Characterization of a functional thrombin
receptor. Issues and opportunities.
S R Coughlin, … , D T Hung, V I Wheaton
J Clin Invest. 1992;89(2):351-355. https://doi.org/10.1172/JCI115592.
Research Article
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Perspectives
Characterization
of a Functional Thrombin Receptor
Issues and Opportunities
ShaunR.Coughlin,**Thien-Khai H. Vu,* David T.
Hung,**
and Virginia1. Wheaton**Cardiovascular ResearchInstitute,andtDepartmentofMedicine, UniversityofCalifornia,
San Francisco, San Francisco, California 94143
Introduction
Howdoes the proteasethrombin activate platelets and other cells? Thisquestionpresentsintriguing issues from both basic
scientificandclinical perspectives. Fromabasic view, thefact
that thrombin isa protease raisesthe possibility ofa novel proteolytic mechanism ofreceptoractivation. Fromaclinical
view, thrombin regulates hemostasis and thrombosis.
More-over, ahostof thrombin actionsoncells have been defined in vitro; in vivo these actionsmay be critical for both normal
responses towounding andfor pathological vascular events. Anunderstandingof the mechanismofthrombin-induced cell
activation promisesreagentsforidentifying thrombin'srole in cellularresponsesinvivo andpossiblynewtherapeutics.
Inthis Perspectives,wereview therecentcloning and
char-acterization ofaplatelet thrombin receptorinourlaboratory (1).Identificationof thethrombinreceptordid revealaunique
proteolytic mechanism ofreceptoractivation, and led tothe
development ofa novel agonist peptide which activates the
thrombin receptorindependent ofthrombin andthrombin's
proteaseactivity. This agonistpeptide isausefulnewtoolfor
addressingtheroleofthrombinreceptoractivationin cellular
responses. It isour hope thathavingthe cloned receptorin hand will also allow the development of thrombin receptor
antagonists. Suchreagentswilldefinethe roleof thrombin re-ceptoractivation in normal anddiseaseprocessesinvivo,and
mayprovide the basis for a new class of antithrombotic or
antiproliferative pharmaceuticals.
Cell-activating
functions of
thrombinInadditiontocleaving fibrinogen tofibrin,thrombin exhibits
important "cell-activating" functions (Fig. 1; reviewed in ref. 2). First and foremost,thrombinis themost potentstimulator of plateletaggregation(3),athrombinactivity probably critical inarterial thrombosis (see below).Avariety of other thrombin activitiesoncellshas beendefined,largely invitro. Thrombin
ischemotactic formonocytes(4), mitogenic for lymphocytes andformesenchymal cellsincludingvascular smooth muscle cells(5,6),and hasanumber of effectsuponthe vascular
endo-Address correspondence to S. R. Coughlin, Box 0524, University of
California, SanFrancisco, San Francisco, CA94143.
Receivedforpublication9October1991.
thelium. These includestimulating endothelial
production
of prostacyclin(7),platelet-activatingfactor(8),plasminogenac-tivator-inhibitor (9),and thepotentsmooth muscle cell
mito-genplatelet-derived growth factor (10).Thrombin also induces
neutrophil adherencetothe vessel wallbyan endothelial-de-pendentmechanism(11),probably by causingsurface
expres-sion ofGMP-140(12).
Teleologically,
thesemultiple
cell-acti-vatingfunctions of thrombinmaybe viewedasorchestratingtheresponse tovascularinjury, potentially mediatingnotonly hemostaticbutperhapsinflammatoryandproliferativeor
re-parativeresponses.Whether thesedisparateactions of throm-binareallimportantinvivoremainstobedefined.
Thrombin activities that would not necessarily be
asso-ciatedwithvascularinjuryhave also been described. The
sur-prising inhibitory effects ofthrombin on neurite outgrowth
(13, 14) and theapparentexpression ofprothrombinmRNA in
brain (15)conjureuphypotheses
regarding
potentialroles forthrombinor arelatedproteaseinneuronal
plasticity
ordevelop-ment,hypotheses whichremaintobetested.
