Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts

20 Pages·2017·10.24 MB·English

RESEARCHARTICLE
Co-ordinated spatial propagation of blood
plasma clotting and fibrinolytic fronts
AnsarS.Zhalyalov1,MikhailA.Panteleev1,2,3,4,MarinaA.Gracheva2,Fazoil
I.Ataullakhanov1,2,3,4,AlexeyM.Shibeko1*
1 CenterforTheoreticalProblemsofPhysicochemicalPharmacologyRAS,Moscow,Russia,2 National
ScientificandPracticalCentreofPediatricHematology,OncologyandImmunology,Moscow,Russia,
3 DepartmentofPhysics,MoscowStateUniversity,Moscow,Russia,4 FacultyofBiologicalandMedical
Physics,MoscowInstituteofPhysicsandTechnology,Dolgoprudny,Russia
a1111111111 *[email protected]
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a1111111111 Abstract
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Fibrinolysisisacascadeofproteolyticreactionsoccurringinbloodandsofttissues,which
functionstodisintegratefibrinclotswhentheyarenomoreneeded.Inordertoelucidateits
regulationinspaceandtime,fibrinolysiswasinvestigatedusinganinvitroreaction-diffusion
OPENACCESS experimentalmodelofbloodclotformationanddissolution.Clottingwasactivatedbyasur-
Citation:ZhalyalovAS,PanteleevMA,Gracheva facewithimmobilizedtissuefactorinathinlayerofrecalcifiedbloodplasmasupplemented
MA,AtaullakhanovFI,ShibekoAM(2017)Co- withtissueplasminogenactivator(TPA),urokinaseplasminogenactivatororstreptokinase.
ordinatedspatialpropagationofbloodplasma
Formationanddissolutionoffibrinclotwasmonitoredbyvideomicroscopy.Computersys-
clottingandfibrinolyticfronts.PLoSONE12(7):
temsbiologymodelofclotformationandlysiswasdevelopedfordataanalysisandexperi-
e0180668.https://doi.org/10.1371/journal.
pone.0180668 mentalplanning.Fibrinclotfrontpropagatedinspacefromtissuefactor,followedbyafront
ofclotdissolutionpropagatingfromthesamesource.Velocityoflysisfrontpropagationline-
Editor:ValderR.Arruda,Universityof
PennsylvaniaPerelmanSchoolofMedicine, arlydependedonthevelocityclottingfrontpropagation(correlationr2=0.91).Computer
UNITEDSTATES modelrevealedthatfibrinformationwasindeedtherate-limitingstepinthefibrinolysisfront
Received:March14,2017 propagation.Thephenomenonoftwofrontswhichswitchedthestateofbloodplasmafrom
liquidtosolidandthenbacktoliquiddidnotdependonthefibrinolysisactivator.Interest-
Accepted:June19,2017
ingly,TPAathighconcentrationsbegantoincreaselysisonsettimeandtodecreaselysis
Published:July7,2017
propagationvelocity,presumablyduetoplasminogendepletion.Spatiallynon-uniformlysis
Copyright:©2017Zhalyalovetal.Thisisanopen
occurredsimultaneouslywithclotformationanddetachedtheclotfromtheprocoagulant
accessarticledistributedunderthetermsofthe
surface.Thesepatternsofspatialfibrinolysisprovideinsightsintoitsregulationandmight
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and explainclinicalphenomenaassociatedwiththrombolytictherapy.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Introduction
Funding:TheprojectwassupportedbyRussian
FoundationforBasicResearchgrants15-51-15008 Bloodcoagulationandfibrinolysisaretwointerconnectednetworksofproteolyticreactions
and17-04-01309toMAP;byRussianAcademyof
thatcontrolformationanddissolutionoffibrinclots,respectively[1].Boththesenetworksare
SciencesgrantstoFIA(MolecularandCellular
organizedascascadeswithnumerouspositiveandnegativefeedbackloops[2,3].Coagulation
Biology,BasicResearchforDevelopmentof
istriggeredbytissuefactor(TF),atransmembraneglycoproteinexpressedatthesitesofvascu-
BiomedicalTechnologies,MolecularMechanisms
ofPhysiologicFunctions)andtoMAP(Basic larinjury.BindingofcirculatingserineproteasefactorVIIatoTFturnsitintoafunctional
PLOSONE|https://doi.org/10.1371/journal.pone.0180668 July7,2017 1/20
Spatialdynamicsoffibrinolysis
ScienceforMedicine);byGrantofPresidentof enzyme,whichstartscoagulationcascadeultimatelyleadingtotheformationofthrombinthat
RussianFederationMK-9245.2016.4toAMSand convertsproteinfibrinogenintofibrinthatpolymerizestoformbloodclot.
