Fibrin Affinity of Erythrocyte-Coupled Tissue-Type Plasminogen Activators Endures Hemodynamic ...

261 Pages·2009·16.31 MB·English

0022-3565/06/3163-1130–1136$20.00
THEJOURNALOFPHARMACOLOGYANDEXPERIMENTALTHERAPEUTICS Vol.316,No.3
Copyright©2006byTheAmericanSocietyforPharmacologyandExperimentalTherapeutics 93450/3080908
JPET316:1130–1136,2006 PrintedinU.S.A.
Fibrin Affinity of Erythrocyte-Coupled Tissue-Type Plasminogen
Activators Endures Hemodynamic Forces and Enhances
Fibrinolysis in Vivo
Kumkum Ganguly, Mukul S. Goel, Tatyana Krasik, Khalil Bdeir, Scott L. Diamond,
Douglas B. Cines, Vladimir R. Muzykantov, and Juan-Carlos Murciano
InstituteforEnvironmentalMedicine(K.G.,T.K.,V.R.M.,J.-C.M.),InstituteforMedicineandEngineering(M.S.G.,S.L.D.),and
DepartmentsofLaboratoryMedicine(K.B.,D.B.C.)andPharmacologyandInstituteforTranslationalMedicineand D
o
Therapeutics(V.R.M.),UniversityofPennsylvaniaSchoolofMedicine,Philadelphia,Pennsylvania;andCentroNacionalde w
n
InvestigacionesCardiovasculares(J.-C.M.),Madrid,Spain lo
a
d
ReceivedJuly28,2005;acceptedNovember10,2005 e
d
fro
m
jp
e
ABSTRACT t.a
s
Plasminogenactivators(PAs;e.g.,tissue-type,tPA)coupledto inlungs,whichresultedinamoreextensivefibrinolysisversus p
e
redbloodcells(RBCs)displayinvivofeaturesusefulforthrom- RBC/rPA(p(cid:1)0.01).Reconcilingthisapparentdivergencebe- tjo
boprophylaxis: prolonged circulation, minimal extravasation, tweeninvitroandinvivobehaviors,RBC/tPA,butnotRBC/rPA urn
and preferential lysis of nascent versus preexisting clots. Yet, perfusedoverfibrininvitroatphysiologicalshearstressbound a
ls
factorscontrollingtheactivityofRBC-boundPAsinvivoarenot to fibrin clots and caused greater fibrinolysis versus RBC/rPA .o
defined and may not mirror the profile of soluble PAs. We (p (cid:1) 0.001). These results indicate that because of high fibrin arg
testedtheroleofRBC/PAbindingtofibrininfibrinolysis.RBC/ affinity, RBC/tPA binding to clots endures hemodynamic t U
tPAandRBC/tPAvariantwithlowfibrinaffinity(rPA)boundto stress, which enhances fibrinolysis. Behavior of RBC/PAs un- n
iv
and lysed plasminogen-containing fibrin clots in vitro compa- der hemodynamic pressure is an important predictor of their e
rs
rably. In contrast, when coinjected in mice with fibrin emboli performanceinvivo. ity
lodging in pulmonary vasculature, only RBC/tPA accumulated o
f P
e
n
n
s
y
lv
a
n
ia
L
Ineffectivedeliveryandpermeationofplasminogenactiva- phylactic agents. RBC carriage 1) markedly prolongs the ib
tors(PAs)intoclotslimitthethrombolyticutility(Holvoetet intravascular circulation of PAs, permitting their inclusion rary
al., 1993; Sakharov and Rijken, 1995). This problem cannot into nascent clots followed by rapid fibrinolysis from within on
besolvedbyescalatingthedosebecauseofanuntowardrisk (“Trojan horse lysis”) (Murciano et al., 2003; Ouriel, 2003); M
a
ofcollateraldamage(e.g.,bleeding).Currentmeanstomod- and 2) precludes the penetration of PAs into existing hemo- y 1
ipfhyaPrmAsachoakvineehtiacds(rteylpaitcivaelllyy(cid:1)li1m-hitecdircimulpaaticotnoinntthheeirblcoloindi)coarl sintagtaicncdloctosllaantedralilmdiatmseaxgterainvatshaeticoenn,trtahlunsemrvinouimsiszyisntgembleaendd- 2, 20
0
theirpropensitytoextravasate,especiallyintodamagedtis- 9
probably other tissues. RBC-coupled tPA (RBC/tPA) circu-
suessuchasthecentralnervoussystem,whichcausesmor-
lates in mice and rats for many hours without harm to the
bidity(Tsirkaetal.,1995;Wangetal.,1998).
carrier RBC or without causing bleeding, while selectively
The use of red blood cells (RBCs) as drug carriers might
dissolving nascent clots resistant to even higher doses of
help solve this problem by converting PAs into thrombopro-
solubletPA(Murcianoetal.,2003).
Given this potential to change how PAs act and can be
ThisstudywassupportedbyAmericanHeartAssociationBugher-Stroke
Award,PENNResearchFoundationAward,andNationalInstitutesofHealth used,itisimportanttodeterminehowbiochemicalproperties
Grants R01 HL66442 (to V.R.M.) and HL60169 (to D.B.C.). J.-C.M. was a andinvivobehaviorofPAsarealteredwhentheyarelinked
recipient of Ramo´n y Cajal Foundation Award and grants from Fondo de
toRBCsandhowtopredictforsuchchanges.Previousinvivo
Investigaciones Sanitarias PI040961 and Comunidad de Madrid GR/SAL/
0325/2004. studies show that RBC carriage overrides differences in
Article, publication date, and citation information can be found at pharmacokineticsamongsolublePAs(Gangulyetal.,2005).
http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.093450. Thus,solubletPAisclearedfromthebloodmorerapidlythan
ABBREVIATIONS: PA, plasminogen activator; RBC, red blood cell; tPA, tissue-type plasminogen activator; rPA, Retavase; Pg, plasminogen;
NC-Pg, noncleavable plasminogen; FDP, fibrin degradation product; PPACK, D-Phe-Pro-Arg-CMK, HCl; PBS, phosphate-buffered saline; ME,
microemboli;RT,roomtemperature;PAI-1,plasminogenactivatorinhibitor-1.
