and Pouyssegur 1986, Rittenhouse-Simmons 1979), a feature vital for normal homeostasis. Classic ligands effect concentration-dependent responses via graded receptor occupancy. How thrombin acting as an enzyme rather than a classic ligand might effect concentrationdependent responses has been a longstanding question (Berndt and Phillips 198 1, Detwiler and Feinman 1973, Martin et al. 1975 and 1976). Specifically, one would predict that even low amounts of thrombin would eventually cleave and activate all cell surface receptors. How then can a concentration-dependent response be achieved? A recent study used
79
tor signaling
has several
implications.
First, it places great importance on the mechanisms that terminate thrombin receptor signaling (see below). Second, it suggests that thrombin receptor antagonists need only slow thrombin receptor activation enough that clearance of second messengers outstrips their generation to block signaling effectively (Ishii et al. 1993). This notion may encourage attempts at antagonist development, which might otherwise be discouraged by the receptor’s intramolecular tethered liganding mechanism. Transmembrane
Signaling
Where the thrombin receptor’s agonist peptide domain binds and how this binding event causes a transmembrane signal that enables the receptor to activate G proteins is unknown. Ligand binding for seven-transmembranedomain receptors is best studied for the &-adrenergic receptor (Dohlman et al. 1992). In this case, the catecholamine ligand interacts with residues predicted to reside within the transmembrane domains. The details of how this binding “switches the receptor on” and even whether peptide agonists bind in an analogous manner are unknown.
Figure 3. Model of thrombin receptor activation. Thrombin, the sphere in this figure, binds to its receptor’s extracellular amino terminal extension (see Figure 2 for detail). After binding to the amino terminal extension, thrombin cleaves the receptor at the LDPR/S cleavage site (junction between open and filled receptor segments), releasing an inactive fragment of the receptor’s amino terminus (open fragment) and exposing a new amino terminus. This newly unmasked amino terminus then functions as a tethered peptide Iigand, binding to as yet undefined sites within the body of the receptor to effect receptor activation. As shown, this binding event presumably translates into a conformational change in the receptor’s cytoplasmic face, effecting G-protein activation. Exactly how ‘news” of ligand binding to a seven-transmembrane-domain receptor is conveyed to the cell’s interior remains a fundamental structure-function question in this field. Reprinted with permission from Nature (Vu et al. 1991b).
antibodies that distinguished the naive receptor from the cleaved and activated form to follow the rate of thrombin receptor cleavage on intact cells (Ishii et al. 1993). The rate of receptor cleavage was proportional to thrombin concentmtion over the range known to elicit concentration-dependent responses, but low thrombin concentrations did ultimately cleave all cell surface thrombin receptors. Cumulative phosphoinositide hydrolysis in response to thrombin correlated precisely with absolute receptor cleavage during a given time interval, not with the integral of receptor cleavage as a function of time. These data strongly suggest that
80
each activated thrombin receptor generates a quantum of second messenger, then ‘shuts off (Ishii et al. 1993). Thrombin concentration would then determine the rate of receptor activation and therefore the rate of second-messenger generation. Second-messenger levels and magnitude of the cellular response are presumably then determined by the balance between the rates of second-messenger generation and clearance (Figure 4). This is unlike the case for classic ligands; there is no ‘equilibrium” binding and the cell cannot utilize graded receptor occupancy to effect graded responses. This formulation
01994,
of thrombin
recep-
Elsevier Science Inc., lOSO-1738/94/$7.00
Mechanisms of Receptor Shutofi Did the Novel Activation Mechanism Beget a Novel Shutoff Mechanism? The formulation of thrombin receptor signaling outlined above suggests that the thrombin receptor’s ‘shutoff mechanism is a critical determinant of the gain of the system and thereby of thrombin responsiveness (Ishii et al. 1993, Vu et al. 1991a). The mechanism of thrombin receptor shut off has not been rigorously defined. By analogy with other seven-transmembrane-domain receptors (Dohlman et al. 1992) it is likely that receptor kinases play an important role in the immediate termination of thrombin receptor signaling. Indeed, recovery of receptor responsiveness to agonist peptide after desensitization was inhibited by phosphatase inhibitors (Brass 1992) consistent with but not proving a role for phosphorylation in desensitization. Receptor internalization (sequestration) and degradation (downregulation) may also be involved in termination of thrombin receptor signaling. Recent work does suggest that the thrombin receptor undergoes agonist-induced internalization (Brass 1992, Ishii et al. 1993) ; the
TCM Vol. 4, No. 2, 1994
relative
contribution
of internalization
to termination of thrombin signaling is unknown. A striking paradox has been noted in thrombin receptor signaling. Based on recent kinetic studies of signaling in receptor-transfected cell lines, it appears that the thrombin receptor stops signaling despite the continued presence of cleaved and “activated” receptors on the cell surface and at a time when cells are refractory to thrombin but sensitive to agonist peptide (Ishii et al. 1993). These observations may be consistent with the earlier finding that responsiveness to agonist peptide recover faster than responsiveness to thrombin in HEL cells (Brass 1992). The finding of an agonistpeptide-responsiv~thmbin-unresponsive state despite the continued presence of cleaved and “activated” thrombin receptor on the cell surface is provocative. A trivial explanation of this observation is the existence of a receptor pool accessible to exogenous agonist peptide but not to thrombin. A more exciting possibility is that the receptor may become modified so that the tethered agonist peptide domain cannot function, but exogenous agonist peptide can. Thus, an as yet uncharacterized and novel shutoff mechanism may have evolved to deal with the tethered ligand and with the obligate relationship of receptor activation to phosphoinositide hydrolysis.
l
Potential Roles for the Thrombin Receptor in Cardiovascular Disease
Synthetic peptides mimicking the thrombin receptor’s agonist peptide domain, blocking antibodies, and the receptor cDNA itself have provided new tools for defining the role of the cloned receptor in both intracellular signaling events and thrombin-induced cellular functions in vitro. However, no human genetic disease caused by a thrombin receptor mutation has yet been described, and “knockout” of the thrombin receptor gene in mice remains to be accomplished. Potent and specific antagonists for the thrombin receptor are not yet available. Because tools to define rigorously the roles of the thrombin receptor in normal physiology and in disease are not yet available, we are left to speculate, Several known thrombin-induced intracellular signaling events and in vitro cellular events have been shown to be
TCM Vol. 4, No. 2, 1994
mediated by the cloned thrombin receptor. A discussion of these events with extrapolation to the receptor’s possible roles in cardiovascular
function
follows.
Intracellular Signaling Thrombin is known to activate both phosphoinositide hydrolysis and to inhibit adenylyl cyclase in platelets (Banga et al. 1988, Brass et al. 1986) and other responsive cells (Jones et al. 1989, Paris and Pouyssegur 1986). In platelets, both of these second-messenger events serve to promote aggregation (Kroll and Schafer 1989). The thrombin receptor agonist peptide has been reported to elicit both of these second-messenger events in fibroblasts (Hung et al. 1992a, VouretCraviari et al. 1992), and transfection of the cloned thrombin receptor conferred both receptor-mediated phosphoinositide turnover and inhibition of adenylyl cyclase to Rat 1 cells (Hung et al. 1992d), suggesting that the cloned receptor can mediate both signaling events. The extent to which the cloned receptor accounts for other thrombin-induced events, in particular, activation of the kinase cascades that mediate longer-term responses such as mitogenesis, remains to
be sorted out (Vouret-Craviari et al. 1992 and 1993, Hung et al. 1992a, McNamara et al. 1992, Reilly et al. 1993).
