Feasibility and Safety of Local Treatment with Recombinant Human Tissue Factor Pathway

13 Pages·2015·3.17 MB·English

RESEARCHARTICLE
Feasibility and Safety of Local Treatment
with Recombinant Human Tissue Factor
Pathway Inhibitor in a Rat Model of
Streptococcus pneumoniae Pneumonia
FlorryE.vandenBoogaard1,3,5*,JorritJ.Hofstra1,Cornelisvan‘tVeer3,5,MarcelM.Levi4,
JorisJ.T.H.Roelofs6,TomvanderPoll3,5,7,MarcusJ.Schultz1,2
1 LaboratoryofExperimentalIntensiveCareandAnesthesiology(L(cid:1)E(cid:1)I(cid:1)C(cid:1)A),AcademicMedicalCenter,
UniversityofAmsterdam,Amsterdam,TheNetherlands,2 DepartmentofIntensiveCareMedicine,
AcademicMedicalCenter,UniversityofAmsterdam,Amsterdam,TheNetherlands,3 Centerfor
ExperimentalandMolecularMedicine(CEMM),AcademicMedicalCenter,UniversityofAmsterdam,
Amsterdam,TheNetherlands,4 DepartmentofInternalMedicine,AcademicMedicalCenter,Universityof
Amsterdam,Amsterdam,TheNetherlands,5 CenterforInfectionandImmunityAmsterdam(CINIMA),
AcademicMedicalCenter,UniversityofAmsterdam,Amsterdam,TheNetherlands,6 Departmentof
Pathology,AcademicMedicalCenter,UniversityofAmsterdam,Amsterdam,TheNetherlands,
7 DepartmentofInfectiousDiseases,AcademicMedicalCenter,UniversityofAmsterdam,Amsterdam,The
Netherlands
OPENACCESS * [email protected]
Citation:vandenBoogaardFE,HofstraJJ,van‘t
VeerC,LeviMM,RoelofsJJTH,vanderPollT,etal.
Abstract
(2015)FeasibilityandSafetyofLocalTreatmentwith
RecombinantHumanTissueFactorPathwayInhibitor
inaRatModelofStreptococcuspneumoniae
Pulmonarycoagulopathyisintrinsictopulmonaryinjuryincludingpneumonia.Anticoagulant
Pneumonia.PLoSONE10(5):e0127261.
strategiescouldbenefitpatientswithpneumonia,butsystemicadministrationofanticoagu-
doi:10.1371/journal.pone.0127261
lantagentsmayleadtosuboptimallocallevelsandmaycausesystemichemorrhage.We
AcademicEditor:JianFu,UniversityofKentucky,
hypothesizednebulizationtoprovideasaferandmoreeffectiverouteforlocaladministration
UNITEDSTATES
ofanticoagulants.Therefore,weaimedtoexaminefeasibilityandsafetyofnebulizationof
Received:September5,2014
recombinanthumantissuefactorpathwayinhibitor(rh-TFPI)inawell-establishedratmodel
Accepted:April14,2015 ofStreptococcus(S.)pneumoniaepneumonia.Thirtyminutesbeforeandevery6hours
Published:May18,2015 afterintratrachealinstillationofS.pneumoniacausingpneumonia,ratsweresubjectedto
Copyright:©2015vandenBoogaardetal.Thisis localtreatmentwithrh-TFPIorplacebo,andsacrificedafter42hours.Pneumoniawasasso-
anopenaccessarticledistributedunderthetermsof ciatedwithlocalaswellassystemicactivationofcoagulation.Nebulizationofrh-TFPIre-
theCreativeCommonsAttributionLicense,which
sultedinhighlevelsofrh-TFPIinbronchoalveolarlavagefluid,whichwasaccompaniedby
permitsunrestricteduse,distribution,and
anattenuationofpulmonarycoagulation.Systemicrh-TFPIlevelsremainedundetectable,
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. andsystemicTFPIactivityandsystemiccoagulationwerenotaffected.Histopathologyre-
vealednobleedinginthelungs.Weconcludethatnebulizationofrh-TFPIseemsfeasible
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. andsafe;localanticoagulanttreatmentwithrh-TFPIattenuatespulmonarycoagulation,
whilenotaffectingsystemiccoagulationinaratmodelofS.pneumoniaepneumonia.
Funding:ThisworkwassupportedbyNovartis,
Switzerland;NetherlandsOrganizationforHealth
ResearchandDevelopment(ZonMW),NWO-VENI
grant2004toMJS(projectnumber016.056.001).
Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or
preparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0127261 May18,2015 1/13
Nebulizedrh-TFPIinStreptococcuspneumoniaePneumonia
CompetingInterests:Novartisdonatedan Introduction
unrestrictedgranttotheAcademicMedicalCenter
Pneumoniaisassociatedwithalocalprocoagulantstateduetoenhancedactivationofcoagula-
Amsterdam,andsuppliedTifacogin(recombinant
humanTFPI)fortheexperiments.Thisdoesnotalter tion,reducedanticoagulantcapacityandinhibitionoffibrinolysisinthealveolarcompartment
theauthors'adherencetoPLOSONEpolicieson [1–3].Intrapulmonaryfibrindepositionandinflammation-inducedcoagulopathyaggravate
sharingdataandmaterials. lunginjuryandultimatelycompromisepulmonaryfunction[4].Asactivationofcoagulationis
bothaconsequenceandacontributortoongoinglunginjury,pulmonarycoagulopathyhas
beensuggestedasatargetfortherapeuticinterventioninpatientswithpneumonia[1].
Tissuefactor(TF)isappreciatedasthemaininitiatorofcoagulationwithinfection.Results
fromanimalandhumanstudiessuggestthatpathologicalexpressionofTFbyinflammatory
cellsplaysadetrimentalroleontheoutcomeofcommunity-acquiredpneumonia(CAP)[3,5,
6].Atthesametime,tissuefactorpathwayinhibitor(TFPI),theendogenousinhibitorofthe
TFpathway,isoverwhelmedbyincreasedTFprocoagulantactivity[7,8].Inseveralmodelsof
lunginjuryitisshownthatblockingtheTFpathwayduringlunginjurypreventslocalcoagula-
tionandpreserveslungfunction[9–11].Moreover,systemicTFinhibitionreducespulmonary
andsystemiccoagulationinrodentmodelsofpneumonia[5,12,13].
Oneimportantdrawbackofsystemicadministrationofanticoagulantsistheincreasedrisk
ofbleedingcomplications[14–16].Inaddition,itisuncertainhowwellthesystemicallyadmin-
istereddrugspenetrateintolungtissueduringpneumonia[17].Notably,TFPIinpulmonary
edemafluidinpatientswiththeacuterespiratorydistresssyndrome(ARDS)hasbeenshown
tobetruncatedandinactive[7].Consideringtheseissues,localtreatmentwithanticoagulant
agentsisanappealingstrategyaslocaltreatmentmaybesafer,whileatthesametimepotential-
lyleadingtohigheralveolarconcentrations[18].
Therearenopreclinicalstudiesthathaveinvestigatedtheeffectsoflocaltreatmentwith
TFPIinpneumoniasofar.Weherehypothesizethatlocaladministrationofrecombinant
human(rh)-TFPIbymeansofnebulizationefficientlyattenuatespulmonarycoagulopathy
whileleavingsystemiccoagulationunaffected.ThereforeweinfectedhealthyratswithStrepto-
coccuspneumoniae,themostcommoncausativepathogenofCAP[19],andtreatedthemlocal-
lywithrh-TFPIorplacebo.
MaterialandMethods
Animals
TheInstitutionalAnimalCareandUseCommitteeoftheAcademicMedicalCenterofthe
UniversityofAmsterdamapprovedallexperiments.Animalswerehandledinaccordancewith
theguidelinesprescribedbytheDutchlegislation,andinternationalguidelinesonprotection,
care,andhandlingoflaboratoryanimals.MaleSprague—Dawleyrats(250–300g;8–10weeks
old)(Harlan,TheHague,TheNetherlands)wereallowedtoacclimatizetolaboratorycondi-
tionsforatleast7days(12:12hday—nightcycleat22°C)beforehandling.