1130
FibrinAffinityofRBC/tPAEnhancesFibrinolysis 1131
rPA(Retavase),atruncatedtPAvariantlackingthegrowth batedfor20minat4°CinplasticwellscontainingPg-depletedfibrin
factor(G),finger(F),andfirstkringle(K1)domains(Kohnert clotspreparedbycoagulatinga0.5-mlsolutionof3mg/mlfibrinogen.
etal.,1992).Yet,bothRBC/tPAandRBC/rPAcirculateforat RBC/PA binding was determined by adding water and measuring
leastmanyhoursinanimals(Murcianoetal.,2003;Ganguly hemoglobin absorbance at 405 nm in lysates. In some wells, fibrin
et al., 2005). Previous studies also revealed that coupling to clotsweresupplementedwithPg(0.2(cid:3)M)orNC-Pg(0.2(cid:3)M)before
additionofRBCs.Todetectfibrinnicking,wemeasuredfibrindeg-
RBCs attenuates tPA inhibition by PAI-1 (Ganguly et al.,
radation products (FDPs) released into the supernatant fluid from
2005).
125I-fibrinclotsasdescribedpreviously(Murcianoetal.,2003;Gan-
Another important property of tPA is its high affinity for
gulyetal.,2005).Inaseparateseriesofexperiments,RBC/PAwas
fibrin,mediatedprimarilythroughbindingofitsFdomainto
preincubated with 10 M excess of the PA inhibitor D-Phe-Pro-Arg-
cryptic binding sites on the fibrin (cid:1)-chain (D region; (cid:1)312–
CMK,HCl(PPACK)andthenaddedtoPg-supplementedfibrinclots.
324, exposed after fibrinogen cleavage by thrombin) in a Fibrinogen,fibronectin,typeIIIcollagen,AnnexinII,andalbumin
lys-independent manner (Medved and Nieuwenhuizen, were immobilized on 24-well polystyrene plates (overnight incuba-
2003). Binding is reinforced by interactions between the K tion at 4°C at protein concentration 0.2 (cid:3)M/well). Protein-coated
domainsoffibrinolyticproteinsandlysineresiduesthatare wellswereblockedwithPBS-bovineserumalbumin(3%)andincu-
exposed upon fibrin polymerization. Plasminogen (via K1 batedwithRBC/PAfor20minonahorizontalshakerat2gatroom
and four domains) and tPA compete for binding to the A(cid:2) temperature. Binding of RBCs was measured by measuring hemo-
D
residues(A(cid:2)148–160)intheDregionoffibrinandalsobind globinat405nminwaterlysateasdescribedabove. o
w
(PtKoaar(cid:2)ctCizamlraperrseoikdteueoetlsyasl(i.A,s1(cid:2)o93f99(cid:2)32C;–M6d1eo0dm)vaeiidnnaoanfdnfiNobnriecinoumw(npeinecthkiutiniivzgee)nbm,y2a0pn0lna3es)r-. 1M25iBIc-iePo.Ad1ic2so5tmIr-itpbPlueAxt,eios1n2(51I–o-2rfP(cid:3)AFg,roe5fe1PCArv-/eRarnBsiCums,alo)RrwBedCru-eaClio-nlnjaebjcuetelgedadtie.v5d.1CinPr-AaRnBeiCsn-/ nloaded
min exposes abundant -COOH terminal lysine residues on thetizedmalemice(C57black).Onehourlater,animalsweresacri- fro
ficed,andtheradioactivityinthebloodandorganswasmeasured. m
fibrinthatincreasethelys-dependentK2-mediatedtPAbind- Fibrin Affinity and Fibrinolytic Activity of RBC/PA in a jp
ing (Kaczmarek et al., 1993; Yakovlev et al., 2000; Medved MouseModelofPulmonaryEmbolism.Weusedamousemodel et.a
andNieuwenhuizen,2003).Retavase(rPA)hastheresidual of thrombosis using suspensions of fibrin microemboli (ME; mean sp
lys-dependent binding site conferred by K2, but it lacks lys- diameter 3–5 (cid:3)m), which embolize into the pulmonary vessels im- etjo
independent binding provided by the F domain (Kohnert mediately after i.v. injection (Murciano et al., 2002). To test the u
rn
etal.,1992). dissolution of ME lodged in the pulmonary vasculature, unlabeled a
It is not known, however, whether 1) RBC-coupled tPA RBC/tPAorRBC/rPA(0.08or0.4mg/kgPA/mouse)wereincubated ls.o
rsetatabilnystoitcsloatfsf;inaintdy 3a)ntdheachciegshsatfofinfiibtyrinfo;r2f)ibRriBnC, p/PrAesebnitndins 1w2i5tIh-M12E5Ir-MadEioasuctsipveitnysiionntsh2e0lumnignsownaiscembeeafsourereidnj1echtiopnoss.tiRnejescitdiuonal. at Urg
tPA and deleted in rPA, is an important factor in RBC/PA RToBCte/srtPtAh,eoradcohnetsrioonl5o1fCRr-BRCB/CPAwatosMincEuibnatveidvow,i5t1hCur-nRlaBbCe/ltePdAM,5E1Cforr- nive
lcolecaalrizhaotwiontoanuds/eorinfibvriitnroolyatsiscaaycstitvoitymiondevlivtoh.eLafasctt,oirtsisthnaott 20 min on ice before injections via the jugular vein, and 51Cr was rsity
measuredinthelungs10minpostinjection(tominimizeembolilysis o
governRBC/PAactivityinvivo. andaccountformostRBC“targeted”tolungsbyclots). f P
We studied fibrin binding and dissolution by RBC/tPA e
RBC/PABindingtoFibrinunderFlow.Afibrinsurfacewas n
versus RBC/rPA in a mouse model of pulmonary embolism formedbyaddingthrombin(2U/ml)totherectangularglasscapil- ns
y
andinvitromodelsandfoundthatthefibrinaffinityoftPA lariesflowchamber(GoelandDiamond,2002)for2hatRT,followed lva
1)isretainedwhenitisboundtoRBC;2)sufficestoanchor byperfusionofafibrinogensolution(3mg/ml)for5minat200s(cid:3)1. nia
RBC/tPAtoclots;and3)facilitatesfibrinolysisbyRBC/tPA. Alayerofnickedfibrinwasformedbyperfusingplasmin(0.01U/ml) L
ib
HemodynamicforcescontrolthebindingofRBC/PAtoclots, over the fibrin for 5 min and then treating the partially degraded ra
alyntiicssaugeenotfs.importance in designing formulations of fibrino- fc(cid:3)ihbMarm)i.nbRseBurrCfoa-vPceeArwloairtyhnerotrhomefapfillbaRrsiBmnCionsriwnniehcrikebeitdthorfeinbHrp-iDne-rVafutaslf-elPodwhtehr-rLaotyuesgs-ChoMfth5Ke0(fa1lon0wd0 ry on M
a
Materials and Methods 250(cid:3)l/min,correspondingtoshearstressesof0.625and3.125dyne/ y 1
Reagents.HumanrecombinanttPA(Alteplase)wasfromGenen- cmR2,BaCn/dPwAaFllisbhreinarolsytrseisssuonfd6e2r.5Falnodw3.1C2a.5lfss(cid:3)k1i,nrceoslplaecgteinve(l1y0.0(cid:3)g/ 2, 20