Cellular Responses The cloned thrombin receptor appears to be capable of mediating many of the known thrombin-induced events described in the introduction. The thrombin receptor clearly plays an important role in platelet activation. Thrombin receptor agonist peptide causes platelet secretion and aggregation (Coller et al. 1992, Vassallo et al. 1992, VouretCraviari et al. 1992, Vu et al. 1991a), and the potency of mutant agonist peptides for platelet activation parallels that for activation of the cloned receptor (Scarborough et al. 1992). The agonist peptide also causes platelet phosphoinositide hydrolysis (Huang et al. 1991). Moreover, antibodies to the cloned receptor block platelet activation by thrombin (Brass et al. 1992, Hung et al. 1992~). These data strongly suggest that the cloned receptor is both sufficient and necessary for platelet activation by thrombin. Because inhibition of platelet function by receptor antibodies was overcome by high concentrations of thrombin, one must
4. Model of thrombin receptor signaling: how does a protease elicit concentrationdependent responses. Thrombin acts as a protease to cleave and activate its receptor. Low concentrations of thrombin can ultimately cleave all cell surface receptors just as high concentrations can. How then does the cell distinguish high from low concentrations of thrombin in its environment? A working model based on recent kinetic studies (Ishii et al. 1993) is shown. Each activated receptor yields some ‘quantum” of second messenger (the liquid in the cups) and then is rapidly shut off. The concentration of thrombin determines the rate of receptor cleavage and activation and hence the rate of second-messenger generation. The balance between rates of second-messenger production and clearance (the spigot) determines the level of second-messenger achieved and hence the magnitude of the cellular response. Figure
01994, Elsevier Science Inc., 1050-1738/94/$7.00
make the caveat that available data do not exclude the existence of a second platelet thrombin receptor. It should be noted, however, that the inhibitory activity of the receptor antibodies was also overcome by high thrombin concentrations in a defined system in which responses are clearly mediated by the cloned receptor (Hung et al. 1992~). A detailed discussion of the role of the clone receptor in mediating thrombin’s many other cellular events is beyond the scope of this review. The thrombin receptor agonist peptide has been reported to mimick thrombin’s actions on U937 cells (a monocytelike cell line), endotbelial cells, vascular smooth muscle, and neuronal cells (Joseph and MacDermot 1993, McNamara et al. 1992, Ngaiza and Jaffe 1991, Suidan et al. 1992). Possible Roles in Cardiovascular Disease While the role of thrombin receptor activation in human hemostasis and thrombosis remains to be rigorously demonstrated, the receptor’s clear role in mediating thrombin-induced platelet activation, the importance of thrombin in platelet-dependentmodels of arterial thrombosis (see above), and the efficacy of ~tith~mbin therapy for unstable angina (Theroux et al. 1989) all suggest that thrombin receptor activation will play an important role. Given thrombin’s known actions on inflammatory and mesenchymal cells and its generation at sites of vascular injury, it is tempting to postulate a role for thrombin receptor activation in inflammatory and proliferative responses. Recently, robust thrombin receptor expression was noted in human atherosclerotic plaques, apparently by smooth muscle cells, mesenchymal-appearing cells of unknown origin, and macrophages (Nelken et al. 1992). In the context of thrombin’s known actions on these cells, the high and selective expression of thrombin receptor in atherosclerotic lesions suggests a possible role for thrombin receptor activation in restenosis and possibly in atherogenesis itself.
0
Acknowledgments
This work was supported in part by National Institutes of Health grants HL44907 and HL43322, and by the University of California’s Tobacco Related Disease Research Program grant 2RT19. 82
The author is an Established Investigator of the American Heart Association. References Banga HS, Walker IX, Winberry LK, Rittenhouse SE: 1988. Platelet adenylate cyclase and phospholipase C are affected differentially by ADP rlbosylation. Biochem J 252:297-300. Bar-Shavit R, Kahn A, Wilner CD, Fenton II Jw: 1983. Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 220~728-731.
DanielTO, GibbsVC, MilfayDF, Garavoy M, Williams LT: 1986. Thmmbin
stimulates
c-sis gene expression in microvascular endothelial cells. J Biol Chem 261:9579-9582. Davey M, Luscher E: 1967. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature 216:857-858. De&viler TC, Feinman RL): 1973. Kinetics of the thrombin-induced release of calcium (II) by platelets. Biochemistry 12:282-289. Dohlman HG, Thorner J, Caron MG, I.&owitz RJ: 1992. Model systems for the study of seven transmembrane segment receptors. Annu Rev Biochem 60:651-688.
Bemdt M, Phillips D: 1981. Platelet membrane proteins: composition and receptor function. In Gordon JL, ed. Platelet Membrane Proteins: Composition and Receptor Function. Amsterdam, Elsevier, pp 43-74.
Ehrlich HJ, Grhmell BW, Jaskunas SR, et al.: 1990. Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin. EMBO J 9:2367-2373.
Brass LF: 1992. Homologous desensitization of HEL cell thrombin receptors. J Biol Chem 267:6044-6050.
Eidt J, Allison P, Nobel S, et al.: 1989. Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury. J Clin Invest 84: 18-27.
Brass LF, Lasposata M, Banga HS, Rittenhouse SE: 1986. Regulation of the phosphoinositide hyantlysis pathway in thrombinstimulated platelets by a pertussis toxinsensitiveguanine nucleotide-bindingpntein. J Biol Chem 261:16,838-16,847. Brass LF, Vassallo RR, Belmonte E, et al.: 1992. 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 267: 13795-l 3798. Camps M, Carozzi A, Schnabel P, et al.: 1993. Isozyme-selective stimulation of phospholipase C-g2 by G protein &subunits. Nature 360~684-686. Chen LB, Buchanan JM: 1975. Mitogenic activity of blood components. I. Thrombin and pro~~rnb~. Proc Nat1 Acad Sci USA 72:131-135.
Fenton II Jw: 1988. Regulation of thrombin generation and functions. Semin Thmmb Hemost 14:234-240. Fitzgerald D, Fitzgerald G: 1989. Role of thrombin and thmmboxane A, in reocclusion following coronary thombolysis. Proc Nat1 Acad Sci USA 867585-7589. Hansen S, Harker L: 1988. Inte~ption of acute platelet-dependent thrombosis by the synthetic antithrombin PPACK. Proc Nat1 Acad Sci USA 853184-3188. Hattori R, Hamilton KK, Fugate RD, McEver RP, Sims PJ: 1989. Stimulated secretion of endothelial vWF is accompanied by rapid redistribution to the cell surface of the intracellular 8ramtle membrane protein GMP-140. J Biol Chem 264:7768-7771.