MD-229.2017.4toMAP.Theoreticalmodellingwas
Whentheclotisnolongerneeded,itisproteolyticallydegradedbyanotherproteaseplas-
supportedbygrantfromRussianScience
minproducedbytheactionoffibrinolyticnetwork.Thissmallercascadecanbeinitiatedby
Foundation16-14-00-224toFIA(sections
eithertissueplasminogenactivator(TPA)releasedbythevascularwallorurokinaseplasmino-
Computationalmodeloffibrinclotgrowthandlysis
andComputersimulationofspatialclotgrowthand genactivator(UPA)presentinaprecursorforminblood[4].Acriticaltriggerandcofactor
lysis;Figures6and7).Thefundershadnorolein fortheiractionisfibrinitselfthatnotonlyprotectsplasminfrominactivation[5],butalso
studydesign,datacollectionandanalysis,decision acceleratesactionofTPAbyanorderof500[6].
topublish,orpreparationofthemanuscript.
Anessentialpropertysharedbycoagulationandfibrinolysisisthatfunctioningofthesesys-
Competinginterests:MAPandFIAarefoundersof temsisspatiallyheterogeneous:enzymesformedinoneplacearetransportedbydiffusionand
HemacoreLLC.Thisdoesnotalterouradherence flowofthefluidtoanotherlocation[7].Moreover,thetaskitselfisspatial:clotsshouldbe
toPLOSONEpoliciesonsharingdataand
formedstrictlyatthesiteofinjury,andtheirdissolutionalsohastobecontrolled[8–10].This
materials.
appearstobeacrucialpointfortheirregulation:therearemanyexamplesthatrolesofindivid-
ualreactionschangeinspaceandtime[11,12],orinthepresenceofflow[13,14].Thereare
somereportsonthecriticalimportanceofdiffusion-controlledprocessinfibrinolysis[15],but
thissystemisunderstoodnotasgoodasclotting.Therearenoreportsonsensitivityanalysis
forfibrinolysis(evenwithouttransportprocess)similartotheonesthatareabundantinclot-
ting[3,16,17],and,ingeneral,themeaningofindividualcomponents,feedbacks,etc.infibri-
nolysisinrespecttoitsspatialdistributionstillremainsnotcompletelyclarified.
Theobjectiveofthepresentstudywastofillthisgapbyinvestigatingspatialfibrinolysis.
Weusedavideomicroscopicreaction-diffusioninvitroexperimentalmodeltomonitorblood
clottinginitiatedbyimmobilizedTF[18–21]inbloodplasmasupplementedwithplasminogen
activator.Acomputationalsystemsbiologymodelofclotformationanddissolutionwasdevel-
opedforanalysisandinterpretationofexperimentaldata.Theobtainedresultssuggestanew
conceptoffibrinolysisregulation,wherethecriticalrate-limitingelementisformationoffibrin
itself.
Materialsandmethods
Materials
Streptokinase(SK)fromStreptococcus(PNS3134)waspurchasedfromSigma(SaintLouis,
MO).TPA(Actilyse)wasfromBoehringer(Ingelheim,Germany).UPAwasfromAmerican
Diagnostica(Stamford,CT).Thrombodynamicsassaykit(HEPES,corntrypsininhibitor
(CTI),Ca(CH COO) TF-bearingsurface)wasfromHemacore(Moscow,Russia).Human
3 2,
recombinantTF(Recombiplastin2G,InstrumentationLaboratoryCompany,Bedford,MA,
USA)wasimmobilizedbyHemacoreLLCusingthemethodthatwasdescribedin[22]with
modifications.Heparin(unfractionatedheparin)wasfromFerane(Moscow,Russia).Acti-
vatedfactorXIwasfromHaematologicTechnologies(EssexJunction,Vermont).Phospholip-
ids(Phospholipid-TGT)werefromRossix(Mo¨lndal,Sweden).
Wholebloodandplasmapreparation
Plasmawaspreparedfromthewholebloodofhealthyvolunteersandpatientsandcollected
withtheapprovaloftheethicscommitteesoftheCenterforTheoreticalProblemsofPhysico-
chemicalPharmacologyandtheNationalScientificandPracticalCentreofPediatricHematol-
ogy,OncologyandImmunology.Bloodwasdrawninto3.8%sodiumcitrate(pH5.5)ata9:1
blood/anticoagulantvolumeratio.Bloodwascentrifugedat100×gfor5minandthesuperna-
tantwascollectedtoobtainplatelet-richplasma(PRP).Plateletcountwasmeasuredandthen
PRPwasdilutedwithplateletfreeplasma(PFP)tosetplateletcountto300,000platelets/μl.