Recently developed potent andspecific thrombin
inhibi-torssuchasrecombinanthirudin, argatroban,and PPACKare
defining the role of thrombin activity in vivo. These agents
bindthrombin itself, blocking notonlythrombin'sabilityto
activate its receptor but all active site-dependent thrombin
functionsincluding thrombin's abilitytocleavefibrinogenand
toactivateproteinC and otherzymogens.Forthisreason,they
do not address whetherthrombin's effects are mediated
di-rectly bythrombin receptoractivationorindirectlyvia other actions of the thrombinprotease.With theselimitations,
avail-able antithrombins provide exciting support for the
impor-tanceofthrombinincertaincellularresponsesinvivo. Animal studies withantithrombinsdosuggestacritical role for throm-bin in platelet thrombus formation in several modelsofarterial
thrombosis(16-20).Ingeneral, theimportanceof thrombin in
mediatingnonthromboticresponsesinvivo remainstobe de-fined. Inparticular, whether thrombinplays asignificantrole
in inflammatory orproliferative responses to vascularinjury remainstobe tested.Arecent studyusingrecombinant hirudin does suggest a role for thrombininmediating restenosisafter
angioplastyin oneanimal model (21).Itishoped thatthe char-acterization of the platelet thrombin receptorwill permitthe
development of thrombinreceptor blockers todirectlyaddress theimportanceof thrombin-inducedcellactivation in vivo.
Identification of
afunctional thrombin receptor
Previous theoriesof possiblemechanisms bywhich thrombin activates platelets included classicalreceptoroccupancy mecha-nisms, mechanisms involving proteolytic cleavage ofthe
recep-CharacterizationofaFunctional Thrombin Receptor 351 J.Clin. Invest.
©D
TheAmerican Society for Clinical Investigation,Inc.0021-9738/92/02/0351/05 $2.00
) -
Thrombin
Monocyte
Platelet
Figure1. Cellular events stimulated by thrombin. Cellulartargets for thrombin are depicted in the context of a blood vessel. Thrombin is the most potentstimulator of platelet aggregation, ischemotacticfor monocytes and mitogenic for lymphocytes andmesenchymalcells. Thrombin also has a numberof effectson thevascular endothelium,including stimulating expression of the neutrophil adhesive protein GMP-140 on the
endothelial surfaceas well astheproduction ofthe potentsmooth muscle cell mitogen, platelet-derived growth factor (PDGF). As discussed in the text, thesemultiple activating functionsof thrombin may be viewed teleologically as orchestrators of hemostatic, inflammatory, and
repara-tiveresponses tovascularinjury.
tor,andcombinations thereof (2, 22). Traditional ligand
bind-ingapproachestoidentifyafunctional thrombinreceptor
iden-tified proteins which bind thrombin but have no apparent
signallingfunction (23, 24). Infact,becausemodified throm-bins that lack bothagonistandantagonist activitydobindto
platelets inamannerindistinguishable from wildtype throm-bin(25-29),it hasnotbeenpossibletoconcludethat thesites identified in binding studies are related to the functional thrombin receptor. Because ofdifficulties with the classical binding approachandthe undefined nature of
thrombin-re-ceptorinteraction,weadoptedanexpression cloningapproach
which followed thrombin-inducedresponses in Xenopus oo-cytesexpressingexogenous mRNA toisolateafunctional
hu-manthrombinreceptorcDNA(1). Thisstrategy wasbasedon
studies by Masu and colleagues who hadsuccessfullyused
ex-pression cloning inXenopus oocytes toisolatea cDNAforthe
substanceK receptor(30). Thecloningof the thrombin recep-tor wasdescribed in detail elsewhere (1). The deduced amino acid sequence of thisclonerevealed a novel member of the
seven transmembrane receptor family. Closest relatives in-cludedreceptorsfor smallpeptides, substancePand substance
K(1), suggesting that the thrombinreceptoris peptide
recep-tor-like. This analogy was a useful hint atthe outset ofthe
structure-functionworkdescribedbelow.
Closerinspection ofthe receptor'saminoacid sequence was
revealing inthelight ofa largebodyof literatureonthrombin action. Thrombincleavespeptidesafterarginines(31). Inhope
offinding a receptor cleavage site, we examined sequences
surroundingextracellular arginines in the receptor sequence.