Chen LB, Teng NNH, Buchanan JM: 1976. Mitogenicity of thmmbin and surface alterations on mouse splenocytes. Exp Cell Res 101:41-46.
Heras M, Chesebro JH, Penny WJ, et al.: 1989. Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Circulation 79:665-667.
Cohen WM, Wu H-F, Featherstone GL, Jenzano SW, Lundblad m 1991. Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kall&rein by thrombin. Biochem Biophys Res Commun 176:3 15-320.
Huang R, Sorisky A, Church WR, Simons E, Rittenhouse SE: 1991. Thrombm receptordirected ligand accounts for activation by thrombin of plateiet phospholipase C and accumulation of 3-phosphorylated phosphoinositides. J Biol Chem 266:18,435-18,438.
Coller BS, Ward P, Ceruso M, et al.: 1992. Thrombin receptor activating peptides: importance of the N-terminal serlne and its ionization state as judged by pH dependence, NMR spectroscopy, and cleavage by aminopeptidase M. Biochemistry 3 1: 11,7 131 1,720.
Hung DT, Vu T-KH, Nelken NA, Coughhn SR: 1992a. Thrombin-induced events in nonplatelet cells are mediated by the novel proteolytic mechanism defined by the cloned platelet thrombin receptor. J Cell Biol 116:827-832.
Coughlin SR, Vu T-KH, Hung DT, Wheaton VI: 1992. Characterization of the cloned platelet thrombin receptor: issues and opportunities. J Clin Invest 89351-355.
Hung DT, Vu T-KH, Wheaton VI, et al.: 1992b. “Mirror image” antagonists of thmmbininduced platelet activation based on thrombin receptor structure. J Clin Invest 89:444450.
81994, Elsevier Science Inc., ~050-1~3~~94~~~.~
TCM k&L4, No. 2, 1994
Hung DT, Vu T-KH, Wheaton VI, I&ii K. Coughlin SR: 1992c. The cloned platelet thrombin receptor is necessary for thrombininduced platelet activation: blocking antiserum to the thrombin receptor’s hirudinlike domain. J Clin Invest 89:1350-1353. Hung DT, Wong YH, Vu T-KH, Coughhn SR: 1992d. The cloned platelet thrombin receptor couples to at least two distinct effecters to stimulate both phosphoinositide hydrolysis and inhibit adenylyl cyclase. J Biol Chem 353:20,831-20,834. Ishii K, Hein L, Kobilka BK, Coughhn SR: 1993. Kinetics of thrombin receptor cleavage on intact cells: relation to signalling. J Biol Chem 268:9780-9786. Jang I-K, Gold HK, Ziskind AA, et al.: 1989. Prevention of platelet-rich arterial thrombosis by selective thrombin inhibition. Circulation 8 1:2 19-225. Jones LG. McDonough PM, Brown JH: 1989. Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization. Mol Pharmacol 36:142-149. Joseph S, MacDermot J: 1993. The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling. Biochem J 290571-577. Kroll MH, Schafer AI: 1989. Biochemical mechanisms of platelet activation. Blood 36:1181-1195. Le Bonniec BF, Esmon CT: 1991. Glu-192Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. Proc Nat1Acad Sci USA 88:7371-7375. Liu L, Vu T-KH, Esmon CT, Coughlin SR: 199 1. The region of the thrombin receptor resembling hitudin binds to thrombin and alters enzyme specificity. J Biol Chem 266: 16.977-16.980. Martin BM, Feinman RD, De&viler TC: 1975. Platelet stimulation by thrombin and other proteases. Biochemistry 14: 1308-l 3 14.
tracellular calcium and stimulates prostacyclin production in human endothelial cells. Biochem Biophys Res Commun 179:16561661. Okamura T, Hasitz M, Jamieson GA: 1978. Platelet glycocalicin: interaction with thrombin and role as thrombin receptor on the platelet surface. J Biol Chem 253:3435-3443.
retraction in neuronal cells through activation of cell surface receptors. Neuron 8:363375. Theroux P, Ouimet H, McCums J: 1989. Aspirin, heparin, or both to treat acute unstable angina.NEnglJMed319:llOF1111.
Vassallo RRJ, Kieber-Emmons T, Cichowski K, Brass LF: 1992. Structure-function relationships in the activation of platelet thromParis S, Pouyssegur J: 1986. Pertussis toxin bin receptors by receptor-derived peptides. inhibits thrombin-induced activation of J Biol Chem 267:6081-6085. phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts. EMBO J Vouret-Craviari V, Van Obberghen-Schilling 5:55-60. VE, Rasmussen UB. et al.: 1992. Synthetic Prescott SM, Zimmerman GA, McIntyre TM: 1984. Human endothelial cells in culture produce platelet-activating factor when stimulated by thrombin. Proc Nat1 Acad Sci USA 81:3534-3538. Reilly CR, Connolly TM, Feng DM, Nutt RF, Mayer EJ: 1993. Thrombin receptor agonist peptide induction of mitogenesis in CCL39 cells. Biochem Biophys Res Commun 190:1001-1008. Rittenhouse-Simmons S: 1979. Production of diglyceride from phosphatidylinositol in activated human platelets. J Clin Invest 63:580-587. Rydel TJ, Rabichandran KG, Tulinsky A, et al.: 1990. The structure of a complex of recombinant hirudin and human alphathrombin. Science 249:277-280. Scarborough RM, Naughton M, Teng W, et al.: 1992. Tethered ligand agonist peptides: structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem 267:13,146-13,149. Suidan HS, Stone SR, Hemmings BA, Monard D: 1992. Thrombin causes neurite
a-thrombin receptor peptides activate Gprotein coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell 3:95-102. Vouret-Craviari V, Van Obberghen-Schilling VE, Scimeca JC, et al.: 1993. Differential activation of p44MAPK(ERKl) by a thrombin and thrombin receptor agonist peptide. Biochem J 289:209-2 14. Vu T-KH, Hung DT, Wheaton VI, Coughlin SR: 1991a. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068. Vu T-KH, Wheaton VI, Hung DT, Coughlin SR: 1991b. Domains specifying thrombinreceptor interaction. Nature 353:67&677. Weksler BB, Ley CW, Jaffe EA: 1982. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A23187. J Clin Invest 62:923-930. Zimmerman GA, McIntyre TM, Prescott SM: 1986. Thrombin stimulates neutrophil adherence by an endothelial celldependent mechanism. Ann NY Acad Sci 485:349-368.
Is this your subscription
TCM
to
Martin BM, Wasiewski WW, Fenton II JW, Detwiler TC: 1976. Equilibrium binding of thrombin to platelets. Biochemistry 15:4886 4893. McNamam CA, Sarembok IJ, Gimple LW, et al.: 1992. Thrombin stimulation of smooth muscle cell proliferation is mediated by a proteolytic, receptor-mediated mechanism. J Clin Invest 91:94-98. Nelken NA, Soifer SJ, O’Keefe J, et al.: 1992. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 90:1614-1621.