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Bloodwascentrifugedat1600×gfor15min,andthesupernatantwascollectedtoobtain
platelet-poorplasma(PPP).Supernatantwascentrifugedagainat10,000×gfor5minto
obtainPFP.PPPandPFPwerepooledandfrozenat-80˚C.Poolsofplasmafrom5donors
eachwerepreparedandusedintheexperiments.
Cellculture
AhumanfetallungfibroblastlinewasobtainedfromtheIvanovskiiResearchInstituteof
Virology(Moscow,Russia).Thepolyethyleneterephthalatefilmswithfibroblastmonolayer
werepreparedasdescribedpreviously[18,20].Thedensityforcellmonolayerswasapproxi-
mately1000cells/mm2,anditsvariation(SD)didnotexceed10%amongeitherthedifferent
filmsforeachseriesordifferentportionsofeachfilm.TheaveragedensityoffunctionalTFon
thefilmswasmeasuredusingtheActichrome-TFkit(SekisuiDiagnostics,Lexington,MA,
USA)anditwas140±15pmole/m2.
Patient
BE,male,1.5yo,withjuvenilemyelomonocyticleukemiaexhibitedanocclusivecatheter-asso-
ciatedthrombosisoftheleftiliacvein,wastreatedwithTPA0.03mg/kg/handheparin10IU/
kg/h.DetaileddescriptionofthepatientcanbefoundinS1Appendix.
Spatialclotgrowthandlysisexperiments
Clotgrowthandlysisexperimentswereperformedusingthevideo-microscopydeviceand
imagecollectingsoftwarefromtheThombodynamicsassayasdescribedin[21]withmodifica-
tions.Frozenplasmawasthawedat37˚Cwithin30minutes;thenitwassupplementedwith
HEPESat30mmol/LtostabilizepHat7.4duringtheexperimentandCTIat0.2mg/mlfinal
concentrationtoinhibitcontactactivation.The15minutesincubationofthesampleat37˚C
wasfollowedbysupplementationwithplasminogenactivator(TPA,UPAorSK).Thesample
wasrecalcifiedbyadditionofCa(CH COO) ,20mmol/Lfinalconcentration,thenitwas
3 2
placedintothemeasuringcuvette.ClottingwasinitiatedwiththeTF-bearingsurface,inserted
inthecuvette.Lightscatteringsignal(625nm)fromfibrinclotwascapturedwithaCCD
camera.
Dataprocessing
Duringthespatialclotgrowthandlysisexperiments,weobtainedasetofmicrophotographs
ofthefibrinclot(Fig1).Asitwasshownpreviously[23]lightscatteringwasproportionalto
fibrinconcentration;thesamelineardependencewasestablishedforourexperimentalsetup
[24],thuswecouldtreattheintensityofthesignalastherelativefibrinconcentration.Asthe
lightscatteringdependsalsoonfiberthickness[25],whichinitsturndependsonthethrombin
concentration,wemayassumethatinourexperimentalsetupthefibrinclotstructureisuni-
form,duetothrombinwhichisgeneratedasapropagatingwavewithpeakconcentration
about50nM[11],makinglightscatteringsignaldependmainlyonfibrinconcentration.We
averagedthelightscatteringsignalinahorizontaldirection(60–80pixelswidth)(Fig1A1,yel-
lowrectangle),obtainingitsdependenceontheverticalcoordinateforeachmomentoftime
(Fig1A4and1B4).Inordertoseparatefibrinclotfromtheliquidplasma,weintroduceda
thresholdlevelofsignal(i.e.fibrinconcentration),abovewhichweconsideredfibrinclottobe
formed.Wemeasuredthemaximalsignalvalueobtainedintheexperiment,andsupposed
thatthefibrinclotwas“formed”atthecertainlevelofthemaximalconcentration.Inallour
experimentsthemaximalsignallevelwasalmostthesame,soweconsideredthesameamount
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Spatialdynamicsoffibrinolysis
Fig1. Picturesoffibrinclotgrowthintheabsenceofplasminogenactivators(2(A1),10(A2)and20(A3)minutesaftertheclottingonset)and
clotgrowthandlysisinthepresenceof30nmol/LofTPA(2(B1),10(B2)and20(B3)minutesaftertheclottingonset).Yellowrectangleonthe
panelA1showstheregionofthedatacollectionforprocessing.Thescalebaris1mmlong.Spatialdistributionoffibrinintheabsenceof
plasminogenactivators(A4)orinthepresenceof30nmol/LofTPA(B4)showsclotpropagationandsimultaneousclotgrowthanddissolution,
respectively.Black,red,andbluelinesshowspatialdistributionoflightscatteringsignal(proportionaltofibrinconcentration)at2nd,10th,and
20thminuteafterinitiationofcoagulation,respectively.Whenthesignalexceededthethresholdlevelinanyarea,weconsideredthattheclot
appearedthere,andwhenthelevelofsignaldecreasedbelowthisthreshold,theclotdissolved.Coordinatesoftheseeventsweredesignated
asfrontsofclotgrowthandlysis.Timecourseofclotgrowthfront(A5)orclotgrowthandlysisfronts(B5)allowstocalculatethevelocitiesof
clotgrowthorlysisastheaveragevelocityofgrowthorlysisfrontpropagationwithinthefirst5minutesaftertheonsetoftheprocess.Inorderto
dothatweuseditslinearapproximationwithinthefirst5minutesaftertheclotting(lysis)lagtime.Clotting(lysis)lagtimewascalculatedasthe
timewhenclotting(lysis)frontcoordinatestartedtoincrease.