Studydesign
Pneumoniawasinducedbyintratrachealinstillationunderlightsedationwith5%isofluraneof
8,5x105colony-formingunits(CFU)S.pneumoniae(serotype3,ATCC6303)inatotalvol-
umeof250μLofbacterialsuspension,whichwasculturedasdescribedpreviously[12,13].Rats
wererandomizedtonebulizationwithrh-TFPI10mg/kg(Tifacogin,Novartis,Chiron,Emery-
ville,CA)orvehicle(300mML-arginine,20mMtrisodiumcitratedihydrate,pH5.5)(n=7
pergroup).Uninfectedratswerenebulizedwithvehicle(n=3)toevaluatetheeffectofnebuli-
zationalone.Uninfecteduntreatedratsservedasnaïvecontrols(n=5).Pergrouprh-TFPIor
vehiclewasadministeredbynebulizationinatotalvolumeof5mLat30minutesbeforeand
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every6hoursafterinductionofpneumonia.Thedosingstrategywasbasedondatafromprevi-
ousstudieswithrh-TFPIadministeredintravenouslyinS.pneumoniaepneumonia[12]andan
estimationoftheefficacyprovidedbythenose-onlyexposuresystem,asdescribedbelow.
Nebulization
Forlocaltreatmentwithrh-TFPIweusedanadapteddynamicairflow,nose-onlyexposuresys-
tem,whichallowsdirectexposureofnebulizedagentstothenosesoftheanimals,asdescribed
before[20].Inshort,thissystemconsistsofaconcentricmanifoldconnectedtothenecksof
bottle-likerestrainttubes(CHT249restrainttube,CHtechnologiesInc.,Westwood,NewJer-
sey)inwhichtheanimalswereconfinedwiththeirnosesadjacenttothebottlenecks.Thebot-
tlesaredetachableallowingdisassemblyofthedeviceforcleaning.Theinhalationchamberis
suitabletoaccommodateupto7ratsatonce.Theaerosolizedagentwassuppliedtotheupper
endofthemanifold,flowedadjacenttothenosesoftheindividualanimals,andthenwas
drawnoutthroughthebottomofthemanifold.Theaerosolatmospherewasgeneratedusing
theAeronebProMicropumpNebulizer(AerogenLtd.).TheAeronebProNebulizerusesavi-
bratingmeshwithmultipleaperturestogenerateafine—particle,low—velocityaerosoland
producesaerosolswithanaveragesizeof2.1μm.Ataconstantoxygenflow(2L/min)theaero-
solsweredirectedtotheinhalationchamber.Theanimalswereaccommodatedtorestraint
tubesatseveraloccasionsintheweekbeforetheexperiments.
Bloodandtissuesampling
At42hoursafterinductionofpneumonia,ratsweresacrificedwithanintramuscularinjection
ofketamine45mg/kg(Eurovet,Bladel,TheNetherlands)andmedetomidine0.25mg/kg
(Novartis,Arnhem,TheNetherlands).Bloodwascollectedfromtheinferiorvenacavain
citrated(0.109M)vacutainertubes.Therightlungwasligated,andtheleftlungwaslavaged
threetimeswith2mLice—coldsaline,0.3%BSA,1mMEDTA.Therightsuperiorlobewas
fixedin10%bufferedformalinandembeddedinparaffin.Theremaininglunglobeswere
weighedandhomogenizedin4volumes(i.e.,4xlungweightinμL)ofsterilesalineusingatis-
suehomogenizer(BiospecProducts,Bartlesville,OK).
Measurements
Plasmaandcell—freesupernatantsfrombronchiallavagefluids(BALF)wereusedformeasur-
inglevelsofrh-TFPI,TFPIactivityandcoagulation.Totalcellnumbersinlavagefluidwerede-
terminedusinganautomatedcellcounter(Z2CoulterPariticleCounter,BeckmanCoulter
Corporation,Hialeah,FL).Neutrophilcountsinlavagefluidswereperformedoncytospin
preparationsstainedwithamodifiedGiemsastain(Diff-Quick;DadeBehringAG,Düdingen,
Switzerland).CommerciallyavailableELISA’swereusedtomeasurelevelsoftumornecrosis
factor(TNF)-α,interleukin(IL)-6,andcytokineinducedneutrophilchemoattractant(CINC)-
3(allR&DSystems,Abingdon,UnitedKingdom)andmyeloperoxidase(MPO;HyCultbio-
technologyb.v.,Uden,TheNetherlands).
Toquantifybacterialnumbersinlungsandblood,serialten—folddilutionsoflunghomoge-
nates,lavagefluidandwholebloodweremadeinsterileisotonicsalineandplatedontosheep—
bloodagarplates.After16hoursofincubationat37°Cin5%CO ,thenumbersofCFU
2
werecounted.