Use the bound-in order card to subscribe to TCM today!
Ngaiza JR, Jaffe EA: 1991. A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates in-
TCM Vol. 4,No.2,1994
D1994, Elsevier Science Inc., lOSO-1738/94/$7.00
83
Viragh S, Challice C: 1982. The development of the conduction system in the mouse embryo heart. IV. Differentiation of the atrioventricular conduction system. Dev Biol89:2540.
plasminogen
Vitadello M, Matteoli M, Gorza L: 1990. Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes. J Cell Sci 97: 1 l-2 1.
sel wall by an endothelial-dependent mechanism, probably by causing surface expression of GMP-140 on the endothelial surface (Hattori et al. 1989, Zimmerman et al. 1986) and directly activates neutrophils themselves (Cohen et al. 1991). These disparate functions of thrombin may be unified by viewing thrombin as an orchestrater of the response to vascular injury or wounding, mediating not only hemostatic but inflammatory and proliferative or reparative responses (Coughlin et al. 1992). Thrombin’s actions on cells also conjure hypotheses regarding possible roles in disease. Several pharmacologic studies demonstrate that thrombin activity is critical forplateletdependent arterial thrombosis in animal models (Eidt et al. 1989, Fitzgerald and Fitzgerald 1989, Hansen and Harker 1988, Heras et al. 1989, Jang et al. 1989). Moreover, the efficacy of antithrombin therapy for unstable angina (Theroux et al. 1989) suggests that thrombin activity is important in this disease. Beyond the platelet, thrombin’s proinflammatory actions on monocytes, neutrophils, and endothelial cells, and its proliferative effect on mesenchymal cells, suggest a possible role in pathologic responses of the vessel wall to injury. Candidate disorders include restenosis, glomerulosclerosis, and perhaps atherogenesis itself. While thrombin’s critical role in hemostasis and thrombosis is well established, the in vivo importance of its proliferative and inflammatory actions has not been defined. This introduction begs an understanding of the mechanisms by which thrombin activates cells. A recently cloned thrombin receptor has provided a framework for understanding how thrombin talks to cells (Vu et al. 1991a) and appears to account for many of the thrombin activities cited above. Structureactivity studies with this receptor have revealed a novel proteolytic mechanism of receptor activation. Moreover, this receptor has provided new tools for defining the role of thrombin-induced cell activation in vivo and may represent a new target for antithrombotic and other therapies (Coughlin et al. 1992). This review describes recent mechanistic
Woodcock-Mitchell J, Mitchell J, Low R, et al.: 1988. a-Smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation 39: 16 l166. Zeller R, Bloch K, Williams B, Arceci R, Seidman C: 1987. Localized expression of atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev 1:693-698. TCM
Thrombin Receptor Function and Cardiovascular Disease Shaun R. Coughlin
Thrombin, a multifunctional protease generated at sites of vascular injury, is a powerful agonist for a variety of cellular processes important in cardiovascular disease. A recently cloned thrombin receptor has provided a framework for understanding how thrombin, a protease rather than a classic ligand, activates cells. It has also yielded new tools for defining the role of thrombin and its receptor in cellular events. This review discusses a working model for how thrombin activates platelets and other cells, and possible roles for the thrombin receptor in thrombotic, proliferative, and inflammatory processes. (Trends Cardiovast bled 1994;4:77-83)
Thrombin
is a multifunctional
generated
at
sites
bin is the most potent activator of plate-
protease
of vascular
injury.
lets in vitro (Bemdt
While best known for its ability to cleave
Davey and Luscher
fibrinogen
also
thrombin variety
and trigger
fibrin formation,
is also a powerful agonist for a of cellular
responses
(Figure
1
chemotactic
and Phillips
1981,
1967). Thrombin for monocytes
Shavit et al. 1983) and is mitogenic lymphocytes,
fibroblasts,
and
is
(Barfor
vascular
[reviewed in Fenton (1988) and Coughlin
smooth muscle cells (Chen and Buchanan
et al. (1992)].
1975, Chen et al. 1976, McNamara
First and foremost,
throm-
1992). Shaun R. Coughlin is at the Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0524, USA.
TCM Vol.4,No. 2,1994
Thrombin
endothelium prostacyclin activating
01994,
acts
et al.
on the vascular
to stimulate
production
of
(Weksler et al. 1982), plateletfactor
(Prescott
et al. 1984),
ElsevierScience Inc., 1050-l738/94/$7.00
activator-inhibitor
(Camps
et al. 1993), and the potent smooth muscle cell mitogen tor (Daniel
platelet-derived
growth fac-
et al. 1986). Thrombin
induces neutrophil
adherence
also
to the ves-
77
Figure 1. Cellular actions of thrombin. Cellular actions of thrombin are illustrated in the context of a blood vessel. Thrombin is the most potent activator of blood platelets, an action probably critical for hemostasis and thrombosis. Thrombin is also chemotactic for monocytes, mitogenic for lymphocytes, activates neutrophils, and has a variety of effects on endothelial cells; these actions of thrombin may contribute to inflammatory responses to vascular injury. Thrombin is a potent mitogen for vascular smooth muscle cells and fibroblasts, actions that may contribute to proliferative and reparative responses to vascular injury or wounding. Reprinted with permission from J Clin Znvest (Coughlin et aI. 1992).
insights provided by the newly cloned thrombin receptor and its possible roles in cardiovascular disease.
l
ThromWm Receptor StructureActivityRelationships: How Does a Protease Talk to a Cell?