0
tech(SouthSanFrancisco,CA),rPA(Reteplase)wasfromCentocor, ml)-coated capillary flow chambers were incubated with citrated 9
Inc. (Horsham, PA), and plasminogen (Pg) was from American Di- platelet-richplasmafor30minatRT.Recalcifiedcitratedplatelet-
agnostica(Greenwich,CT).cDNAencodingaplasmincleavagesite- free plasma containing RBC-tPA, RBC/rPA, and/or normal RBCs
resistantmutantofplasminogen,referredasnoncleavableplasmin- and corn trypsin inhibitor (50 (cid:3)g/ml; inhibitor of factor XIIa) was
ogen (NC-Pg [R561A]; hereafter NC-Pg) was a kind gift from Dr. perfusedovercollagen-adherentplateletsatawallshearrateof62.5
Castellino(UniversityofNotreDame,NotreDame,IN)(Grellaand s(cid:3)1untilaprominentRBC-containingfibrinclotwasdeposited.The
Castellino,1997).FibronectinwasfromRochePaloAltoLLC(Palo chamberwasimmediatelyperfusedwithHanks’balancedsaltsolu-
Alto, CA). Calf skin type III collagen, plasmin, plasmin inhibitor tionatacontrolledflowrateof50or250(cid:3)l/minfor60minoruntil
(H-D-Val-Phe-Lys-CMK), plasminogen activator inhibitor (D-Phe- thefibrincompletelydissolved.ImagesofadherentRBCsandfibrin
Pro-Arg-CMK, HCl), and streptavidin were from Calbiochem (San undergoinglysiswereacquiredusingacharge-coupleddevicecam-
Diego, CA). Thrombin, fibrinogen, and components of buffer solu- era(Hamamatsu,Bridgewater,NJ).Afterimageacquisition,bound
tionswerefromSigma-Aldrich(St.Louis,MO).AnnexinIIwasfrom RBCsperunitareawerecountedinmultiplefieldsofview(n(cid:4)10).
BiodesignInternational(Kennebunk,ME). LysisofFibrinClotsafterContinuousandTransientExpo-
RBC/PABindingtoFibrinClots,ImmobilizedPlasmaCom- suretoFreePAversusRBC-PA.SolublePAorRBC/PA(2.5nM)
ponents,andVascularProteins.Approximately6(cid:2)104tPAor wasappliedtothesurfaceofPg-containing125I-fibrinclots(500(cid:3)l).
rPAmoleculeswereboundperRBCwithoutlossofenzymaticactiv- Clots were exposed to PA either continuously for 2 h at 37°C or
ityandRBCbiocompatibility,asdescribedpreviously(Murcianoet transientlyfor5minfollowedbywashing,additionof200(cid:3)lofPBS
al.,2003;Gangulyetal.,2005).SuspensionsofRBC/PAwereincu- tothetopoftheclot,andthena2-hincubationat37°C.Fibrinolysis
1132 Gangulyetal.
was assessedbymeasuring thereleaseofradioisotopeintothesu- RBC/PA indeed occurred (Fig. 1B). Naive RBCs did not re-
pernatants. lease FDP even in the presence of Pg (dashed line). In con-
trast, both RBC/tPA and RBC/rPA caused significant FDP
Results release from Pg-treated ((cid:5)5%; p (cid:1) 0.001), but not from Pg-
depleted fibrin clots (Fig. 1B). Substitution of Pg by NC-Pg
RBC/PABindingtoFibrinClots.Weincubatedsuspen- andinhibitionofPAactivitybyPPACKblockedFDPrelease
sionsofRBCsorRBC/PAinwellscontainingclotsprepared induced by RBC/PA. Thus, Fig. 1 shows that in this in situ
from Pg-depleted fibrin for 20 min at 4°C. Binding of naive model, RBC/PA interaction with Pg generates plasmin,
RBCsinclot-containingwells(Fig.1A,dashedline)exceeded which nicks fibrin, exposing abundant -COOH terminal ly-
bindingtoalbumin-coatedwells((cid:5)6(cid:6)0.9(cid:2)104versus2(cid:6) sineresiduesandthusenhancedRBC/PAbinding.
0.6(cid:2)103RBCs/well;p(cid:1)0.01),probablybecauseofmechan- BindingofRBC/PAtoImmobilizedPlasmaandVas-
ical trapping of RBCs between the clots and the chamber cular Proteins. Binding of RBC/rPA to wells coated with
walls.However,weobserved10-foldgreater(p(cid:1)0.01)bind- immobilizedfibrinogen,fibronectin,typeIIIcollagen,orAn-
ingofRBC/tPAtoclots(designated100%),whereasRBC/rPA nexin II did not exceed binding of either RBC/rPA or naive
bindingdidnotdifferfromcontrol.RBC/tPAbindinginwells RBCs(4.9(cid:6)0.2(cid:2)103RBCs/well)toalbumin-coatedwells.In
containingfibrinclots(8.1(cid:6)0.9(cid:2)105RBC/well)was(cid:5)50% contrast,RBC/tPAdisplayedsignificantlygreaterbindingto
D
ofthetheoreticalmaximumnumberofRBCthatcouldbind immobilized fibrinogen (85 (cid:6) 22 (cid:2) 103 RBCs/well) (p (cid:1) o
w
asamonolayerinthissizewell(Samokhinetal.,1983). 0.001). However, RBC/tPA binding to Annexin II did not nlo
SupplementationoffibrinclotswithPgenhancedthebind- differ from background binding of naive RBCs to albumin- ad
ingofRBC/tPA(butnotcontrolRBCs)by(cid:5)25%abovebind- coatedwells(Fig.2). ed
ing to Pg-depleted fibrin clots (Fig. 1A). RBC/rPA bound to DistributionofInjectedRBC/tPAversusRBC/rPAin fro
m
Pg-supplementedfibrinclotstothesameextentasRBC/tPA. Mice.InvivobindingofbothRBC/tPAandRBC/rPAtofibrin jp
IPng,cornedtruacsetd,PthgerebpinladcienmgeonftRbByCit/tsPnAonbcyle5a0v%abalendmueltiamnitn,aNteCd- wteosutldthbiseeaxspsuecmtepdtiboenc,awuseetorfafciebdrindunaiclkiisnogtobpye-pllaabsemleadPRgB.TCo/ et.as
p
brPinAdiwnigthofthReBPCA/riPnAh.ibPitroerinPcPuAbaCtKionsuopfprReBssCe/dtPtAheairnbdinRdBinCg/ tPFAirasntd,wReBtCra/rcPeAdtihnejetcitsesduei.vd.isintrimbuicteio.nofRBC/tPAversus etjou
to Pg-supplemented fibrin clots by (cid:5)25 and (cid:5)75%, respec- RBC/rPA1hafteri.v.injectioninnaivemice.51Crwasfound rna
tively. Thus, RBC/PA-mediated Pg conversion into plasmin primarilyinthebloodatlevelssimilartocontrol 51Cr-RBC, ls.o
enhances the RBC/tPA binding to fibrin and permits the excluding adverse effects of PA on RBC survival (Fig. 3A). arg
bindingofRBC/rPA,probablybecauseoffibrinnicking. Neither RBC/tPA nor RBC/rPA showed substantial renal or t U