Wenoted that thereceptor'srelativelylong(100residue)
extra-cellular amino-terminal extension contained the putative
thrombincleavagesiteLDPR/S (singleletteramino acidcode, / representingthepoint ofcleavage afterarginine41 [R41]in
the receptor, Figs. 2 and 3). This site resembled the known
thrombin cleavagesite found in thethrombin-activated zymo-genproteinC(LDPR/I) (32). Carboxyltothisputative
cleav-agesiteliesareceptordomainresemblingthecarboxyltailof the polypeptide hirudin, aleech-derivedanticoagulantwhich binds thrombin with remarkable avidity. To put these
se-quenceobservationsinto context,thrombin possesses an
ex-tended substratebindingsurface thatrecognizesresidues both amino andcarboxylto asubstrate'scleavage site(33)(Fig.
2).
Partofthisextended substratebinding surface,the anion-bind-ing exosite(Fig.2), isimportant for thrombin'sabilityto
acti-vate itsreceptor(1, 34). Becausethecarboxyl tail of hirudin interactswith thrombin'sanion-binding exosite (35), the pres-ence ofthe hirudin-like sequence carboxyl to the putative
thrombincleavagesite within thethrombin receptor'samino terminal extension suggested thatreceptorproteolysisatthis cleavage sitemightbeimportantinreceptoractivation(Fig.2).
Weevaluatedtheimportanceof theputativecleavage site
Cleavage
Agonist peptide
Anion
binding
exosite site _ region binding domain
HumanTR35-63NATLDPR/SFLLRNPNDK-YE--PFWEDEE--KNE MouseTR35-68 DATVNPR/SFFLRNPSENTFELVPLGDEEEEEKNE Hirudin 55-65 DFEEIP--EEYLQ
B Anion-binding
exosite
S2-S4
Subsites
Thrombinreceptor's
amino terminal extension
Figure2.Thrombin-receptor inter-action. Functional domains within thethrombinreceptor's amino-ter-minalextension aredepictedinA. Thecleavagerecognition sequence
(LDPR),thrombincleavage site,
ag-onistpeptide domain,and
anion-bindingexositebinding do-mainasdefinedbystructureactivity
studies(38,39)withthe human
re-ceptorareshown. These arealigned
with the murine thrombinreceptor sequenceand theanion-binding
exositebinding sequenceofthe leech
anticoagulanthirudin. A model for interaction of these domains with thrombin is shown in B. Thrombin has an extended substratebinding
surface(represented bythecanyon
running laterally)whichrecognizes
residues both amino andcarboxylto itssubstrate'scleavagesite.Structure function studies(38, 39) suggestthat thereceptor'shirudin-like domain
(YEPFWEDEE)interacts with thrombin'sanion-binding exosite,
while itscleavagesite (LDPR/S) in-teractswiththrombin's S1-S4 sub-sites.Thismodel hasimportant
im-plicationsforthedevelopmentof
blocking monoclonal antibodiesand
receptor-peptidebasedthrombin
in-hibitors,asdiscussed in the text (re-printedwithpermissionfrom Na-ture[38]).
wereexpressed in Xenopusoocytesandtheir abilitytorespond
to thrombin assessed. The putative cleavage site mutant
(R4 lA) could not be activated by thrombin. The other
mu-tants,R46A andR70A,werefully activatable. Asan indepen-dent means of rendering the putative thrombin cleavage site uncleavable, serine 42atthe site'sP1'positionwaschangedto
aproline. Like the R4 lA mutant,this S42Pmutantreceptor
couldnotbe activated by thrombin. These data strongly sug-gested that the receptor's putative thrombin cleavage sitewas critical for thrombin-inducedreceptoraction (1).
How might proteolysis atthis site leadtoreceptor activa-tion?The favored hypothesiswasthat proteolysis atthis site would unmaska new amino terminus that mightserve as a
peptide ligand for the receptor(1). Precedent for proteolytic unmasking ofa"ligand" existed in trypsinogen activationto
trypsin (36), and in fibrinogen cleavage to fibrin monomer (37). Indeed,apeptide mimicking thenewreceptoramino
ter-minus that would be revealed byreceptorcleavageattheR4 1
positionwasafull agonist for the clonedreceptorexpressed in
oocytes.Not only could the wildtype receptorbe activated, the R4IAreceptor,unactivatable by thrombin, responded fullyto thenewamino terminus "agonist peptide." Thus the agonist
peptide could bypass the requirement for proteolysis and
acti-vate the receptor directly (1). This agonist peptide not only activated the thrombinreceptorexpressed inoocytes;itwasa
full agonist for platelet activation (1). Thissuggeststhat
activa-tion of the cloned thrombinreceptoror ahighly related
recep-toris sufficient for platelet activation.