https://doi.org/10.1371/journal.pone.0180668.g001
offibrintoformtheclot.Assoonasthisthresholdwasexceededinanyarea,weconsideredthat
theclotappearedthere,andwhenthelevelofsignaldecreasedbelowthisthreshold,theclotdis-
solved.Wedeterminedthecoordinatesofthesetwoeventsforeachtimestepandplottedthem
overtime(Fig1A5and1B5)toestimatethelagtimeofclotting(lysis)andthevelocityofclot
growth(lysis).Theclotting(lysis)lagtimewasdeterminedasthetimepassedsincetheactiva-
tionofcoagulationuptothemomentwhentheclotgrowth(lysis)frontstartedtomove.Fig15
showsthatthevelocityofclotgrowth(lysis)frontpropagationdecreasedovertime;tocharac-
terizeitweuseditslinearapproximationwithinthefirst5minutesaftertheclotting(lysis)lag
time.Wecompareddifferentthresholdlevels(10%-50%frommaximalsignal,FigureAinS1
Appendix)andreceivedverysimilarresults;wechosethethresholdlevelofclotformationas
20%fromthemaximallevelandusedittoprocessalldata,bothinvitroandinsilico.
Statisticalanalysisofexperimentaldata
WeusedMann-Whitneytestwiththeleveloferrorp=0.05toexaminethedifferencesindata
obtainedunderdifferentconditions.
Computationalmodeloffibrinclotgrowthandlysis
Computationalsystemsbiologymodelofbloodclotformationanddissolutionwasdesigned
toreproducetheexperimentaldesignofFig1.ThemodelarrangementisshowninFig2.
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Spatialdynamicsoffibrinolysis
SimulationswithComsol4.3(Comsol,Burlington,MA)wereperformedina1-dimensional
regionthatwas3mmlong.Clotgrowthwasinitiatedbytissuefactorlocatedatthex=0.TPA
wasevenlydistributedintheareaofsimulation(FigureDinS1Appendix).
Modelequationswerereaction-diffusionequationsbasedonthelawsofMichaeliskinetics
andofmassactionfortheschemeinFig2D.Adetaileddescriptionofthemodelequations
canbefoundinS1Appendix(equationsS1-S58).Modelparametersweremostlyfromexperi-
mentsandaregiveninTablesH,IandJinS1Appendix.
The"coagulation"partwasbasedonacomputationalmodelofone-dimensionalspatialclot
growthpreviouslydevelopedandvalidatedbyourgroup[17,18].Briefly,thispartincluded
severaldistinctmodules:a)binding/dissociationofVIIa/VIItoTFandfeedbackactivationof
Fig2. Spatialkineticsoffibringenerationintheabsence(A)orinthepresence(B)of50nmol/LTPA.Spatialfibrindistributionisshownfor10th(blackline),
30th(blueline)and60th(redline)minuteofsimulation.(C)Timecourseofclotgrowth/lysisfrontduringsimulation.(D)Schemeofbloodcoagulationcascade
mainreactions.Zymogensareshownasbluecircles,activatedproteinsareshownasyellowcircles.Inactivecofactorsareshownasbluerectangles,
activatedcofactorsareshownascyanrectangles.Redarrowsshowactivation,blackarrowsshowtransitionfrominactivetoactiveform,andformationof
complexes.Greenarrowsshowinhibition.Doublearcshowsphospholipidsurfacethatisrequiredforcomplexformationoractivation.PgAstandsfor
plasminogenactivator;FDPstandsforfibrindegradationproducts.