The thrombin receptor is a member of the seven-transmemb~e-domain receptor family, but is activated by a novel proteolytic mechanism (Vu et al. 1991 a). Thrombin binds to and cleaves its receptor’s extracellular amino terminal domain to unmask a new amino terminal. This new amino terminal then functions as a tethered peptide ligand, binding to as yet undefined sites within the body of the receptor to effect receptor activation (Figures 2 and 3). The studies that developed and extended this model are described below. Molecular Basis for Thrombin-Receptor Interaction Examination of the thrombin receptor’s amino acid sequence revealed a putative thrombin cleavage site resembling the known tbrombin cleavage site in protein C within the receptor’s amino terminal exodomain (Figure 2). Carboxyl to this 78
site was a sequence resembling the carboxy1 tail of hirudin, a structure known to interact with thrombin’s anionbinding exosite (Rydel et al. 1990). These analogies suggested that the receptor might interact with thrombin in the manner shown in Figure 2b and serve as a thrombin substrate (Vu et al. 199 la). This model has been supported by multiple studies: 1. Mutation of the thrombin receptor cleavage site to block cleavage rendered the receptor unactivatable by thrombin (Vu et al. 1991a). 2. Replacing the thrombin cleavage recognition sequence with that for enteropeptidase switched receptor specificity; cells (Hung et al. 1992d) or oocytes (Vu et al. 1991b) expressing this construct responded to enteropeptidase but not to thrombin. 3. Synthetic peptides mimicking the receptor’s cleavage site were cleaved by thrombin (Vu et al. 199 1b) and uncleavable “mutant“ peptides mimicking this region bound thrombin and inhibited its activity against synthetic substrates, fibrinogen, and its receptor (Hung et al. 1992b, Liu et al. 1991, Vu et al. 1991b). 4. Recent studies used an antibodybinding method to demonstrate receptor
01994, Else&r Science Inc., 1050-1738~4/$7.~
cleavage directly on intact cells and to show that receptor cleavage correlated with signaling [Ishii et al. (1993); see below]. 5. The bidentate interaction shown in Figure 2b is supported by the observations that receptor peptides require both the primary cleavage recognition sequence LDPR and the receptor’s hirudinlike domain sequence KYEPF for avid interaction with thrombin (Liu et al. 1991, Vu et al. 199ib). Moreover, alanine-scanning mutants of the KYEPF sequence suggest that this sequence bmds thrombin’s anionbinding exosite in a manner grossly analogous to the DFEEI sequence in hirudin’s carboxyl tail (Vu et al. 199lb). x-ray crystallographic studies of cocrystals of thrombin with receptor-based peptides are in progress and promise to reveal the details of this interaction. Several important questions regarding thrombin-receptor interaction remain to be answered. First, do other receptor domains beyond those described above participate in thrombin-receptor interaction? Second, do other thrombinbinding proteins on the surface of platelets and other cdls play an important role in presenting tbrombin to its receptor (either directly by formation of a ternary complex with thrombin and its receptor, or indirectly by protecting locally produced thrombin from inactivation)? Such a role has been suggested for the platelet surface glycoprotein GPIb (Okamura et al. 1978). Lastly, does the thrombin receptor cause a conformational change in thrombin to enable cleavage of the LDPR site? This question is raised by the identity of the human thrombin receptor’s thrombin cleavage site (LDPR) with that in bovine protein C (Vu et al. 1991a). When bound to thrombomodulin, thrombin gains the ability to cleave and activate the anticoagulant protein C. Thrombomodulin effects this change in thrombin substrate specificity in part by inducing a conformational change in thrombin such that it can accommodate the normally unfavorable LDPR cleavage recognition sequence (Ehrlich et al. 1990, Le Bonniec and Esmon 1991). Whether the thrombin receptor causes a similar conformational change in thrombin to accommodate the receptor’s own LDPR sequence and promote efficient receptor cleavage is unknown. TCM Vol. 4, No. .?z1994
\
HUMAN TR38-60 MOUSE TRB-62 a
A”IoN-8INDING BlH”ING DOM~I”
AGOVLST PEPriDE DaHarN
CLEavaGE SITE
I
Mechanism
ElOIiTE
Silent in the Vncleaved Receptor?
I
LOPR/SFLLRN/PND-K/YEPFWE-DEE.. VNPR/SFFLRN/PSENT/::L'IPLGDEE..
HIRUDIN 55-65
The model just described casts the throm-
. ..DFEEIPEEY'LO-Coo
Figure 2. (a) Functional domains within the thrombin receptor’s amino terminal extension. The cleavage recognition sequence (LDPR), thrombin cleavage site, agonist peptide domain, and anion-binding exosite binding domain as defined by structure-activity studies with the human receptor are shown. These are aligned with the murine thrombin receptor sequence and the anion-binding exosite-binding sequence of the leech anticoagulant hirudin. (b)A model for interaction of these domains with thrombin. Thrombin has an extended substrate-binding surface (represented by the canyon running laterally) that recognizes residues both amino and carboxyl to its substrate’s cleavage site. Structure-function studies suggest that the receptor’s hirudinlike domain (KYEPF) interacts with thrombin’s anion-binding exosite, while its cleavage site (LDPRIS) interacts with thrombin’s Sl-S4 subsites. This model has important implications for the development of blocking antibodies and receptor peptide-based thrombin inhibitors. Reprinted with permission from Nature (Vu et al. 1991b).
Proteolytic Unmasking of a Tethered Peptide Ligand: A Novel Mechanism of Receptor Activation How might proteolysis within the thrombin receptor’s amino terminal extension activate the receptor? There are several precedents for a protease activating a target protein by unmasking a new amino terminus within that protein. In particular, proteolytic activation of the zymogen trypsinogen occurs when enteropeptidase cleaves it to unmask a new amino terminus that then binds intramolecularly to effect a conformational change and create an active trypsin molecule. A grossly analogous mechanism exists for the thrombin receptor (Figure 3) (Vu et al. 1991a). Synthetic peptides that mimic the new amino
TCM Vol. 4, No. 2, 1994
terminus
created
when
cleaves its receptor receptor
thrombin
are full agonists
activation,
quirement
and bypass
for receptor
for
the re-
proteolysis
(Fig-
ures 2 and 3) (Vu et al. 1991a).
This
observation
suggests
the model
shown
in Figure 3. Thrombin
cleaves its recep-
tor’s amino
extension
terminal
mask a new amino amino
terminus
terminus. then
to un-
This new
functions
as a
tethered peptide ligand, binding to an as yet undefined receptor
site within the body of the
to effect
receptor
activation
(Vu et al. 1991a).
As discussed
synthetic
mimicking
peptides
onist peptide
domain” receptor
below, this “ag-
(Figure
vide a new tool for defining the thrombin
2) pro-
the role of
in various
cellu-
lar events.
01994.
of the “Proteolytic Switch’:
How Does the Tethered Ligand Remain
ElsevierScienceInc., lOSO-1738/94/$7.00
bin receptor as a peptide receptor that contains its own ligand. This ligand remains cryptic until unmasked when thrombin cleaves the receptor. How might this “proteolytic switch” work? Recent structure-function studies of the receptor’s agonist peptide domain suggest a possible answer. The first five amino acids of the receptor’s agonist peptide domain (SPLLR in the single-letter code) are sufficient to specify agonist activity (Scarborough et al. 1992, Vassallo et al. 1992, Vouret-Craviari et al. 1992). The protonated amino group of Serl and the Phe2 side chain are vital for agonist function; the Leu4 and At-g5 side chains play less important roles (Coller et al. 1992, Scarborough et al. 1992, Vassal10 et al. 1992). The importance of Serl’s protonated amino group is particularly appealing, as this group is created by receptor cleavage. This may explain in part how the agonist peptide domain’s activity is masked when the receptor is in the uncleaved state (Scarborough et al. 1992). Steric and structural contributions to maintaining the agonist peptide silent in the uncleaved receptor remain to be defined. Kinetics of Thrombin Receptor Cleavage and Relation to Signaling: How Does a Protease Elicit Concentration-Dependent Responses? Like other important signaling molecules, thrombin effects concentration-dependent and graded responses in its target cells (Berndt and Phillips 1981, Detwiler and Feinman 1973, Martin et al. 1975 and 1976, Paris and Pouyssegur 1986, Rittenhouse-Simmons 1979), a feature vital for normal homeostasis. Classic ligands effect concentration-dependent responses via graded receptor occupancy. How thrombin acting as an enzyme rather than a classic ligand might effect concentrationdependent responses has been a longstanding question (Berndt and Phillips 198 1, Detwiler and Feinman 1973, Martin et al. 1975 and 1976). Specifically, one would predict that even low amounts of thrombin would eventually cleave and activate all cell surface receptors. How then can a concentration-dependent response be achieved? A recent study used
79
tor signaling
has several
implications.