Assays. Todeterminetheefficacyoflocaldeliveryofrh-TFPI,bymeasuringtotalrh-TFPI
immunogenlevelsinthelungandinplasmaofratsnebulizedwithrh-TFPI,wedevelopedan
enzyme-linkedimmunosorbentassay(ELISA)usingmonoclonalmouseanti-humanTFPIdi-
rectedagainsttheKunitzdomain2(Sanquin,Amsterdam,theNetherlands)asacoating
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antibodyandpolyclonalrabbitanti-humanTFPI(kindgiftofDr.WalterKisiel,Universityof
NewMexico,AlbuquerqueNM,USA)asadetectingantibody.
Furthermore,todetermineifnebulizationwithrh-TFPIaffectedtheoverallsystemicTFPI
activity,weemployedthetwo-stagechromogenicTFPIassayoriginallydescribedbySandset
etalbymeasuringitsinhibitoryactivityinafactorXa-generationassay[21].Inbrief,heat—in-
activatedplasmawasincubatedwithamixtureofrecombinantFVIIa(Novoseven,NovoNor-
diskA/S,Bagsvaerd,Denmark),alimitedamountofFX(kindgiftofDr.WalterKisiel,
UniversityofNewMexico,AlbuquerqueNM,USA)andrelipidatedrecombinantTF(Innovin,
DadeBehring,Surrey,UK)inordertoformTF-FVIIa-FXa-TFPIcomplexes.Tomeasureresid-
ualTFactivityexcessFXwasaddedinthesecondstageandFXagenerationwasdetermined
usingS2222.Standardcurveswerepreparedbyserialdilutionofcitratednormalratplasma.
Thrombin—antithrombincomplexes(TATc)andfibrindegradationproducts(FDP)were
measuredusingcommerciallyavailableELISA(TATc:BehringwerkeAG,Marburg,Germany,
FDP;AsserachromD—Di,DiagnosticaStago,Asnières—sur—Seine,France);antithrombin
(AT),plasminogenactivatoractivity(PAA),andplasminogenactivatorinhibitor(PAI)-1ac-
tivityweremeasuredbyautomatedamidolyticassays[22].
Histopathology
Immediatelyafterratswerekilled,lungsampleswerefixedin10%bufferedformalinfor24h
andembeddedinparaffininaroutinefashion.Four—micrometersectionswerestainedwith
hematoxylinandeosin(H&E).Allslideswerecodedandscoredforthefollowingparameters:
interstitialinflammation,endothelialitis,bronchitis,edema,pleuritisandthrombusforma-
tion,andbleedingbyapathologistwhowasblindedforgroupidentity.Confluent(diffuse)
inflammatoryinfiltratewasquantifiedseparatelyandexpressedaspercentageofthelung
surface;thenumberofthrombiwascountedin5randommicroscopicfields.Theremaining
parameterswereratedseparatelyonascalefrom0(conditionabsent)to4(presentinmassive
amounts).
Statisticalanalyses
Comparisonsbetweentheexperimentalratgroupsandvehicle—treatedplaceboratgroup
wereperformedusingKruskal—Wallistests,followedbyMann—WhitneyU—testswhereap-
propriate.Dataareexpressedasindividualdataorasmedianwithinterquartileranges.Ap-
value<0.05wasconsideredstatisticallysignificant.Statisticalanalyseswereperformedwith
GraphPadPrism(GraphPadSoftware,SanDiego,CA).
Results
Levelsofrh-TFPIinBALFandplasma
Inpreviousstudiesnebulizationofanticoagulantsattenuatedpulmonarycoagulopathy,but
alsoaffectedsystemiccoagulation[18].Toverifywhetherrh-TFPIdeliverybynebulization
wasrestrictedtothelungcompartment,wemeasuredrh-TFPIlevelsinlavagefluidandplas-
ma.Rh-TFPIlevelsweresignificantlyincreasedinlavagefluidofrh-TFPItreatedrats,andno
rh-TFPIwasdetectedinlavagefluidofvehicletreatedrats(Fig1A).Rh-TFPIlevelsremained
undetectableinplasmaofrh-TFPItreatedrats(Fig1B),confirmingcontainmentofrh-TFPI
deliverywithinthelungcompartmentwhennebulized.SystemicTFPIactivitywasalsonotal-
teredbynebulizationofrh-TFPI(Fig1C),furthersuggestinglocaladministrationofrh-TFPI
doesnothaveasystemiceffect.