Plasminogen-DependentFibrinNickingbyRBC/PA. cardiac uptake ((cid:1)0.5% ID) and only modestly elevated (10– niv
Released FDPs from 125I-labeled fibrin clots was measured 15% ID) splenic uptake, the site of elimination of senescent ers
after incubation with RBC/PA for 20 min at 4°C to test RBCs. Neither RBC/tPA nor RBC/rPA accumulated in the ity
whether plasmin-mediated fibrin nicking initiated by lungs to a significant extent compared with naive RBCs, of P
excludingaggregationandmechanicalretentioninthealve- e
n
olarcapillaries. ns
y
Thebloodleveloffree125I-tPAand125I-rPAwas(cid:1)10and lv
a
(cid:5)25% ID 1 h post-i.v. injection, respectively (Fig. 3B). Be- nia
cause of rapid clearance and metabolism, residual 125I-PA L
ib
was found only in liver and kidney at this time point. In ra
contrast, the organ distribution of RBC-bound tPA and rPA ry o
followed that of the carrier RBCs: 50 to 70% of injected n M
a
y
1
2
, 2
0
0
9

Fig.1.BindingofRBC/tPAversusRBC/rPAtostaticfibrinclots.RBC/
tPA(blackcolumns)versusRBC/rPA(hatchedcolumns)wasincubated Fig.2.BindingofRBC/PAtoimmobilizedplasmacomponentsandvas-
for20minat4°Cinwellswithfibrinclots(Fn).A,RBCbindingtoclots. cularproteins.RBC,RBC/tPA,andRBC/rPAwereincubatedwithalbu-
RBC/tPA binding to fibrin clots (8.1 (cid:6) 0.9 (cid:2) 105 RBCs/well; left black min (black columns), fibrinogen (hatched columns), fibronectin (gray
column)isdesignated100%relativetoothercolumnsshowingeffectsof columns), collagen (stripped columns), and Annexin II (gray checker-
Pg,NC-Pg,orPAinhibitorPPACK.Dashedline,bindingofnaiveRBCs. board columns) for 20 min at RT on a slow-speed horizontal shaker.
B,releaseof125I-labeledFDP.Dashedline,basaleffectofnaiveRBCs. BindingofRBCswasmeasuredbyhemoglobinanalysisinwaterlysateat
Thedatainthisandotherfiguresshowthemean(cid:6)S.E.M.,withn(cid:7)5, 405nm.RBC/tPAshowedsignificantbindingwithfibrinogen((cid:1)(cid:1)(cid:1),p(cid:1)
unlessspecifiedotherwise. 0.001versusRBCbindingtobovineserumalbuminandfibrinogen).
FibrinAffinityofRBC/tPAEnhancesFibrinolysis 1133
D
o
w
n
lo
a
d
e
d
fro
m
Fig.3.Distributionofdual-labeledRBC/PAinjectedinmice.A,distri- jp
bauntdioRnBoCf/r5P1CAr-(lhaabteclhededRcBoClum(wnhsi)t.eBc,odluismtrnibs)u,tRioBnCo/ftP1A25I(-blalabcekledcosluolmunblse) et.as
tPA(whitecolumns),rPA(graycolumns),RBC-coupledtPA(blackcol- Fig.4.RBC/tPAbindstoandlysespulmonaryembolimoreeffectively pe
umns),andRBC-coupledrPA(hatchedcolumns).Thedataareshownas versusRBC/rPA.A,residualpulmonarylevelof125I-fibrinME,preincu- tjo
panerdcemnatajogrevoifscienrjeacltoerdgadnosse1(%haIDft)erofi.lva.bienljeedctRioBnC.sorPAfoundinblood b(haattecdhfeodrc2o0lumminnso)naitc0e.0w8itohre0i.t4hemrgR/kBgC,/1tPhAaf(tbelraci.kv.coinlujemctniosn)oorfRmBixCt/urrPeAs urna
throughthejugularvein.DifferencesbetweentPAversusrPAgroups((cid:1)(cid:1)) ls
and0.08versus0.4mg/kggroups(##)werestatisticallysignificantatp(cid:1) .o
RBC/125I-tPAandRBC/125I-rPAwasfoundintheblood(Fig. 0.01.B,pulmonaryuptakeof51Cr-labeledRBC,51Cr-RBC/tPA,and51Cr- arg
3B). Importantly for the results that follow, the amount of RBC/rPA (double hatched, black, and hatched columns) injected alone t U
RBC/tPAfoundinthelungsofnaivemicedidnotexceedthat (cloelfutmtnhsr)e.eTchoeludmatnas)aroersphroewinncausba%teIdDwofit5h1Curn-llaabbeelleeddRMBECr(reicgohvtertehdreine niv
of RBC/rPA, as assessed by measuring either 51Cr (Fig. 3A) thelungs10minafteri.v.injection. ers
or125I(Fig.3B). ity
o
RBC/tPA, but Not RBC/rPA, Binding to Fibrin Clots level of 51Cr-RBC and 51Cr-RBC/rPA was no greater than f P
FacilitatesLysisofPulmonaryMicroemboli.Totestthe that seen in control animals (Fig. 4B), whereas the pulmo- e
n
adhesionofRBC/PAtofibrinclotsandtheirfibrinolyticeffect naryuptakeof51Cr-RBC/tPApreincubatedwithmicroemboli ns
y
in vivo, we injected Pg-containing fibrin microemboli into was 3-fold greater than 51Cr-RBC/tPA alone (13.1 (cid:6) 2.9 lv
a
mice i.v. ME rapidly become invested with blood elements, versus4.1(cid:6)0.8%IDincontrolmice;p(cid:1)0.05)or51Cr-RBC nia
enlarge, and deposit in precapillary pulmonary arterioles or 51Cr-RBC/rPA alone (p (cid:1) 0.05 each). Therefore, high af- L
ib
(Murcianoetal.,2002). finity to fibrin promotes RBC/tPA retention within the pul- ra
Equal doses of unlabeled RBC/tPA and RBC/rPA were monaryclots,whereasRBC/rPAisdislodged. ry o
incubatedwith125I-labeledmicroembolionicefor20min,the RBC/tPABindingtoFibrinunderFlow.Oneofthekey n M
mixtureswereinjectedi.v.,andtheresidualradioactivityin differences between the in vitro (Fig. 1) and in vivo (Fig. 4) a
y
the lungs was measured 1 h later. In agreement with our models is the presence of hemodynamic forces in the latter. 1
2
previousresults,(cid:5)30%oftheisotoperemainedinthelungs To test whether fibrin affinity suffices to anchor RBC/PA to , 2
0
of control mice, corresponding to the fraction of emboli that intact versus nicked fibrin under flow conditions, we per- 0
9
had withstood spontaneous fibrinolysis (Murciano et al., fusedsuspensionsofRBC,RBC/tPA,andRBC/rPAfor5min
2002). Both RBC/tPA and RBC/rPA enhanced clot lysis, re- atawallshearrate62.5s(cid:3)1acrossafibrinmonolayerformed
ducingresidual125Iinthelungsto(cid:1)10%ofbaseline(i.e.,at fromPg-depletedfibrinogen(Fig.5).