First, it places great importance on the mechanisms that terminate thrombin receptor signaling (see below). Second, it suggests that thrombin receptor antagonists need only slow thrombin receptor activation enough that clearance of second messengers outstrips their generation to block signaling effectively (Ishii et al. 1993). This notion may encourage attempts at antagonist development, which might otherwise be discouraged by the receptor’s intramolecular tethered liganding mechanism. Transmembrane
Signaling
Where the thrombin receptor’s agonist peptide domain binds and how this binding event causes a transmembrane signal that enables the receptor to activate G proteins is unknown. Ligand binding for seven-transmembranedomain receptors is best studied for the &-adrenergic receptor (Dohlman et al. 1992). In this case, the catecholamine ligand interacts with residues predicted to reside within the transmembrane domains. The details of how this binding “switches the receptor on” and even whether peptide agonists bind in an analogous manner are unknown.
Figure 3. Model of thrombin receptor activation. Thrombin, the sphere in this figure, binds to its receptor’s extracellular amino terminal extension (see Figure 2 for detail). After binding to the amino terminal extension, thrombin cleaves the receptor at the LDPR/S cleavage site (junction between open and filled receptor segments), releasing an inactive fragment of the receptor’s amino terminus (open fragment) and exposing a new amino terminus. This newly unmasked amino terminus then functions as a tethered peptide Iigand, binding to as yet undefined sites within the body of the receptor to effect receptor activation. As shown, this binding event presumably translates into a conformational change in the receptor’s cytoplasmic face, effecting G-protein activation. Exactly how ‘news” of ligand binding to a seven-transmembrane-domain receptor is conveyed to the cell’s interior remains a fundamental structure-function question in this field. Reprinted with permission from Nature (Vu et al. 1991b).
antibodies that distinguished the naive receptor from the cleaved and activated form to follow the rate of thrombin receptor cleavage on intact cells (Ishii et al. 1993). The rate of receptor cleavage was proportional to thrombin concentmtion over the range known to elicit concentration-dependent responses, but low thrombin concentrations did ultimately cleave all cell surface thrombin receptors. Cumulative phosphoinositide hydrolysis in response to thrombin correlated precisely with absolute receptor cleavage during a given time interval, not with the integral of receptor cleavage as a function of time. These data strongly suggest that
80
each activated thrombin receptor generates a quantum of second messenger, then ‘shuts off (Ishii et al. 1993). Thrombin concentration would then determine the rate of receptor activation and therefore the rate of second-messenger generation. Second-messenger levels and magnitude of the cellular response are presumably then determined by the balance between the rates of second-messenger generation and clearance (Figure 4). This is unlike the case for classic ligands; there is no ‘equilibrium” binding and the cell cannot utilize graded receptor occupancy to effect graded responses. This formulation
01994,
of thrombin
recep-
Elsevier Science Inc., lOSO-1738/94/$7.00
Mechanisms of Receptor Shutofi Did the Novel Activation Mechanism Beget a Novel Shutoff Mechanism? The formulation of thrombin receptor signaling outlined above suggests that the thrombin receptor’s ‘shutoff mechanism is a critical determinant of the gain of the system and thereby of thrombin responsiveness (Ishii et al. 1993, Vu et al. 1991a). The mechanism of thrombin receptor shut off has not been rigorously defined. By analogy with other seven-transmembrane-domain receptors (Dohlman et al. 1992) it is likely that receptor kinases play an important role in the immediate termination of thrombin receptor signaling. Indeed, recovery of receptor responsiveness to agonist peptide after desensitization was inhibited by phosphatase inhibitors (Brass 1992) consistent with but not proving a role for phosphorylation in desensitization. Receptor internalization (sequestration) and degradation (downregulation) may also be involved in termination of thrombin receptor signaling. Recent work does suggest that the thrombin receptor undergoes agonist-induced internalization (Brass 1992, Ishii et al. 1993) ; the
TCM Vol. 4, No. 2, 1994
relative
contribution
of internalization
to termination of thrombin signaling is unknown. A striking paradox has been noted in thrombin receptor signaling. Based on recent kinetic studies of signaling in receptor-transfected cell lines, it appears that the thrombin receptor stops signaling despite the continued presence of cleaved and “activated” receptors on the cell surface and at a time when cells are refractory to thrombin but sensitive to agonist peptide (Ishii et al. 1993). These observations may be consistent with the earlier finding that responsiveness to agonist peptide recover faster than responsiveness to thrombin in HEL cells (Brass 1992). The finding of an agonistpeptide-responsiv~thmbin-unresponsive state despite the continued presence of cleaved and “activated” thrombin receptor on the cell surface is provocative. A trivial explanation of this observation is the existence of a receptor pool accessible to exogenous agonist peptide but not to thrombin. A more exciting possibility is that the receptor may become modified so that the tethered agonist peptide domain cannot function, but exogenous agonist peptide can. Thus, an as yet uncharacterized and novel shutoff mechanism may have evolved to deal with the tethered ligand and with the obligate relationship of receptor activation to phosphoinositide hydrolysis.
l
Potential Roles for the Thrombin Receptor in Cardiovascular Disease
Synthetic peptides mimicking the thrombin receptor’s agonist peptide domain, blocking antibodies, and the receptor cDNA itself have provided new tools for defining the role of the cloned receptor in both intracellular signaling events and thrombin-induced cellular functions in vitro. However, no human genetic disease caused by a thrombin receptor mutation has yet been described, and “knockout” of the thrombin receptor gene in mice remains to be accomplished. Potent and specific antagonists for the thrombin receptor are not yet available. Because tools to define rigorously the roles of the thrombin receptor in normal physiology and in disease are not yet available, we are left to speculate, Several known thrombin-induced intracellular signaling events and in vitro cellular events have been shown to be
TCM Vol. 4, No. 2, 1994
mediated by the cloned thrombin receptor. A discussion of these events with extrapolation to the receptor’s possible roles in cardiovascular
function
follows.
Intracellular Signaling Thrombin is known to activate both phosphoinositide hydrolysis and to inhibit adenylyl cyclase in platelets (Banga et al. 1988, Brass et al. 1986) and other responsive cells (Jones et al. 1989, Paris and Pouyssegur 1986). In platelets, both of these second-messenger events serve to promote aggregation (Kroll and Schafer 1989). The thrombin receptor agonist peptide has been reported to elicit both of these second-messenger events in fibroblasts (Hung et al. 1992a, VouretCraviari et al. 1992), and transfection of the cloned thrombin receptor conferred both receptor-mediated phosphoinositide turnover and inhibition of adenylyl cyclase to Rat 1 cells (Hung et al. 1992d), suggesting that the cloned receptor can mediate both signaling events. The extent to which the cloned receptor accounts for other thrombin-induced events, in particular, activation of the kinase cascades that mediate longer-term responses such as mitogenesis, remains to
be sorted out (Vouret-Craviari et al. 1992 and 1993, Hung et al. 1992a, McNamara et al. 1992, Reilly et al. 1993).