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VII;b)activationofXandIXbyVIIa-TF,c)activationofIIbyXa;d)feedbackstimulationof
VandVIIIandXIbythrombin;e)enhancedactivationofIX,X,IIbyXIa,IXa-VIIIa,Xa-Va
respectively;f)actionofallmajorstoichiometricinhibitorsandnegativefeedbackofthepro-
teinCpathwaythatdestroyedactivefactorsVaandVIIIa.Thevariablesoftheofthemodel
weretotalconcentrationsoffactorsVIIa,VII,IXa,IX,Xa,X,IIa,II,VIIIa,VIII,Va,V,XIa,XI,
zymogenproteinC,activatedproteinC,Xa-TFPI,TFPI,AT-III,fibrinogen,fibrin.Tomimic
surfaceactivation,weusedsurfacedensitiesofTF,VIIa-TF,andVII-TF,andcouplessurface
reactionswiththevolumereactionsusingboundaryconditions.
The"fibrinolytic"partofthemodeliscompletelynovel.Thevariablesincludetotalconcen-
trationsoffreeTPA,plasminogenintheglu-andlys-forms,plasmin,alltheminthefibrin-
boundform,andalsoa2M,a2AP,andPAI-1.Fibrinolysisistriggeredbybindingofplasmino-
genandTPAtofibrinleadingtoplasminformationandfibrindegradation.
Themajormodelassumptionsinclude:a)equlibriumbindingofallmembranecomplexes
[26];b)platelet-derivedmicroparticlesandlipoproteinsprovidemostofthemembranesurface
inplasma[27];c)nofeedbackformationoftwo-chainTPA.;d)fibrinformationanddestruc-
tionareassumedtobesimpleone-stagereactions;e)wedonotconsiderThrombinActivata-
bleFibrinolysisInhibitor(TAFI)-andfactorXIII-dependentfeedforwardreactions.
AtypicalequationofsurfacereactionsforTF-mediatedinitiationofclotting:
ds kVII(cid:0) TF;IIa(cid:1)s (cid:1)½IIaF(cid:138)j
VII(cid:0) TF ¼kVII;TF(cid:1)½VII(cid:138)j (cid:1)s (cid:0) kVII(cid:0) TF(cid:1)s (cid:0) cat VII(cid:0) TF x¼0(cid:0) kVII(cid:0) TF;Xa
dt a x¼0 TF d VII(cid:0) TF KVII(cid:0) TF;IIaþ½IIaF(cid:138)j eff
M x¼0
(cid:1)s (cid:1)½XaF(cid:138)j ð1Þ
VII(cid:0) TF x¼0
Ontheleft,therateofinactiveextrinsictenase(σ )concentrationchangewithtimeis
VII–TF
shown.Accordingtotheequation,itisdeterminedbytwoprocessesgivenontheright:1)
associationanddissociationofTFwithFVII(thefirstandthesecondterms);2)activationof
inactiveextrinsictenasebythrombin(thethirdterm)andFXa(thefourthterm).
Atypicalequationofvolumereactions:
d½IIa(cid:138) @2½IIaF(cid:138) klocalII;Xa(cid:0) Va (cid:1)½Xa(cid:0) VaB(cid:138)(cid:1)½IIB(cid:138)
¼D (cid:1) þkII;Xa(cid:1)N (cid:1)½XaF(cid:138)(cid:1)½II(cid:138)þ cat (cid:0) ðkIIa;AT(cid:0) III
dt IIa @x2 eff a KlocalII;Xa(cid:0) Va=k(cid:1)N a
M a
(cid:1)½AT (cid:0) III(cid:138)þkIIa;a2M(cid:1)½a M(cid:138) þkIIa;a1AT (cid:1)½a AT(cid:138) þkIIa;PCI(cid:1)½PCI(cid:138) Þ(cid:1)½IIaF(cid:138)ð2Þ
a 2 0 a 1 0 a 0
Ontheleft,therateofthrombinconcentrationchangewithtimeisshown.Accordingto
theequation,itisdeterminedbythreeprocessesgivenontheright:1)diffusionofthrombin
(thefirstterm);2)activationofprothrombinbyFXa(thesecondterm)orprothmobinase
complex(thethirdterm);3)inhibitionofthrombinbyantithrombin,a2-macroglobulin,
alpha1-antithrypsinandproteinCinhibitor(thefourthmember).