least3-fold)comparedwithcontrolanimals.However,equal Inthismodel,whichallowsforfarlessnonspecificentrap-
dosesofRBC/tPAcausedsubstantiallygreaterdissolutionof ment compared with the static model, naive RBCs showed
emboliversusRBC/rPA(p(cid:1)0.01)(Fig.4A). comparably low adhesion to either immobilized fibrin or al-
We then investigated the mechanism underlying this sig- bumin(Fig.5B).PerfusedRBC/tPAadheredtofibrin,butnot
nificant difference in the biological activity of RBC/tPA ver- toalbumin-coatedflowchambers(Fig.5,AandB),similarto
sus RBC/rPA in vivo. This disparity could not be attributed their behavior in the “static” model (Fig. 1). Nicking of the
eithertoPAdetachmentfromRBCoraneffectofconjugation fibrin by plasmin before RBC/tPA perfusion increased the
on RBC biodistribution and biocompatibility (Fig. 3). Thus, binding of RBC/tPA 4-fold (p (cid:1) 0.001). Yet, perfused RBC/
weaskedwhethertheenhancedactivityofRBC/tPAinvivo rPAdidnotbindtoeitheranaiveornickedfibrinmonolayer
might be because of, at least in part, its greater affinity for (Fig. 5). Therefore, the affinity of RBC/rPA to nicked fibrin
fibrin. To test this possibility, we injected 51Cr-RBC, 51Cr- wassufficientforadhesiontoclotsinastaticmodel(Fig.1A),
RBC/tPA,or51Cr-RBC/rPAthathadbeenpreincubatedwith but it was not enough to withstand the forces generated by
unlabeled microemboli for 20 min on ice. The pulmonary physiologicallyrelevantlevelsofflow(Fig.5).
1134 Gangulyetal.
took more than 40 min for RBC/rPA to initiate lysis. An
increaseinshearratefrom62.5to312.5s(cid:3)1didnotchange
fibrinolysisinducedbyRBC/tPA(datanotshown).Therefore,
RBC/tPAbindingtofibrinacceleratesthesubsequentdisso-
lutionofnascentclotsunderflow.
Binding to Fibrin after Transient Exposure En-
hances Clot Lysis by RBC/tPA. A second in vitro model
was used to examine whether RBC/tPA binding to clots fa-
cilitates fibrinolysis. Soluble or RBC-bound tPA versus rPA
(2.5 nM) was added to Pg-containing fibrin clots in the ab-
sence of shear forces. When PA or RBC/PA was incubated
withclotscontinuouslyfor2hat37°C,nosignificantdiffer-
enceintPA-versusrPA-mediatedlysiswasobserved,either
byfreeorRBC-bound(Fig.7A).
In the set of experiments, we eliminated unbound drugs
after a 5-min incubation with the clots, followed by a 2-h D
o
incubationat37°C(Fig.7B).Inthissetting,imitatingthein w
n
vivo condition in which lysis is produced by residual clot- lo
a
d
associatedPA(whereasunboundmaterialisrapidlycleared), e
d
freerPAinducedgreaterfibrinolysisthandidfreetPA.This fro
result probably reflects impedance of tPA diffusion into the m
fibrin meshwork because of fibrin affinity, leading to its jpe
retentiononthesurfaceofclots(FisherandKohnert,1997). t.a
s
Incontrast,RBC/tPAcaused2-foldgreaterfibrinolysisthan pe
RBC/rPAaftertransientcontact(p(cid:1)0.01),whichisconsis- tjo
u
Fig.5.PerfusedRBC/tPA,butnotRBC/rPA,bindstofibrinandnicked tentwiththenotionthatRBC/tPAbindingtofibrinleadsto rn
fibrin under flow conditions. RBC suspensions were perfused in flow a
chambers (5 min at 0.6 dyne/cm2, followed by washing with PBS for 5 greaterdrugretentionandsubsequentclotlysis. ls.o
min).A,microscopicimagesofboundRBCs.B,quantitativeanalysisof rg
RBCbindinginthechamberscoatedwithalbumin(gray),fibrin(white; a
###),ornickedfibrin((cid:1)(cid:1)(cid:1))(blackcolumns;p(cid:1)0.001). t U
n
iv
e
RBC/tPABindingtoFibrinAcceleratesFibrinolysis rs
underFlowConditions.WethentestedwhetherRBC/tPA ity
o
bindingtofibrinenhancesfibrinolysisunderflowconditions. f P
Recalcified citrated platelet-free plasma containing RBCs, e
n
n
RBC/tPA, or RBC/rPA was applied to an adherent layer of s
y
platelets in the flow chambers to form clots (Fig. 6, left, lv
a
showing time 0), which were then perfused with Hanks’ nia
balancedsaltsolutionfor0to60minatawallshearrateof L
ib
62.5s(cid:3)1,whichisclosetotheshearforcespresentinthelung ra
microcirculation(GoelandDiamond,2003). ry o
WhenclotswereformedinthepresenceofnaiveRBCs,the n M
fibrinfibrilsremainedintactforatleast60min(Fig.6,top). a
y
Initial fibrin formation was not impaired in the presence of 1
2
RBC/PA (Fig. 6, time 0). However, whereas RBC/tPA com- , 2
0
pletelydegradedthefibrinfibrilswithin20min(bottom),it 0
9

Fig.7.InvitrodissolutionoffibrinclotsbysolubleandRBC-coupledtPA
Fig. 6. RBC/tPA adhesion to fibrin clots under flow accelerates subse- versus rPA. tPA (black columns) or rPA (gray columns) was incubated
quentfibrinolysis.Dissolutionoffibrinformedinflowchambersinthe with Pg-containing 125I-fibrin clots for 2 h at 37°C (top) or for 5 min
presence of perfused control RBCs (A), RBC/rPA (B), or RBC/tPA (C). followedbywashingand2-hincubationat37°C.Theamountofreleased
Fibrinfibrils(arrows)disappearedinchambersperfusedat0.6dyne/cm2 125I-labeledFDPswasmeasured.(cid:1)(cid:1),differenceintPAversusrPAwas
withRBC/tPAversusRBC/rPAby25versus40min.Scalebar,10(cid:3)m. statisticallysignificant;p(cid:1)0.01.