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Fig1.Nebulizationwithrh—TFPIincreaseslevelsofrh-TFPIinbronchoalveolarlavagefluidbutnotinplasma.Levelsofrh-TFPImeasuredin
bronchoalveolarlavagefluid(BALF)(A),andplasma(B),andTFPIactivityinplasma(C)afternebulizationofrh-TFPI(rh-TFPI)orvehicle(control)inrats42
hoursafterintratrachealinstillationofS.pneumoniae.Dataarerepresentedasindividualdatawithmedian.***p<0.001versuscontrol.
doi:10.1371/journal.pone.0127261.g001
Pulmonaryandsystemicactivationofcoagulation
Pneumoniaisassociatedwithanincreaseincoagulationactivity[2,3].Accordingly,wedem-
onstratedaprocoagulantstateininfectedratsasreflectedbyincreasedlevelsofTATcandFDP
withaconcurrentdropinATlevelsinBALF(Fig2A–2C)andelevatedTATclevelsinplasma
(Fig2D).Systemictreatmentwithrh-TFPIisknowntoreduceactivationofcoagulationin
bothpre-clinicalandclinicalpneumoniastudies[9,12,13,23].Nebulizationofrh-TFPIsignif-
icantlyattenuatedtheinfection-inducedincreaseinTATcandFDPlevelsandreductionofAT
levelsinlavagefluidcomparedtoratstreatedwithvehicle(Fig2A–2C).Atthesametime,neb-
ulizationofrh-TFPIdidnotaltersystemiccoagulation(Fig2D).Nodifferencesincoagulation
wereobservedbetweenuninfectednaïveanduninfectedvehiclenebulizedrats(datanot
shown).Together,thesedataconfirmtheefficacyofnebulizedrh-TFPItoattenuatepulmonary
coagulopathyinpneumoniaandsuggestthatlocaladministrationofrh-TFPIdoesnotposea
riskofsystemichemorrhage.
Pulmonaryandsystemicfibrinolysis
Theusedmodelofpneumococcalpneumoniaisassociatedwithinhibitionoffibrinolysisdue
toenhancedreleaseofPAI-1[12,20],whichresembleschangesinpatientswithpneumonia[2,
24,25].Inaccordance,wemeasuredelevatedlevelsofPAI-1inBALFanddecreasedlevelsof
PAAinBALFandinplasma.Nebulizationwithrh-TFPIdidnotinfluencelocalorsystemicfi-
brinolysis(Fig3).Nodifferencesinfibrinolysiswereobservedbetweenuninfectednaïveand
uninfectedvehicletreatedrats(datanotshown).
Pulmonaryandsystemicbacterialloads
Toexaminetheeffectofnebulizedrh-TFPIonlocalbacterialloadsanddisseminationwe
quantifiedbacterialnumbersinBALF,lunghomogenateandwholeblood.Bacterialloadswere
notdifferentbetweenthestudygroups(Fig4).
Inflammatoryresponse
Apulmonaryinflammatoryresponsewaselicitedbypneumococcalpneumonia42hoursafter
intratrachealchallengewithS.pneumoniaeinthelungsofrats,asshownbyasignificantincrease
incellinfluxinBALF,mainlyconsistingofneutrophilscomparedtouninfectedcontrolrats
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Fig2.Nebulizationwithrh-TFPIattenuatescoagulationinBALFbutnotinplasma.Levelsofthrombin-antithrombincomplexes(TATc)(A),antithrombin
activity(AT)(B),fibrindegradationproducts(FDP)(C)measuredinBALF,andlevelsofTATcmeasuredinplasma(D)ofratsnebulizedwithrh-TFPI(SPrh-
TFPI,openbars)orvehicle(SPcontrol,blackbars)42hoursafterintratrachealinstillationofS.pneumoniaeandinnaïvecontrolrats(control,greybars).
Barsdepictmedian±IQR;**p<0.01versusSPcontrol,##p<0.01versusnaïvecontrols.
doi:10.1371/journal.pone.0127261.g002
(Table1)andthepresenceofinterstitialinflammation,bronchitisandedemaseeninlunghisto-
pathologyslidesrepresentedastotalhistopathologyscores(Fig5).Inhalationofnebulizedrh-
TFPIdidnotaffecttotalcellnumbersorabsoluteorrelativeneutrophilcountsinBALF.However
thelevelofMPO,amarkerofneutrophilactivity,wasreducedinBALFofratsnebulizedwithrh-
TFPIcomparedtoratstreatedwithvehicle(p=0.03,Table1).Furthermore,lungweightandcy-
tokines/chemokines(TNF-α,IL-6,CINC-3)werenotinfluencedbynebulizationofrh-TFPI(S1
Fig).Nodifferencesincellcountswereobservedbetweenuninfectednaïveanduninfectedvehicle
treatedrats(datanotshown).Nolungtissueprotectiveeffectwasseenfromnebulizationwithrh-
TFPIcomparedtovehicletreatedratsreflectedbyunalteredtotalhistopathologyscores(Fig5).
