Activation of factor VII-activating protease in human inflammation: a sensor for cell death

Van, Stephan Femke, Hazelzet, Bulder Ingrid, Boermeester, Wuillemin, Aarden, Zeerleder, Zeerleder Sacha

Description

Cell death is a central event in the pathogenesis of sepsis and is reflected by circulating nucleosomes. Circulating nucleosomes were suggested to play an important role in inflammation and were demonstrated to correlate with severity and outcome in sepsis patients. We recently showed that plasma can release nucleosomes from late apoptotic cells. Factor VII-activating protease (FSAP) was identified to be the plasma serine protease responsible for nucleosome release. The aim of this study was to investigate FSAP activation in patients suffering from various inflammatory diseases of increasing severity. Methods We developed ELISAs to measure FSAP-C1-inhibitor and FSAP-α 2 -antiplasmin complexes in plasma. FSAP-inhibitor complexes were measured in the plasma of 20 adult patients undergoing transhiatal esophagectomy, 32 adult patients suffering from severe sepsis and 8 from septic shock and 38 children suffering from meningococcal sepsis. Results We demonstrate plasma FSAP to be activated upon contact with apoptotic and necrotic cells by an assay detecting complexes between FSAP and its target serpins α 2 -antiplasmin and C1-inhibitor, respectively. By means of that assay we demonstrate FSAP activation in post-surgery patients, patients suffering from severe sepsis, septic shock and meningococcal sepsis. Levels of FSAP-inhibitor complexes correlate with nucleosome levels and correlate with severity and mortality in these patients. Conclusions These results suggest FSAP activation to be a sensor for cell death in the circulation and that FSAP activation in sepsis might be involved in nucleosome release, thereby contributing to lethality.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 3
• Langue: English

Extrait

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RESEARCH

OpenAccess

ActivationoffactorVII-activatingproteasein
humaninflammation:asensorforcelldeath
FemkeStephan
1
,JanAHazelzet
2
,IngridBulder
1
,MarjaABoermeester
3
,JWOliviervanTill
3
,TomvanderPoll
4
,
WalterAWuillemin
5
,LucienAAarden
1
andSachaZeerleder
1,6*

Abstract
Introduction:
Celldeathisacentraleventinthepathogenesisofsepsisandisreflectedbycirculating
nucleosomes.Circulatingnucleosomesweresuggestedtoplayanimportantroleininflammationandwere
demonstratedtocorrelatewithseverityandoutcomeinsepsispatients.Werecentlyshowedthatplasmacan
releasenucleosomesfromlateapoptoticcells.FactorVII-activatingprotease(FSAP)wasidentifiedtobetheplasma
serineproteaseresponsiblefornucleosomerelease.TheaimofthisstudywastoinvestigateFSAPactivationin
patientssufferingfromvariousinflammatorydiseasesofincreasingseverity.
Methods:
WedevelopedELISAstomeasureFSAP-C1-inhibitorandFSAP-
a
2
-antiplasmincomplexesinplasma.
FSAP-inhibitorcomplexesweremeasuredintheplasmaof20adultpatientsundergoingtranshiatal
esophagectomy,32adultpatientssufferingfromseveresepsisand8fromsepticshockand38childrensuffering
frommeningococcalsepsis.
Results:
WedemonstrateplasmaFSAPtobeactivateduponcontactwithapoptoticandnecroticcellsbyanassay
detectingcomplexesbetweenFSAPanditstargetserpins
a
2
-antiplasminandC1-inhibitor,respectively.Bymeans
ofthatassaywedemonstrateFSAPactivationinpost-surgerypatients,patientssufferingfromseveresepsis,septic
shockandmeningococcalsepsis.LevelsofFSAP-inhibitorcomplexescorrelatewithnucleosomelevelsand
correlatewithseverityandmortalityinthesepatients.
Conclusions:
TheseresultssuggestFSAPactivationtobeasensorforcelldeathinthecirculationandthatFSAP
activationinsepsismightbeinvolvedinnucleosomerelease,therebycontributingtolethality.