FibrinAffinityofRBC/tPAEnhancesFibrinolysis 1135
Discussion clotsinsituinstaticmodel(Fig.1),butnotinvivo(Fig.4B).
Furthermore,intheclotperfusionmodelonlyRBC/tPA,but
Patients with multiple transient ischemic attacks, stroke
not RBC/rPA, bound to naive and nicked fibrin (Fig. 5) and
in evolution, organ transplant rejection, poststenting cre-
showed greater fibrinolytic activity than RBC/rPA (Fig. 6)
scendo angina, among others, have a high propensity for
under flow. Interestingly, adherence to exposed lysine resi-
recurrent thrombosis. The vulnerable period is often of de-
dues in nicked fibrin, albeit insufficient to anchor perfused
fined and of relatively limited duration (Taylor et al., 1999;
RBC/rPA, reinforces the RBC/tPA anchorage (Fig. 5), al-
Johnston, 2002), yet current means for thromboprophylaxis
thoughagainthiseffectwasnotasevidentinastaticmodel
havelimitedefficacyandconsiderablesafetyconcerns,which
(Fig.1A).
thwart dose escalation or combined therapy (Throckmorton,
RBC/tPA, but not RBC/rPA, displayed plasminogen-inde-
2001;LangeandHillis,2004;Patronoetal.,2004).Fibrino-
pendent binding to the cryptic tPA-binding sites that are
lyticdrugshavethepotentialtocomplementexistingthrom-
exposed in the immobilized fibrinogen because of partial
boprophylaxis (Hong et al., 2003), if their pharmacokinetic
polymerizationoftheprotein(Yakovlevetal.,2000)(Fig.2).
andactivitiescanbemodifiedtoenhancedrugdeliveryinto
Of interest, RBC/tPA did not display elevated (versus naive
clots and overcome safety concerns. Vascular occlusion by
RBC) binding to other extracellular vascular proteins, in-
fibrinistheproximatemorbideventinthemostthrombotic
cludingAnnexinII,whichisanendothelialcellsurfacecore-
D
disorders. Thus, prophylactic use of PAs capable of rapidly ceptor for tPA and plasminogen (Hajjar et al., 1998; Kim, o
w
dissolving nascent, highly susceptible clots from within be- 2002). Furthermore, neither RBC/tPA nor RBC/rPA showed nlo
fore vascular occlusion (Murciano et al., 2003; Ouriel, 2003) elevated (versus naive RBC) binding to endothelial cells in ad
isconceptuallyattractive,iffeasible. culture (data not shown), either control or thrombin-acti- ed
ImpedimentstotheprophylacticuseofexistingPAsseem vated (treatment that induces exposure of Annexin II on fro
m
insurmountable. PAs penetrate clots poorly (Rijken et al., endothelial surface) (Peterson et al., 2003). Thus, it is un- jp
2a0ff0in4i)t.yIritosneilcfailmly,petdheesacdlvotanpteargmeoeuastiopnrobpyerttPyAof(Fhiigg.h6)fib(Srian- lainkcehlyortRhBatC/AtPnAneaxnindpIrIomseortveeistsaasctaivnitya,uuxnilliiakreysoalduabpletotrPAto. et.as
p
kishtearrionvgaPnAdsRbijekfoerne,1th9r9o5m;FbiosshiserwaonudldKaolhlenveiratt,e1t9h9i7s)p.rAodbmleimn-, enHhaonwceevdera,catidvhiteysioofnRtBoCcl/toPtsAisvenrostusthReBoCn/lryPrAeainsonvivfoor. Fthoer etjou
if 1) their half-lives can be prolonged beyond what is cur- example,RBC/tPA,butnotRBC/rPA,retainstheabilitytobe rna
rentlyattainablebymolecularmodifications(Dawsonetal., activatedbyfibrin(Gangulyetal.,2005).Moreover,binding ls.o
1994;Thomasetal.,1994;Liberatoreetal.,2003;Moreadith of tPA to RBCs renders tPA more resistant to PAI-1 than arg
and Collen, 2003; Hagemeyer et al., 2004); and 2) bleeding solubletPA(Gangulyetal.,2005).Itisdifficulttoweighthe t U
andcollateraltissuedamageareavoided(Wangetal.,1998). contribution of these features of RBC/PA formulations to niv
Thus,adrugdeliverysystemfundamentallychangingphar- theirperformancesinvivo.Yet,thecorrespondencebetween ers
macokineticsisrequiredforprophylacticuseofPAs. theadherenceofRBC/tPAtofibrininstaticsystems(Figs.1 ity
Our recent results showed that coupling PAs to RBCs, an and7)andunderhemodynamicforces(Figs.5and6)andits of P
attractiveprospectivecarrierforintravasculardrugdelivery effectiveness in vivo (Fig. 4) shows that fibrin affinity is e
n
(Magnanietal.,2002),mayprovidesuchaplatform(Trojan intrinsictoRBC/tPAfibrinolyticactivity. ns
y
horsefibrinolysis)(Murcianoetal.,2003;Ouriel,2003).Cou- TheimportantroleofhemodynamicforcesintheRBC/PA lv
a
pling to RBCs also alters some biological properties of PAs, behaviorisconcordantwitheffectsofshearstressoncoagu- nia
e.g.,enhancestPAresistancetoPAI-1(Gangulyetal.,2005). lation and clot lysis, adhesion of factor VIII to collagen, L
ib
These encouraging data justify characterization of RBC/PA platelet activation, and adhesion of blood cells to endothe- ra
moresystematically,forwhichinvitromethodsthatreliably lium(AnandandDiamond,1996;FisherandKohnert,1997; ry o
simulatetheirinvivobehaviorareneeded. Falati et al., 2002; Einav and Bluestein, 2004; Frenette, n M
To test relationships between fibrin affinity and fibrino- 2004;Leytinetal.,2004;Tomokiyoetal.,2005).Mostinvitro a
y
lytic activity of RBC/PA, we compared in vivo fibrinolytic studies of antithrombotic drugs have been performed in 1
2
activityofRBC/tPAandRBC/rPA,atruncatedvariantwith staticmodels.Yet,discordancebetweeninvivoandstaticin , 2
prolongedhalf-lifeinthecirculation((cid:5)25versus(cid:5)5minfor vitro models justifies the need to develop more relevant 00
9
tPA) (Kohnert et al., 1992) and reduced fibrin affinity. We model systems in which to test the behavior of antithrom-
found that RBC/tPA retains its high affinity for fibrin clots, botic agents and perhaps other drugs intended to work
evident in both static and perfusion systems in vitro and in withinthevasculature.