Surfacereactionsgenerateaninwardfluxofactivatedfactors,whichissetasboundarycon-
ditionforthevolumereactions.Basedontheknownconcentrationsofallparticipantsofthese
reactionsatsometime-pointandonthekineticconstants,wecalculatefurtherdynamicsof
coagulationusingasetofequations(S1Appendix).
Results
Spatiallysisoffibrinclotformedinthepresenceofplasminogen
activatorsstartsfromtheareaofclottingactivation
Spatialdynamicsofclotformationinplasmafromhealthyvolunteers[28,29]orpatientswith
hemophiliaA[28]orsepsis[21]showedthatnoclotlysisoccurredwithinthetimeofobserva-
tion(upto1hour)intheabsenceoffibrinolysisactivators.Exvivospatialclotgrowthinthe
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PFPfromthepatientunderfibrinolytictherapywithrecombinantTPA(Actilyse,Boehringer
Ingelheim,Germany)wasaccompaniedbyasimultaneousclotlysis:thefrontoflysispropaga-
tionappearedontheclottingactivationsurfaceandfollowedclotgrowth(S2Video,Fig3A).
Whilepreviousstudiesinvestigatedexternalspatiallysis[30,31],oursetupwasfocusedon
internalspatiallysis,whenclottingandlysiswereactivatedsimultaneously.Weattemptedto
obtainthisphenomenoninasimpleandreproduciblemodelofspatialclotlysis,wherewe
couldsetanyinitialconditions.WewereabletoobserveitinPFPfromhealthyvolunteers,
supplementedwithTPAinvitro(S3Video,Fig3B).
Inordertoinvestigatewhetherthisphenomenonwouldpersistundermorephysiological
conditions,wealsoperformedtheseexperimentsinplatelet-richplasma(PRP,300,000plate-
lets/μl)andfoundtheverysamepatternofspatialclotlysis(althoughthevelocitiesofclot
growthandlysispropagationwerehigherinPRP)(S4Video,Fig3C).Alternatively,weacti-
vatedclottinginPFPwithaconfluentlayeroffibroblasts(S5Video,Fig3D)andobservedthe
samespatiallysis(alsowithincreasedvelocitiesofbothclotgrowthandlysis).Finally,the
sameresultwasinPRPwhereclottingwasactivatedwithfibroblasts(FigureBinS1Appendix,
S6Video).Basedonthesedata,allfurtherresearchwasconductedinPFPwithimmobilized
TFclottingactivation,asthissetupwasthemostreproducibleandstraightforward.
Characterizationofspatialclotgrowthandlysis
FibrinclotgrowthintheabsenceofplasminogenactivatorstartedinthevicinityoftheTF-
bearingsurfaceandpropagatedfromitinthebulkofplasma(Fig1A1–1A3).Fig1A4shows
Fig3.Picturesoffibrinclotgrowthandlysisinbloodplasma5,15and30minutesafterthestartofexperiment.Greyrectangleonthe
topofeachimageisaninsetwithimmobilizedTF.Fibrinclotiswhite,whileliquidplasmaisblack/darkgrey.(A)PFPfromapatientunder0.03
mg/kg/hTPAtherapy.(B)PooledPFPfromhealthyvolunteerssupplementedwith14nmol/LTPAinvitro.(C)PRPfromhealthyvolunteer
supplementedwith14nmol/LTPAinvitro.(D)PooledPFPfromhealthyvolunteerssupplementedwith14nmol/LTPAinvitro,clottingwas
initiatedwithaTF-bearingfibroblastsconfluentmonolayer.Clotgrowthandlysisweremonitoredinplasma,treatedasdescribedinMethods
section.Individualexperimentsareshown.
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Spatialdynamicsoffibrinolysis
thedependenceoffibrinclotlightscatteringintensityonthedistancefromtheclottingactivat-
ingsurface.Clotgrowthischaracterizedbyasteepfrontofthesignalchange,whichmovesin
timewiththegraduallydecreasingspeed(Fig1A5).Inthepresenceof30nmol/LTPA(Fig
1B1–1B3),clotgrowthstartedsimilarlybutwasfollowedbythedecreaseofthelightscattering
signal,whichstartedfromthesamesiteasthegrowthdid,andpropagatedinthesamedirec-
tion.Weusedclottingandlysislagtimes(seeMaterialsandMethods)todeterminetheonset
ofclottingandlysis;clottingandlysispropagationvelocitiestodeterminetheinitialspatial
aspectsofclottingandlysis.