Cellular Responses The cloned thrombin receptor appears to be capable of mediating many of the known thrombin-induced events described in the introduction. The thrombin receptor clearly plays an important role in platelet activation. Thrombin receptor agonist peptide causes platelet secretion and aggregation (Coller et al. 1992, Vassallo et al. 1992, VouretCraviari et al. 1992, Vu et al. 1991a), and the potency of mutant agonist peptides for platelet activation parallels that for activation of the cloned receptor (Scarborough et al. 1992). The agonist peptide also causes platelet phosphoinositide hydrolysis (Huang et al. 1991). Moreover, antibodies to the cloned receptor block platelet activation by thrombin (Brass et al. 1992, Hung et al. 1992~). These data strongly suggest that the cloned receptor is both sufficient and necessary for platelet activation by thrombin. Because inhibition of platelet function by receptor antibodies was overcome by high concentrations of thrombin, one must
4. Model of thrombin receptor signaling: how does a protease elicit concentrationdependent responses. Thrombin acts as a protease to cleave and activate its receptor. Low concentrations of thrombin can ultimately cleave all cell surface receptors just as high concentrations can. How then does the cell distinguish high from low concentrations of thrombin in its environment? A working model based on recent kinetic studies (Ishii et al. 1993) is shown. Each activated receptor yields some ‘quantum” of second messenger (the liquid in the cups) and then is rapidly shut off. The concentration of thrombin determines the rate of receptor cleavage and activation and hence the rate of second-messenger generation. The balance between rates of second-messenger production and clearance (the spigot) determines the level of second-messenger achieved and hence the magnitude of the cellular response. Figure
01994, Elsevier Science Inc., 1050-1738/94/$7.00
make the caveat that available data do not exclude the existence of a second platelet thrombin receptor. It should be noted, however, that the inhibitory activity of the receptor antibodies was also overcome by high thrombin concentrations in a defined system in which responses are clearly mediated by the cloned receptor (Hung et al. 1992~). A detailed discussion of the role of the clone receptor in mediating thrombin’s many other cellular events is beyond the scope of this review. The thrombin receptor agonist peptide has been reported to mimick thrombin’s actions on U937 cells (a monocytelike cell line), endotbelial cells, vascular smooth muscle, and neuronal cells (Joseph and MacDermot 1993, McNamara et al. 1992, Ngaiza and Jaffe 1991, Suidan et al. 1992). Possible Roles in Cardiovascular Disease While the role of thrombin receptor activation in human hemostasis and thrombosis remains to be rigorously demonstrated, the receptor’s clear role in mediating thrombin-induced platelet activation, the importance of thrombin in platelet-dependentmodels of arterial thrombosis (see above), and the efficacy of ~tith~mbin therapy for unstable angina (Theroux et al. 1989) all suggest that thrombin receptor activation will play an important role. Given thrombin’s known actions on inflammatory and mesenchymal cells and its generation at sites of vascular injury, it is tempting to postulate a role for thrombin receptor activation in inflammatory and proliferative responses. Recently, robust thrombin receptor expression was noted in human atherosclerotic plaques, apparently by smooth muscle cells, mesenchymal-appearing cells of unknown origin, and macrophages (Nelken et al. 1992). In the context of thrombin’s known actions on these cells, the high and selective expression of thrombin receptor in atherosclerotic lesions suggests a possible role for thrombin receptor activation in restenosis and possibly in atherogenesis itself.
0
Acknowledgments
This work was supported in part by National Institutes of Health grants HL44907 and HL43322, and by the University of California’s Tobacco Related Disease Research Program grant 2RT19. 82
The author is an Established Investigator of the American Heart Association. References Banga HS, Walker IX, Winberry LK, Rittenhouse SE: 1988. Platelet adenylate cyclase and phospholipase C are affected differentially by ADP rlbosylation. Biochem J 252:297-300. Bar-Shavit R, Kahn A, Wilner CD, Fenton II Jw: 1983. Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 220~728-731.
DanielTO, GibbsVC, MilfayDF, Garavoy M, Williams LT: 1986. Thmmbin
stimulates
c-sis gene expression in microvascular endothelial cells. J Biol Chem 261:9579-9582. Davey M, Luscher E: 1967. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature 216:857-858. De&viler TC, Feinman RL): 1973. Kinetics of the thrombin-induced release of calcium (II) by platelets. Biochemistry 12:282-289. Dohlman HG, Thorner J, Caron MG, I.&owitz RJ: 1992. Model systems for the study of seven transmembrane segment receptors. Annu Rev Biochem 60:651-688.
Bemdt M, Phillips D: 1981. Platelet membrane proteins: composition and receptor function. In Gordon JL, ed. Platelet Membrane Proteins: Composition and Receptor Function. Amsterdam, Elsevier, pp 43-74.
Ehrlich HJ, Grhmell BW, Jaskunas SR, et al.: 1990. Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin. EMBO J 9:2367-2373.
Brass LF: 1992. Homologous desensitization of HEL cell thrombin receptors. J Biol Chem 267:6044-6050.
Eidt J, Allison P, Nobel S, et al.: 1989. Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury. J Clin Invest 84: 18-27.
Brass LF, Lasposata M, Banga HS, Rittenhouse SE: 1986. Regulation of the phosphoinositide hyantlysis pathway in thrombinstimulated platelets by a pertussis toxinsensitiveguanine nucleotide-bindingpntein. J Biol Chem 261:16,838-16,847. Brass LF, Vassallo RR, Belmonte E, et al.: 1992. 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 267: 13795-l 3798. Camps M, Carozzi A, Schnabel P, et al.: 1993. Isozyme-selective stimulation of phospholipase C-g2 by G protein &subunits. Nature 360~684-686. Chen LB, Buchanan JM: 1975. Mitogenic activity of blood components. I. Thrombin and pro~~rnb~. Proc Nat1 Acad Sci USA 72:131-135.
Fenton II Jw: 1988. Regulation of thrombin generation and functions. Semin Thmmb Hemost 14:234-240. Fitzgerald D, Fitzgerald G: 1989. Role of thrombin and thmmboxane A, in reocclusion following coronary thombolysis. Proc Nat1 Acad Sci USA 867585-7589. Hansen S, Harker L: 1988. Inte~ption of acute platelet-dependent thrombosis by the synthetic antithrombin PPACK. Proc Nat1 Acad Sci USA 853184-3188. Hattori R, Hamilton KK, Fugate RD, McEver RP, Sims PJ: 1989. Stimulated secretion of endothelial vWF is accompanied by rapid redistribution to the cell surface of the intracellular 8ramtle membrane protein GMP-140. J Biol Chem 264:7768-7771.
Chen LB, Teng NNH, Buchanan JM: 1976. Mitogenicity of thmmbin and surface alterations on mouse splenocytes. Exp Cell Res 101:41-46.