vivo.PlasminformedfromPgbyRBC/PAenhancesitsbind-
ing to clots (Fig. 1), probably by nicking the (cid:2)C domain of
References
fibrin,exposing-COOHterminallysines(MedvedandNieu-
AnandSandDiamondSL(1996)Computersimulationofsystemiccirculationand
wenhuizen,2003)andperhapsalsoviaformingternarycom-
clotlysisdynamicsduringthrombolytictherapythataccountsforinnerclottrans-
plexesbetweenRBC/PA,Pg,andfibrin(Yakovlevetal.,2000; portandreaction.Circulation94:763–774.
DawsonKM,CookA,DevineJM,EdwardsRM,HunterMG,RaperRH,andRoberts
Medved and Nieuwenhuizen, 2003). RBC/tPA is a more ef-
G (1994) Plasminogen mutants activated by thrombin. Potential thrombus-
fectivethrombolyticagentinvivothanisRBC/rPA(Fig.3A), selectivethrombolyticagents.JBiolChem269:15989–15992.
which binds only to nicked fibrin clots in a static model in EinavSandBluesteinD(2004)Dynamicsofbloodflowandplatelettransportin
pathologicalvessels.AnnNYAcadSci1015:351–366.
vitro(Fig.1A). FalatiS,GrossP,Merrill-SkoloffG,FurieBC,andFurieB(2002)Real-timeinvivo
This study reveals the limitations of inferring how RBC- imagingofplatelets,tissuefactorandfibrinduringarterialthrombusformationin
themouse.NatMed8:1175–1181.
coupledPAwillbehavebasedonthepropertiesofitssoluble FisherSandKohnertU(1997)Majormechanisticdifferencesexplainthehigherclot
counterpart and in vitro studies. Thus, rPA has sufficient lysispotencyofreteplaseoveralteplase:lackoffibrinbindingisanadvantagefor
bolusapplicationoffibrin-specificthrombolytics.FibrinolysisProteolysis11:129–
affinityfornickedfibrintoanchorRBC/rPAonPg-containing 135.
1136 Gangulyetal.
FrenettePS(2004)Sicklecellvasoocclusion:heterotypic,multicellularaggregations Medved L and Nieuwenhuizen W (2003) Molecular mechanisms of initiation of
drivenbyleukocyteadhesion.Microcirculation11:167–177. fibrinolysisbyfibrin.ThrombHaemostasis89:409–419.
GangulyK,KrasikT,MedinillaS,BdeirK,CinesDB,MuzykantovVR,andMur- MoreadithRWandCollenD(2003)ClinicaldevelopmentofPEGylatedrecombinant
cianoJC(2005)Bloodclearanceandactivityoferythrocyte-coupledfibrinolytics. staphylokinase(PEG-Sak)forbolusthrombolytictreatmentofpatientswithacute
JPharmacolExpTher312:1106–1113. myocardialinfarction.AdvDrugDelivRev55:1337–1345.
Goel MS and Diamond SL (2002) Adhesion of normal erythrocytes at depressed MurcianoJC,HarshawD,NeschisDG,KoniarisL,BdeirK,MedinillaS,FisherAB,
venousshearratestoactivatedneutrophils,activatedplateletsandfibrinpoly- Golden MA, Cines DB, Nakada MT, et al. (2002) Platelets inhibit the lysis of
merizedfromplasma.Blood100:3797–3803. pulmonarymicroemboli.AmJPhysiol282:L529–L539.
GoelMSandDiamondSL(2003)NeutrophilcathepsinGpromotesprothrombinase MurcianoJC,MedinillaS,EslinD,AtochinaE,CinesDB,andMuzykantovVR
and fibrin formation under flow conditions by activating fibrinogen-adherent (2003)Prophylacticfibrinolysisthroughselectivedissolutionofnascentclotsby
platelets.JBiolChem278:9458–9463. tPA-carryingerythrocytes.NatBiotechnol21:891–896.
GrellaDKandCastellinoFJ(1997)Activationofhumanplasminogenbystaphy- OurielK(2003)Thrombi–bewareofredcellsbearinggifts.NatBiotechnol21:871–
lokinase.Directevidencethatpreformedplasminisnecessaryforactivationto 872.
occur.Blood89:1585–1589. Patrono C, Coller B, FitzGerald GA, Hirsh J, and Roth G (2004) Platelet-active
HagemeyerCE,TomicI,JaminetP,WeirichU,BasslerN,SchwarzM,RungeMS, drugs:therelationshipsamongdose,effectivenessandsideeffects:theSeventh
BodeC,andPeterK(2004)Fibrin-targeteddirectfactorXainhibition:construc- ACCPConferenceonAntithromboticandThrombolyticTherapy.Chest126:234S–
tionandcharacterizationofarecombinantfactorXainhibitorcomposedofan 264S.
Peterson EA, Sutherland MR, Nesheim ME, and Pryzdial EL (2003) Thrombin
antifibrinsingle-chainantibodyandtickanticoagulantpeptide.ThrombHaemo-
inducesendothelialcell-surfaceexposureoftheplasminogenreceptorannexin2.
stasis92:47–53.
JCellSci116:2399–2408.
HajjarKA,MauriL,JacovinaAT,ZhongF,MirzaUA,PadovanJC,andChaitBT
RijkenDC,Barrett-BergshoeffMM,JieAF,CriscuoliM,andSakharovDV(2004)
(1998)TissueplasminogenactivatorbindingtotheannexinIItaildomain.Direct
Clotpenetrationandfibrinbindingofamediplase,achimericplasminogenacti-
modulationbyhomocysteine.JBiolChem273:9987–9993.
vator(K2tu-PA).ThrombHaemostasis91:52–60.