Threeregimesofspatiallysis
Inordertodeterminethedosedependenteffectsofspatialclotlysisparameters,wespiked
plasmawithincreasingconcentrationsofTPA.WefoundthatTPAdoseescalationcauseddif-
ferentscenariosofspatiallysis:
1. clotlysisstartedabout200μmawayfromtheclottingactivator,and2frontsoflysispropa-
gatedinoppositedirections(TPA6nmol/L(Fig4A,FigureCinS1Appendix)).For6
nmol/LofTPAthechanceofthiseventwas100%(n=4),for14nmol/LofTPAitwas73%
(n=19),for30nmol/LofTPAitwas11%(n=18);forTPAconcentrationshigherthan30
nmol/L,clotlysisalwaysstartedattheTF-bearingsurface.
2. clotlysisstartedattheTF-bearingsurfaceandthelyticfrontmettheclottingfrontandthe
clotwascompletelydissolved(TPA100nmol/L(Fig4B));
3. clotlysisstartedattheTF-bearingsurfaceandstoppedatsomedistancefromit,whilethe
clotgrowthcontinued(TPA800nmol/L(Fig4C)).
Shapeofspatiallysisfrontpropagationdoesnotdependon
plasminogenactivatortype
Wechoseconcentrationofplasminogenactivatorstofittherangeusedduringfibrinolytic
therapy(TPA50nmol/L(60nmol/Lforacutemyocardialinfarction[32]),UPA30nmol/L
(180IU/ml[33]),SK1200nmol/L(200IU/ml[34])).Thepatternofclotlysisfrontpropagation
forUPA(Fig5A)wassimilartotheoneforlowTPAconcentration(Fig4A):lysisstarted
about100μmawayfromtheTF-bearingsurfaceandtwofrontsoflysispropagatedfromthe
pointofonset,towardstheclottingactivatorandawayfromit.Thesingle-chainUPAthatwe
usedhadlimitedactivitytowardsplasminogen,whichcanexplainthisresult.Thepatternof
Fig4.Threeregimesofspatialclotlysis.PooledPFPfromhealthyvolunteerssupplementedwith
increasingconcentrationofTPA.Clotlysisstarted200μmawayfromtheclottingactivatingsurfaceinthe
presenceof6nmol/LTPA(A);completeclotdissolutionwasobservedinthepresenceof100nmol/LTPA(B);
spatialclotlysisstopped300μmawayfromclottingactivatingsurfaceinthepresenceof800nmol/LTPA(C).
Clotgrowthandlysisweremonitoredinfreshfrozennormalpooledplasma,treatedasdescribedinMethods
section.Individualexperimentsareshown.
https://doi.org/10.1371/journal.pone.0180668.g004
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Spatialdynamicsoffibrinolysis
Fig5. Kineticsofclotgrowthandlysisfrontscoordinateinthepresenceof30nmol/LUPA(A)or1200nmol/LSK(B).Spatialclotlysislagtime
andvelocitydependedonthetypeofplasminogenactivator,whilespatialclotgrowthdidnotchangecomparedtocontrol.Thelagtimesoflysis
(C)weresignificantlydifferentforallplasminogenactivators(2.4±0.3minforSK,3.4±0.2minforTPAand11±1minforUPA).Thevelocityoflysis
(D)wasthesameforUPA(48±4μm/min)andSK(44±3μm/min),butitwasabout1.5timeshigherforTPA(78±5μm/min).Clotgrowthvelocity
andlagtimewerenotsignificantlydifferentfromcontrol(74±2μm/minand0.77±0.14min,respectively).Clotgrowthandlysisweremonitoredin
freshfrozennormalpooledplasma,treatedasdescribedinMethodssectionandsupplementedwithvehicle(control,n=7),50nmol/LTPA
(n=9),30nmol/LUPA(n=7)or1200nmol/LSK(n=7).
https://doi.org/10.1371/journal.pone.0180668.g005
clotlysisforSK(Fig5B)wassimilartohighTPAconcentration(Fig4C):lysisstoppedatsome
distancefromtheclottingactivatorwhiletheclotgrowthcontinued.