LocalBleeding
Toexaminetheeffectofnebulizedrh-TFPIonlocalbleeding,wespecificallylookedforbleed-
inginhistopathologyslides.Nebulizationofrh-TFPIdidnotresultinlocalbleedings,neither
inunchallengedrats,norinratswithpneumonia.
Discussion
Thepresentstudyshowsthatlocaladministrationofrh-TFPIbynebulizationisfeasibleandsafe
inawell-establishedratmodelofS.pneumoniaepneumonia.Indeed,nebulizationofrh-TFPI
inhibitedbronchoalveolarcoagulation,andthistreatmentdidnotresultinmeasurablelevelsof
rh-TFPIinplasma,didnotaffectsystemiccoagulation,anddidnotresultinlocalbleedings.
Coagulationisanessentialpartofhostdefensethatinteractswiththeinflammationsystem
tomountanadequateresponse.Thiswasreflectedbyalocalprocoagulantstateandsystemic
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Fig3.Nebulizationwithrh-TFPIdoesnotinfluencelocalorsystemicfibrinolysis.Plasminogenactivatoractivity(PAA)andplasminogenactivator
inhibitor(PAI)-1activityinBALF(AandB)andPAAinplasma(C)inratsnebulizedwithrh-TFPI(SPrh-TFPI,openbars)orvehicle(SPcontrol,blackbars)
42hoursafterintratrachealinstillationofS.pneumoniaeandinnaïvecontrolrats(control,greybars).Barsdepictmedian±IQR;##p<0.01versus
naïvecontrols.
doi:10.1371/journal.pone.0127261.g003
Fig4.Nebulizationwithrh-TFPIdoesnotinfluencebacterialloadsinthepulmonaryorsystemiccompartment.NumbersofS.pneumoniaecolony
formingunits(CFU)werequantifiedinbronchoalveolarlavagefluid(BALF)(A),lunghomogenates(B),andwholeblood(C)ofratsnebulizedwithrh-TFPI
(rh-TFPI)orvehicle(control)42hoursafterintratrachealinstillationofS.pneumoniae.Barsdepictmedian±IQR.Nostatisticaldifferencebetweengroups
wasobserved.
doi:10.1371/journal.pone.0127261.g004
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Table1. Influenceofnebulizedrh-TFPIonlunginflammationandinjuryduringratStreptococcus
pneumoniaepneumonia.
Control SPcontrol SPrh-TFPI
Totalcells 28(12–50) 123(91–451)## 112(72–187)
PMN 0(0–0) 88(24–666)## 62(49–143)
MPO[pg/ml] 88(86–89) 131(125–150) 87(73–99)*
NOTE:Totalcellandneutrophil(PMN)countsx104/mlinbronchoalveolarlavagefluidand
myeloperoxidase(MPO)levelsinlunghomogenatesofratsnebulizedwithrh-TFPI(SPrh-TFPI)orvehicle
(SPcontrol)42hoursafterintratrachealinstillationofS.pneumoniae(n=7pergroup),orofnaïverats
(control,n=5).Dataareexpressedasmedian(interquartilerange)
##p<0.01versuscontrol
*p<0.05versusSPcontrol.
doi:10.1371/journal.pone.0127261.t001
Fig5.Effectsofnebulizedrh-TFPIonhistopathology.Totalhistopathology(PA)scoresoflungtissue(A)fromratsnebulizedwithrh-TFPI(rh-TFPI)or
vehicle(control)42hoursafterintratrachealinstillationofS.pneumoniaewithrepresentativemicrophotographs(BandC).Nobleedingswereobserved.Bars
depictmedian±IQR.Scalebar=200μm.Nostatisticaldifferencewasobservedbetweengroups.
doi:10.1371/journal.pone.0127261.g005
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coagulopathycausedbyinfectionwithS.pneumoniaeinthepresentratpneumoniamodel,
whichisinaccordancewithobservationsfrompreviousstudies[5,12,13,26].Notably,these
findingsresembletheclinicalsituation,asincreasedpulmonarycoagulation[2,5,27]andsys-
temiccoagulationabnormalities[28]arecommonfindingsinpatientswithpneumonia.