Modelfor thrombin
receptoractivationThe studies described above suggest thatthrombin receptor activation proceeds by the novel mechanism depicted in Fig. 3. Thrombin cleaves itsreceptorafter arginine 41 in the receptor's amino terminal extension, exposing a new amino terminus thatfunctionsas atethered peptide ligand for thereceptor.The newamino terminus then bindstoan asyetundefinedreceptor site, effectingreceptoractivation (1).
Recent studies buttress this model (38, 39). Replacing the receptor'sthrombin cleavage site (LDPR/S)with that for
en-terokinase (DDDDK/S) completely switched the receptor's
specificity, creatingafunctional "enterokinasereceptor"that was fully activatable by enterokinase but unresponsive to thrombin (38). These results stronglysuggestthatreceptor
pro-teolysis that unmasks the amino terminus beginningaposition
42(SFLL. . .)issufficient forreceptoractivation (38).No
spe-cial"hormone-like" properties ofthrombin needtobeinvoked for activation of thisreceptor.
Additional studies have shown the receptor's hirudin-like sequence can indeed bind thrombin's anion-binding exosite
(38,39). Detailed mutagenesis studies have defined the specific residues important in this interaction and strengthened the analogy between the receptor's anion binding exosite binding
CharacterizationofaFunctionalThrombinReceptor 353
coo
-WH3+t-_
-.-+
NH
31
11fl1W~too*1
COO-Figure 3. Model of thrombin recep-toractivation.Thrombin binds its receptor via the receptor's amino terminal extension as detailed in Fig. 2;whether additional receptor do-mains may participate in thrombin
binding is unknown.After binding totheamino terminal extension, thrombincleaves the receptor at the LDPR/S cleavage site(junction be-tween openandfilledreceptor
seg-ments), releasingan inactive frag-mentofthe receptor's amino
ter-minus(openfragment)andexposing
anew aminoterminus.This newly unmasked amino terminus then
functionsas a tethered peptide ligand, binding to an as yet undefined pocket,thereby effecting receptor activation (1)(reprinted withpermission fromNature[38]).
domain and hirudin's carboxyl tail(38). These relationships
arecartoonedinFig.2 B.
Issues and opportunities
Thestudies presentedaboveraise provocative questionsand present opportunities atboth basic mechanistic and clinical
levels. Someofthesearediscussedbelow.
From a basic perspective, we have learned a great deal about how the receptor senses thrombin in itsenvironment, i.e., via the binding and
proteolytic
eventsdescribed above. Moredetailed informationregarding
thrombin-receptor inter-action willundoubtedly come from crystallographic studies.The rapid unravelling ofthemechanism bywhichthrombin activates itsreceptor"fell out" ofananalysisof thethrombin
receptor'samino acidsequence.Existingliteratureson
throm-bin
structure/function
andonseven transmembrane domain receptorsallowedustoidentify
thereceptor'sthrombin cleav-age site andbinding domainsandtoframe the thrombinrecep-tor as a"peptide receptor", leading naturallytothe model
de-pictedinFigs.2and 3.
Manyquestions remain unanswered.Wheredoes the
ago-nistpeptide domain unmasked byreceptor
proteolysis bind,
and how doesthisbindingeventleadto atransmembrane
sig-nal that allowsthe receptortotalkto intracellular
signalling
molecules(Fig. 3)?Theseintracellular
signalling
moleculesarecertainly
G-proteins;
thespecific
G-proteins
remaintobe de-fined.Theunique
proteolytic
mechanismbywhich thethrombinreceptor becomes liganded prompts a number of
questions.