Introduction
Moreover,nucleosomescouldbedetectedinpatients
Sepsisischaracterizedbyanextensiveinflammatorywithsevereperitonitis[7].Levelsofcirculatingnucleo-
responseincludingcytokinegeneration,activationofsomesandpulmonarynucleosomelevelsweredemon-
plasmaticcascadesystemsandinflammatorycellslead-stratedtocorrelatewithseverityandoutcomeinsepsis
ingtoorgandysfunctionandinmanycasestodeath[1].patients[6,7].Recentfindingssuggestthatthesecircu-
Extensivecelldeathasadownstreameffectoftheselatingnucleosomesplayacrucialroleininflammation.
mediatorswaspostulatedtobecriticallyinvolvedintheCirculatinghistones3and4turnedouttobehighly
developmentoforgandysfunction[2].Indeed,severalcytotoxicandtomediatelethaleffectsinsepsis[8].We
studiesinanimalmodelsforsepsisandinsepsisrecentlyshowedthatFactorVII-activatingprotease
patientsdemonstratedwidespreadapoptosisoflymphoid(FSAP)inplasmacanremovenucleosomesfromlate
tissueandtoalesserextentofparenchymalcells[3-5].apoptoticcells[9,10].
Asaresultofextensivecelldeathcirculatingnucleo-FSAP,alsoknownasplasmahyaluronicacidbinding
somescouldbemeasuredinsepsispatients[6].protein2(HABP2),isaserineproteasewhichcirculates
inplasmaasaninactivesingle-chainmoleculeof64
*Correspondence:[e-mail]
kDa.Itisproteolyticallyconvertedinitsactivetwo-
1
DepartmentofImmunopathology,SanquinResearchatCLBand
chainformconsistingofa50kDaheavyanda28kDa
LandsteinerLaboratoryoftheAMC,Plesmanlaan125,1066CXAmsterdam,
lightchainconnectedbyadisulfidebond[11].Purified
TheNetherlands
Fulllistofauthorinformationisavailableattheendofthearticle
plasma-derivedFSAPisdescribedtobesusceptibleto
A©tt2ri0b1u1tiSotnepLihcaennseteahl.t;tlpic:/e/ncrseeaetiBvieocMoemdmCoennst.roarlgL/tlidceTnhisseiss/bayn/2o.p0,enwhaiccchespsearrmtiictlseudnisrtersitbriuctteedduunsde,erditshtreibteurtimosn,oafnthdereCprreoatdivuectiCoonminmaonnys
medium,providedtheoriginalworkisproperlycited

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autoactivation[12].Recentlypublisheddatasuggestthat
purifiedFSAPcanbindandbeactivatedbynegatively
chargedpolyanionssuchasheparin,polyphosphates,
RNAandDNA[11,13-15].Inpurifiedsystems,various
serineproteaseinhibitors(serpins)suchasC1-inhibitor
(C1-inh),
a
2
-antiplasmin(AP),antithrombinIII(AT-III)
andplasminogenactivatorinhibitor-1(PAI-1)[11,16-19]
werereportedtoinhibittheamidolyticactivityof
plasma-derivedactivatedFSAP.Inplasma,C1-inhhas
beenreportedtobethemaininhibitorofactivated
FSAP[16].
Sincecompoundsofcirculatingnucleosomesinduce
lethalityinsepsis[8],wesuggestFSAPactivationinsep-
sistobeinvolvedinnucleosomerelease.Theaimofthis
studyistoinvestigateFSAPactivationinpatientssuffer-
ingfrominflammatorydiseasesofincreasingseverity.
Duetoalackofspecificsubstrateanditssusceptibility
forautoactivationmeasurementofFSAPactivationis
troublesome.Therefore,wesetupassaystofollow
FSAPactivationinplasma.Wemadeuseofthefact
thatuponactivationFSAPquicklyformsstablecovalent
complexeswithitsplasmainhibitors.WesetupELISAs
tomeasureFSAP-serpincomplexes.Bymeansofthese
assayswemeasuredFSAPactivationinpatientsafter
surgery,patientswithseveresepsis,septicshockand
meningococcalsepsis.
Materialsandmethods
Patients
Thestudywasapprovedbytheinstitutionalmedical
ethicscommitteesofthecentersinvolved,andfromall
studyparticipantsorlegalrepresentativeswritten
informedconsentwasobtained.
Healthycontrols
Citratedplasmawascollectedfrom20healthyDutchlab
workers.
Post-operativeacute-phaseresponse
Twentyconsecutivepatientswithresectableadenocarci-
nomaofthemiddleordistalesophagusoresophagogas-
tricjunctionwerestudied.Pre-operativeand
peroperativeinvestigationsrevealednodistantmetas-
tases,andnoneofthepatientsreceived(neo-)adjuvant
chemotherapyorradiotherapy[20].EDTAandcitrated
bloodwassampledpre-operatively(Day0)andondays
1,3,5,7,and10aftersurgeryandthebloodsamples
werestoredat-80°Cuntilanalysis.
Severesepsisandsepticshock
PatientsofthemedicalandsurgicalICUwereeligibleif
theymettheinclusioncriteriaforseveresepsisandsep-
ticshockaccordingtothedefinitionsoftheAmerican
CollegeofChestPhysisians(ACCP)consensusconfer-
ence[21].Patientswerefollowedfor90daysoruntil
death.Thesepsispatientsparticipatedinarandomized,