CoagulationactivationisprimarilydrivenbytheTFpathway,and,interferencewiththeTF
pathwayconsistentlyreducescoagulopathy,bothinstudiesofsystemicinfectionandinstudies
oflocalinfection,likepneumonia[5,6,9,10,12,14,29–36].Moreover,inpreviousrodent
studiesofpneumococcalpneumonia,blockingtheTFpathwaystronglyinhibitedalveolar
thrombingeneration[5,12,13].Inthesestudies,TFpathwayinhibitorswereadministeredsys-
temically,bearingtheriskofbleedingcomplications.Recently,studieshavebeenundertaken
toexplorethefeasibilityoflocaldeliveryofanticoagulantagentsanditseffectivenessinthe
pulmonarycompartment.
Inthepresentstudy,wedemonstratethatnebulizedrh-TFPIattenuatedFDPandTATclev-
elsandlargelypreservedATlevelsinlavagefluid,withoutaffectingTATclevelsinplasma,indi-
catingthattheanticoagulanteffectofnebulizedrh-TFPIisrestrictedtothealveolar
compartment.Likewise,inotherpreclinicalstudiesoflocaltreatmentwithanticoagulantagents,
rh-activatedproteinC,anti-thrombin,andheparinreducedpulmonarycoagulationwithoutaf-
fectingsystemiccoagulation,however,nebulizeddanaparoidalsoexertedsystemiceffectsonco-
agulation[26,37].Furthermore,localrh-TFPItreatmentdidnotinfluencefibrinolysis,as
reflectedbyunalteredPAI-1orPAAlevelsinlavagefluidandplasma,whichisinaccordance
withearlierreports[13].Interferingwiththeprocoagulantresponseelicitedbyinfectionhas
showninconsistenteffectsonhostdefense.InratmodelsofdirectlunginjuryblockingtheTF
pathwayattenuatedvascularleakage,neutrophilinfluxandlevelsofcytokinesandchemokines
[10,38].However,areducedcoagulantresponsemayunderminehostdefense,aslowTFmice
demonstratedincreasedlunghemorrhagewithconcurrentincreasedinflammationduringacute
lunginjury[39].Inthepresentstudywedidnotobservelunghemorrhageineitherstudygroup
andnebulizationofratswithrh-TFPIdidnotimportantlyaffectinflammationorlungpatholo-
gy,whichwasinlinewithpreviousreportsonpneumococcalpneumoniainrodents[5,12].Fur-
thermore,thrombicouldraiseabarrierforbacteria.Althoughsystemicdisseminationhasbeen
suggestedasapotentialdrawbackofanticoagulanttreatmentinpneumonia[40],localtreatment
withrh-TFPIdidnotinfluencebacterialcountsinthelungorsystemiccompartment.
Largeclinicaltrialshavebeencarriedouttostudytheeffectofrestorationofimpairedanti-
coagulantpathwaysasadjunctivetreatmentinhumansepsis[14–16].Althoughinflammation-
inducedcoagulationwasattenuatedbytreatmentwithanticoagulantagents,thesestudiesdid
notconvincinglyshowabeneficialeffectonoutcome[41].TheOPTIMISTtrial,investigating
rh-TFPIinsepsispatients,suggestedaprotectiveeffectfromrh-TFPIinasubgroupofCAPpa-
tients,especiallywhennottreatedwithheparin[14].TFPIwasfoundtobeinamainlytruncat-
edandinactiveforminlavagefluidofpatientswithARDS[7],andTFPIactivitywasreduced
inpatientswithpneumonia,suggestingrh-TFPImaybeoftherapeuticvalueinthissetting[12,
42].TheensuingCAPTIVATEtrial,specificallydesignedtoinvestigatetheeffectofrh-TFPIin
severeCAP,demonstratedattenuationofcoagulation,howeverfailedtoshowabeneficialeffect
onoutcome[23].Intheseclinicaltrialsanticoagulantagentswereadministeredsystemically,
consequentlyincreasingtheincidenceofbleedingcomplications.Indeed,theOPTIMISTtrial
reportedmoreadverseeventswithbleedinginpatientstreatedwithrh-TFPIthaninplacebo
treatedpatients(24%versus19%)[14],whichmayhavecounterweighedpotentiallyfavorable
effects.Recently,thefirstsmallstudiesofnebulizedheparintreatmenthavebeenconductedin
patientswithARDSandreportinconsistentanticoagulanteffects[18,43,44].