This mechanism
produces
aligand
that is tetheredtotherecep-torandcannotdiffuseaway. How is the receptor turned off? We have shown that theagonist peptide can desensitize the receptor, thusatleastone
specific
mechanismindependent of proteolysisbythrombin existstoshutoffthis receptor.By anal-ogy with otherseventransmembrane receptors, it isprobable
that several mechanisms
including
receptorphosphorylation
by a beta-adrenergic receptor kinase-like enzyme(40)
and otherprotein kinasesregulatethrombin receptor desensitiza-tion.Perhapsmoreinterestingbutentirely speculative
isapossi-ble second mechanismin whichthrombin might cleave the receptorata second sitecarboxyl totheR41/S42 activation site, relievingthe receptor of its
agonist peptide
domain.Theunusual activation mechanism ofthrombin receptor
activationrequiresinessencea
peptide
receptor thatcontainsits own ligand. Where did this mechanism come from? Was it anevolutionary specialization ofaprevious peptidereceptor?
Orisit
representative
ofamoreprimitive heritage from which peptidereceptors andligandsmay haveevolved?Willthisclone sire a familyof thrombinor protease
recep-tors?And how manyofthe assortedcell-activation functions of thrombindoesthisreceptor accountfor?Theagonist peptide describedabove, whichactivatesthe clonedthrombinreceptor
directlyandindependent of thrombinanditsproteaseactivity,
will be useful in this regard. Specifically, thrombin responses
mimickedby the agonist peptide are likely mediated by the unusualmechanism describedabove via the cloned receptor or
ahighly relatedreceptor. Thrombinresponses notmimicked bytheagonist
peptide
may bemediatedbyadifferentmecha-nism.
What is therelationshipof knownthrombinbinding
pro-teins,inparticular, platelet GPIb,tothrombin signallingand to
thisreceptor? Bernard-Soulier syndrome plateletsaredeficient
in GPIb and have aselectiveandpartial defectintheir
respon-sivenesstothrombin(reviewedin3, 22). DoesGPIbpromote
activation ofthe cloned receptor bybinding thrombinand
lo-calizing ittothecellsurface (3, 20, 22)?Abodyof
circumstan-tialevidencedoes supportthis model,butdirect demonstration ofan
adjunctive
roleforGPIb inthrombinsignalling using,
for example,GPIbexpressedin null cellsislackingatthis time.As analternativehypothesis,
could GPIbserve as asink forthrom-bin?Perhaps,atlowambientthrombin concentrations,GPIb might bindthrombinlongenough to allow inactivation by
an-tithrombin III. By preventing thrombin interaction with the receptor and receptordesensitizationby trace levels of
throm-binin the normal circulation, such amechanism might also
explainthedefect in thrombin signalling seen in
Bernard-Sou-her.
Lastly, it ispossiblethat thrombin binding to GPIb servesanindependent, as yetundefinedfunction.
Fromthepathophysiologicand clinical perspective, these
studiessuggestanumber ofpaths to novel reagents that will be useful indefiningthe roleofthrombin receptor activation in vivo. Receptor-based peptides that bind and inhibit various thrombin functionshavebeenproduced (38, 39, 41). These are novelamongthrombin inhibitorpeptides (42) in that they are basedon humansequence; whether this will translate into any
practical clinical benefitoverotheragents in development is unknown.
blockers. Blockingmonoclonalantibodies mightbe obtained
inanumberofways.Antibodiestothe receptor'shirudin-like
sequenceorthrombincleavagesiteshouldinhibit thrombin-re-ceptorinteraction andreceptorproteolysis.Antibodiesbinding
these regions ortheagonist peptide domainitselfmight also
interfere with the"liganding" ofthereceptorafter proteolysis.
Asimilar function would beserved byantibodiesto receptor regionscomprising oraffecting the asyet undefinedbinding
site for the agonist peptide. Whether nonantibody receptor
blockers,i.e., competitiveornoncompetitive antagonistsofthe agonistpeptide,canbeobtained remainstobe explored.Such reagentswilldefine theroleofthrombinreceptoractivationin vivo, andmayleadtothedevelopmentofa newclassof
phar-maceuticals.
Acknowledgments
WethankDr.HenryBournefor critical review of this manuscript. ThisworkwassupportedinpartbyNationalInstitutes of Health
grantsHL-44907 and HL-43821,and byUniversityofCalifornia To-bacco-RelatedDisease ResearchProgramgrant2RT-19, andwasdone duringthetenure ofanawardtoS.R.Coughlin from the American HeartAssociationandSmithKline Beecham.
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