PlasmasupplementedwithSKorUPAgeneratedfibrinclotsjustlikethecontrol,andthere
wasnosignificantdifferenceinclotgrowthvelocityorlagtimebetweendifferenttypesofplas-
minogenactivators(Table1,Fig5C&5D).Thelagtimeofclotlysiswas2.4±0.3minforSK,
3.4±0.2minforTPAand11±1minforUPA.Thevelocityofspatialclotlysiswas43.7±3.2μm/
minforSK,48.4±4.7μm/minforUPA(nosignificantdifference),butforTPAthevelocitywas
significantlyhigher:78.7±4.8μm/min.Despitesomedifferencesintheplasminogenactivator’s
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Spatialdynamicsoffibrinolysis
Table1. Parametersofclotgrowthandlysisintheabsenceandpresenceofthrombolytics.
Lysisactivator Clotgrowth Clotlysis
Lagtime(min) Velocity(μm/min) Lagtime(min) Velocity(μm/min)
None 0.8±0.1 73.6±2.1 - -
UPA(30nmol/L) 0.8±0.1 69.4±3.5 11±1 48.4±4.7
SK(1200nmol/L) 0.7±0.2 70.1±4.9 2.4±0.3 43.7±3.2
TPA(6nmol/L) 0.7±0.1 73.9±4.2 22.3±1.8 55.3±3.4
TPA(50nmol/L) 0.7±0.1 75.2±5.6 3.4±0.2 78.7±4.8
TPA(200nmol/L) 0.6±0.1 78±3 2.0±0.2 77.3±4.4
TPA(800nmol/L) 0.7±0.1 76.6±4.3 2.5±0.4 41.2±5.6
https://doi.org/10.1371/journal.pone.0180668.t001
activities,allofthemgeneratedspatiallysisfrontpropagationandnotaffectingtheinitiation
ofclotting.
TPAconcentrationdeterminestheonsetoflysisanditsspatialvelocity
ThelagtimeofspatialclotgrowthdidnotchangewithTPAdoseescalation(Table1,Fig6A).
Thevelocityofclotgrowthwas73–78μm/minwithintheTPAconcentrationrangeof0–800
nmol/L(nosignificantdifference)(Table1,Fig6B).Thus,clottinginitiationwasnotaffected
byTPA.
Thelagtimeofclotlysisdecreasedfrom22±2minforTPA6nmol/Ldownto2.0±0.2min
forTPA200nmol/Landslightlyincreasedupto2.5±0.4minforTPA800nmol/L(signifi-
cantlydifferent)(Fig6A).Thespatialclotlysisvelocityincreasedfrom55±3μm/minforTPA
6nmol/Lupto75–78μm/minfortheTPAconcentrationrangeof30–200nmol/Land
decreaseddownto41±5μm/minforTPA800nmol/L(Fig6B).
Computersimulationofspatialclotgrowthandlysis
SpatialkineticsoffibrinformationintheabsenceofTPAwassimilartotheoneweobserved
invitro(Fig2).Inoursimulations,clottinglagtimewas2.5minutes(0.77±0.14mininvitro)
andclotgrowthvelocitywas77μm/min(74±2μm/mininvitro).Itmaymeanthatourunder-
standingofclottinginitiationthatwasembodiedinthecorrespondingpartofthemodel
requiresfurtherimprovement.Inaddition,weneedtonotethatascommerciallyavailable
immobilizedTFwaspoorlycharacterized,theexactconstantsofclottinginitiationmaydiffer
fromthatweobtainedfromliterature.However,thisdiscrepancyinsimulationsandinvitro
datahadminimalimpactonthesubjectofthiswork.
Wefoundthatspatialclottinginplasmasupplementedwithplasminogenactivatorgener-
atedtwowavesthatswitchedplasmafromliquidstatetosolidstateandbacktoliquid(FigureE
inS1Appendix).Thewaveofthrombincausedfibringenerationwhichstartedonthevery
frontofitandgelatedplasma.Thewaveofplasmincausedfibrindegradationwhichliquefied
plasma.Theinitiationsignal(TF)waslocatedontheboundaryoftheareaandthesewaves
werepropagatinginaself-sustainingmanner,whichmeantthatplasminogenactivatorsupple-
mentedplasmawasanactivemediumsupportingtwodifferentautowave-likemodes.
Simulations(Fig6A&6B)showedgoodcorrelationwiththeexperimentaldata.Spatialclot
lysislagtimedecreasedfrom74.5minforTPA4.5nmol/Ldownto13minforTPA400nmol/
Landincreasedupto19.5minforTPA1600nmol/L(Fig6A),showingthesamedependence
oflysislagtimeonTPAconcentrationasinvitro.Thevelocityofspatialclotlysisincreased
from50μm/minforTPA4.5nmol/Lupto60μm/minforTPA15nmol/L,slightlydecrease
withintherangeofTPAconcentrationof15–400nmol/L.Thevelocitydecreaseddownto
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