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double-blindplacebocontrolledpilotstudytostudythe
efficacyofC1-inhibitorinsepsis[22].EDTAand
citratedbloodwassampledatinclusionintothestudy
beforetheadministrationofC1-inhbitororplacebo,
respectively.Thebloodsampleswerestoredat-80°C
untilanalysis.Clinicalparameters,organdysfunction
scoresandacutephaseparameterswereassessedas
recentlydescribed[6].
Meningococcalsepsis
Childrenbetween1monthand18yearsofagewith
septicshockandpetechiae/purpurawereenrolledin
thisstudy.Thechildrenwereincludedinarando-
mized,double-blindplacebocontrolleddose-finding
studytotesttheefficacyofplasma-derivedproteinC
(PC)insepsis.Twenty-eightreceivedPCinescalating
doses,whereas10receivedaplacebo.Arterialcitrated
andEDTAbloodsampleswerecollectedwithintwo
hoursafteradmission(beforethestartofPCorthe
placebo)andseveraltimepointsafterwardsandstored
at-80°Cuntilanalysis.Theclinicalcharacteristicsof
thesepatientsaredescribedindetailelsewhere[23].
Clinicalparameters,organdysfunctionscoresand
acutephaseparameterswereassessedasrecently
described[23].
Reagents
MousemonoclonalantibodiestoFSAP(anti-FSAP-4
andanti-FSAP-9),tocomplexedC1-inhibitor(KOK-12),
to
a
2
-antiplasmin(AAP-20),andacontrolantibody
(anti-IL6)werepreparedatourdepartment(allIgG1

)
[10,24].PE-labeledrabbit-anti-mouseF(ab
’
)2antibody
wasobtainedfromDako(Glostrup,Denmark).Iscove
’
s
modifiedDulbecco
’
smediumwasobtainedfromBio-
WhittakerEurope(Verviers,Belgium).Fetalcalfserum
wasobtainedfromBodincoBV(Alkmaar,TheNether-
lands).Penicillinandstreptomycinwereobtainedfrom
Gibco/Invitrogen(Groningen,TheNetherlands).Etopo-
side,ß-mercaptoethanol,RNase,andnitrobluetetrazo-
liumand5
’
-bromo-4
’
-chloro-3
’
-indolylphosphate(NBT/
BCIP)wereobtainedfromSigma(Zwijndrecht,The
Netherlands).NuPage4to12%polyacrylamidegels,
samplebuffer,dithiothreitol(DTT)andnitrocellulose
membraneswereobtainedfromInvitrogen(Groningen,
TheNetherlands).Westernblockingreagentwas
obtainedfromRocheDiagnostics(Mannheim,Ger-
many).Streptavidin-alkalinephosphatasewasobtained
fromMabtech(NackaStrand,Sweden).Highperfor-
manceELISAbuffer(HPE)andPoly-HRP-labeledstrep-
tavidinwereobtainedfromSanquin(Amsterdam,The
Netherlands).(3,5,3
’
,5
’
)-tetramethylbenzidine(TMB)was
obtainedfromMerck(Darmstadt,Germany).CNBr-acti-
vatedsepharoseandProteinGSepharosewasobtained
fromPharmaciaBiochem(Uppsala,Sweden).

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