Thereareseverallimitationstoourexperimentalstudy.Thechosendosagefornebuliza-
tionwasbasedondatafrompreviousstudiescombinedwithacalculationoftheefficacyof
PLOSONE|DOI:10.1371/journal.pone.0127261 May18,2015 9/13
Nebulizedrh-TFPIinStreptococcuspneumoniaePneumonia
thenose-onlyexposuresystem[12,13].Importantly,rh-TFPIwasonlydetectableinthepul-
monarycompartmentanddidnotinfluencesystemicTFPIactivity.However,lowerconcen-
trationsofrh-TFPImaysufficeandyieldthesameanticoagulanteffect,whereashigher
treatmentconcentrationsmaybeneededforlocalanti-inflammatoryorantibacterialeffects.
Furthermore,wedidnotinvestigatefunctionalendpoints,suchasalveolargasexchange,nor
theeffectonoveralloutcome.Inourstudyweinvestigatedtheeffectofpre-treatmentwith
localrh-TFPIintheabsenceofantibiotictreatment,whichdoesnotresembletheclinicalsit-
uationandmayelicitdifferenteffects.Forinstance,inamurinemodelofongoingpneumo-
nia,delayedrh-TFPItreatmentinhibitedaccumulationofneutrophilsinlungtissueand
reducedcytokineandchemokinelevels,suggestingthatTF-mediatedcoagulationmightonly
influenceinflammationduringanongoingprocoagulant/proinflammatoryresponse,and
thiseffectbecameonlyapparentwithoutconcurrentantibiotictreatment[13].Assuch,a
pilotphaseIItrialinvestigatingpost-pneumoniatreatmentwithconcurrentantibiotictreat-
mentwouldbenecessarytotranslatetheresultstotheclinicalpractice.Inaddition,itwould
beofinteresttocombinenebulizationofrh-TFPIwithotheranti-inflammatoryandantico-
agulantagentsthatarefrequentlyusedinaclinicalsetting,suchasheparinorcorticosteroids.
Ofnote,alsoduringnon-infectiouslunginjury,forinstanceinducedbymechanicalventila-
tion,pulmonarycoagulationisenhanced[45],whichimpliespatientsmaybenefitfromneb-
ulizationwithrh-TFPIinthissetting.Notably,delayedrh-TFPItreatmentshowedamodest
antibacterialeffectinthelung,andagrowthinhibitingeffectonS.pneumoniaeinthepres-
enceofhumanseruminvitrowasobserved[13].Recentstudiesdescribeantimicrobialactivi-
tyofC-terminalpeptidesoftherh-TFPImoleculeagainstseveralpathogensviathe
complementsystem[46,47].Inaddition,rh-TFPIincombinationwithantibiotictreatment
improvedsurvivalinmicechallengedwithGram-negativebacteria[48].Inourstudypre-
treatmentwithnebulizedrh-TFPIdidnotinfluencebacterialloadsinthelungorplasmain
ourmodel.Therefore,itwillbeofinteresttoinvestigatethetherapeuticvalueofdelayed
treatmentwithnebulizedrh-TFPIorC-terminalderivedpeptides,andtheeffectofconcomi-
tantantibiotictreatment,mimickingtheclinicalsituation.
Conclusions
Inconclusion,theTF-mediatedprocoagulantenvironmentwithinthelungcompartmentdur-
ingpneumoniaprovidesarationaleforlocaltreatmentwithrh-TFPI.Weshowthatlocaltreat-
mentwithrh-TFPIhasclearanticoagulanteffects,whicharerestrictedtothelung
compartment,therebyminimizingtheriskofpotentialharmfuladverseeffects.Finally,local
treatmentwithrh-TFPIdidnotinducelocalbleedings.
SupportingInformation
S1Fig.Nebulizationwithrh-TFPIdoesnotinfluencelungweightorcytokine/chemokine
levelsinlungsinratpneumococcalpneumonia.
(TIF)
AuthorContributions
Conceivedanddesignedtheexperiments:FvdBJJHCvtVTvdPMJS.Performedtheexperi-
ments:FvdBJJH.Analyzedthedata:FvdBJJTHR.Contributedreagents/materials/analysis
tools:MML.Wrotethepaper:FvdBMJS.
PLOSONE|DOI:10.1371/journal.pone.0127261 May18,2015 10/13