Heras M, Chesebro JH, Penny WJ, et al.: 1989. Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Circulation 79:665-667.
Cohen WM, Wu H-F, Featherstone GL, Jenzano SW, Lundblad m 1991. Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kall&rein by thrombin. Biochem Biophys Res Commun 176:3 15-320.
Huang R, Sorisky A, Church WR, Simons E, Rittenhouse SE: 1991. Thrombm receptordirected ligand accounts for activation by thrombin of plateiet phospholipase C and accumulation of 3-phosphorylated phosphoinositides. J Biol Chem 266:18,435-18,438.
Coller BS, Ward P, Ceruso M, et al.: 1992. Thrombin receptor activating peptides: importance of the N-terminal serlne and its ionization state as judged by pH dependence, NMR spectroscopy, and cleavage by aminopeptidase M. Biochemistry 3 1: 11,7 131 1,720.
Hung DT, Vu T-KH, Nelken NA, Coughhn SR: 1992a. Thrombin-induced events in nonplatelet cells are mediated by the novel proteolytic mechanism defined by the cloned platelet thrombin receptor. J Cell Biol 116:827-832.
Coughlin SR, Vu T-KH, Hung DT, Wheaton VI: 1992. Characterization of the cloned platelet thrombin receptor: issues and opportunities. J Clin Invest 89351-355.
Hung DT, Vu T-KH, Wheaton VI, et al.: 1992b. “Mirror image” antagonists of thmmbininduced platelet activation based on thrombin receptor structure. J Clin Invest 89:444450.
81994, Elsevier Science Inc., ~050-1~3~~94~~~.~
TCM k&L4, No. 2, 1994
Hung DT, Vu T-KH, Wheaton VI, I&ii K. Coughlin SR: 1992c. The cloned platelet thrombin receptor is necessary for thrombininduced platelet activation: blocking antiserum to the thrombin receptor’s hirudinlike domain. J Clin Invest 89:1350-1353. Hung DT, Wong YH, Vu T-KH, Coughhn SR: 1992d. The cloned platelet thrombin receptor couples to at least two distinct effecters to stimulate both phosphoinositide hydrolysis and inhibit adenylyl cyclase. J Biol Chem 353:20,831-20,834. Ishii K, Hein L, Kobilka BK, Coughhn SR: 1993. Kinetics of thrombin receptor cleavage on intact cells: relation to signalling. J Biol Chem 268:9780-9786. Jang I-K, Gold HK, Ziskind AA, et al.: 1989. Prevention of platelet-rich arterial thrombosis by selective thrombin inhibition. Circulation 8 1:2 19-225. Jones LG. McDonough PM, Brown JH: 1989. Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization. Mol Pharmacol 36:142-149. Joseph S, MacDermot J: 1993. The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling. Biochem J 290571-577. Kroll MH, Schafer AI: 1989. Biochemical mechanisms of platelet activation. Blood 36:1181-1195. Le Bonniec BF, Esmon CT: 1991. Glu-192Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. Proc Nat1Acad Sci USA 88:7371-7375. Liu L, Vu T-KH, Esmon CT, Coughlin SR: 199 1. The region of the thrombin receptor resembling hitudin binds to thrombin and alters enzyme specificity. J Biol Chem 266: 16.977-16.980. Martin BM, Feinman RD, De&viler TC: 1975. Platelet stimulation by thrombin and other proteases. Biochemistry 14: 1308-l 3 14.
tracellular calcium and stimulates prostacyclin production in human endothelial cells. Biochem Biophys Res Commun 179:16561661. Okamura T, Hasitz M, Jamieson GA: 1978. Platelet glycocalicin: interaction with thrombin and role as thrombin receptor on the platelet surface. J Biol Chem 253:3435-3443.
retraction in neuronal cells through activation of cell surface receptors. Neuron 8:363375. Theroux P, Ouimet H, McCums J: 1989. Aspirin, heparin, or both to treat acute unstable angina.NEnglJMed319:llOF1111.
Vassallo RRJ, Kieber-Emmons T, Cichowski K, Brass LF: 1992. Structure-function relationships in the activation of platelet thromParis S, Pouyssegur J: 1986. Pertussis toxin bin receptors by receptor-derived peptides. inhibits thrombin-induced activation of J Biol Chem 267:6081-6085. phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts. EMBO J Vouret-Craviari V, Van Obberghen-Schilling 5:55-60. VE, Rasmussen UB. et al.: 1992. Synthetic Prescott SM, Zimmerman GA, McIntyre TM: 1984. Human endothelial cells in culture produce platelet-activating factor when stimulated by thrombin. Proc Nat1 Acad Sci USA 81:3534-3538. Reilly CR, Connolly TM, Feng DM, Nutt RF, Mayer EJ: 1993. Thrombin receptor agonist peptide induction of mitogenesis in CCL39 cells. Biochem Biophys Res Commun 190:1001-1008. Rittenhouse-Simmons S: 1979. Production of diglyceride from phosphatidylinositol in activated human platelets. J Clin Invest 63:580-587. Rydel TJ, Rabichandran KG, Tulinsky A, et al.: 1990. The structure of a complex of recombinant hirudin and human alphathrombin. Science 249:277-280. Scarborough RM, Naughton M, Teng W, et al.: 1992. Tethered ligand agonist peptides: structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem 267:13,146-13,149. Suidan HS, Stone SR, Hemmings BA, Monard D: 1992. Thrombin causes neurite
a-thrombin receptor peptides activate Gprotein coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell 3:95-102. Vouret-Craviari V, Van Obberghen-Schilling VE, Scimeca JC, et al.: 1993. Differential activation of p44MAPK(ERKl) by a thrombin and thrombin receptor agonist peptide. Biochem J 289:209-2 14. Vu T-KH, Hung DT, Wheaton VI, Coughlin SR: 1991a. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068. Vu T-KH, Wheaton VI, Hung DT, Coughlin SR: 1991b. Domains specifying thrombinreceptor interaction. Nature 353:67&677. Weksler BB, Ley CW, Jaffe EA: 1982. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A23187. J Clin Invest 62:923-930. Zimmerman GA, McIntyre TM, Prescott SM: 1986. Thrombin stimulates neutrophil adherence by an endothelial celldependent mechanism. Ann NY Acad Sci 485:349-368.
Is this your subscription
TCM
to
Martin BM, Wasiewski WW, Fenton II JW, Detwiler TC: 1976. Equilibrium binding of thrombin to platelets. Biochemistry 15:4886 4893. McNamam CA, Sarembok IJ, Gimple LW, et al.: 1992. Thrombin stimulation of smooth muscle cell proliferation is mediated by a proteolytic, receptor-mediated mechanism. J Clin Invest 91:94-98. Nelken NA, Soifer SJ, O’Keefe J, et al.: 1992. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 90:1614-1621.
Use the bound-in order card to subscribe to TCM today!
Ngaiza JR, Jaffe EA: 1991. A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates in-
TCM Vol. 4,No.2,1994
D1994, Elsevier Science Inc., lOSO-1738/94/$7.00
83