HolvoetP,DewerchinM,StassenJM,LijnenHR,TollenaereT,GaffneyPJ,and D
Collen D (1993) Thrombolytic profiles of clot-targeted plasminogen activators. SapklhaasmroavcDloVtalynsdisR.iCjkiercnuDlaCti(o1n99952):1S8u8p3e–r1f8ic9ia0l.accumulationofplasminogenduring ow
1P0a0ra7m–1e0t1e6rs. determiningpotencyandinitialandmaximalrates.Circulation87: Sa(m19o8k3h)inReGdPb,lSoomdircneollvtaMrDge,tiMnguztyokcaonltlaovgeVn-Rc,oaDteodmosguarftaskceys.SFPE,BanSdLSetmti1r5n4ov:25V7N– nloa
HongTT,DriscollEM,WhiteAJ,SherigillA,GiboulotTA,andLucchesiBR(2003) 261. de
Glycoprotein IIb/IIIa receptor antagonist (2S)-2-[(2-naphthyl-sulfonyl)amino]-3- Taylor RL, Borger MA, Weisel RD, Fedorko L, and Feindel CM (1999) Cerebral d
[p[a2n-(o[4ic-(a4c-ipdipdeirhiyddinroycl)h-l2o-r[i2d-e(4(-CpiRpLer4i2d7in96y)l),eitnhcyol]mbbuintaatniooynl]waimthinaos)paicreitnyal]nadm/oinroe]nporxo-- minitcerroveemntbioolnisd.uArninngTcahrodriaocpuSlumrgon6a8r:y89by–p9a3s.s:increasedemboliduringperfusionist from
atrpeaartimn,enptrebvyernetcsomcobrionnaanrtytiasrstueeryplarestmhirnoomgebnosaisctiavfatetorr.suJcPcehsasrfumlactholroEmxpboTlyhteicr ThBoemnanseGttRW,TFh(i1b9o9d4e)auAxlHon,gE-hrraeltft-lCifeJ,aBnaddfiilbloriJnM-s,pKeceiyfticBfAor,mReoffintoisCsuJe,ZpilvaisnmJiAn,oagnend jpe
306:616–623. activatorinrabbitmodelsofembolicstrokeandperipheralbleeding.Stroke25: t.a
Johnston SC (2002) Clinical practice. Transient ischemic attack. N Engl J Med 2072–2079. sp
347:1687–1692. ThrockmortonDC(2001)Futuretrialsofantiplateletagentsincardiacischemia. e
KaczmarekE,LeeMH,andMcDonaghJ(1993)Initialinteractionbetweenfibrin NEnglJMed344:1937–1939. tjo
afinbdrint(iosgsuene)pislaismmpionrotgaenntfaocrtitv-aPtAorac(tt-iPviAty)..JThBeioGlClyh-Pemro-2A6r8g:-2P4r7o4b–i2n4d7i9n.g site on ToNmaokkaiygoakKi,TK,aanmdikMuobrooiYM,H(2a0n0a5d)aVoTn,WAriallkeibrTa,nNdafakcattoormaiccYe,leOragtaetsapYla,tJeluentgadShMe-, urna
KimJHK(2002)AnnexinII:aplasminogen-plasminogenactivatorco-receptor.Front sionandthrombusformationonacollagensurfaceinplatelet-reducedbloodunder ls
KoMkBhrinaionerstgcriitlne7UU:2d,,3aRL4n1iuld–ldodHpl3pr,4ohP8tre.Rians,zeVHde,rohmLeeaicjihennnseJarHrMe,,mWetaeiaenlnt.ia(ni1ng9-e9Vd2e)rinhBoiotehcffheeErmeJif,coalSdlteepdrrnotp-APer,AtiOedspeioltefzttihoUne, TsfnNilreoakuwtaruocrSnoeEna(,dlLGidotenuiogdanel)sna3.ne7Brda7lrot:i3isoo4dAn0,1–a0A3n54md:41as.0er7iaz8lu–Dr1eG0a,8ra4en.mdeSdtiraictekdlabnydtSiss(1u9e9p5l)aEsmxciintoogtoexnina-citnivdautcoerd. at U.org
n
variantBM06.022.ProteinEng5:93–100. WangYF,TsirkaSE,StricklandS,StiegPE,SorianoSG,andLiptonSA(1998) iv
Lange RA and Hillis LD (2004) Antiplatelet therapy for ischemic heart disease. Tissueplasminogenactivator(tPA)increasesneuronaldamageafterfocalcerebral e
LeNytiEnnVgl,AJlMlenedD3J5,0M:2y7k7h–a2y8l0o.vS,MisL,LyubimovEV,GarveyB,andFreedmanJ YaikscohvelemviaS,inMwaikldo-gtoynpeenaknodEt,PAK-udreofcichikeinntamNic,eN.NieautwMenedhu4i:z2e2n8–W2,31In.gham K, and rsity
(B2i0o0ch4)emPaBthiooplohgyiscRhiegshCsohmemarusntr3e2s0s:i3n0d3u–c3e1s0a.poptosiseventsinhumanplatelets. ManeddvpeladsLm(i2n0o0g0en)C-boinnvdeinrsgiosniteosf.fiBbiroinchoegmenisttoryfib3r9i:n1:5m73e0ch–a1n57is4m1.ofexposureoftPA- of P
e
LiberatoreGT,SamsonA,BladinC,SchleuningWD,andMedcalfRL(2003)Vam- n
pirebatsalivaryplasminogenactivator(desmoteplase):auniquefibrinolyticen- Address correspondence to: Dr. Vladimir R. Muzykantov, Institute for ns
zymethatdoesnotpromoteneurodegeneration.Stroke34:537–543. EnvironmentalMedicine,UniversityofPennsylvaniaSchoolofMedicine,One ylv
MagnaniM,RossiL,FraternaleA,BianchiM,AntonelliA,CrinelliR,andChiaran- John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068. a
tiniL(2002)Erythrocyte-mediateddeliveryofdrugs,peptidesandmodifiedoligo- E-mail:[email protected] nia
nucleotides.GeneTher9:749–751. L
ib
ra
ry
o
n
M
a
y
1
2
, 2
0
0
9
Functional Phenotyping of Human Plasma
Using a 361-Fluorogenic Substrate
Biosensing Microarray
DhavalN.Gosalia,1WilliamS.Denney,2CleoM.Salisbury,3JonathanA.Ellman,3
ScottL.Diamond1,2
1DepartmentofBioengineering,InstituteforMedicineandEngineering,
UniversityofPennsylvania,Philadelphia,PA19104-6281;
telephone:[phone];fax:[phone];e-mail:[email protected]
2DepartmentofChemicalandBiomolecularEngineering,InstituteforMedicineand
Engineering,UniversityofPennsylvania,Philadelphia,PA19104-6281
3DepartmentofChemistry,CenterforNewDirectionsinOrganicSynthesis,
UniversityofCalifornia,Berkeley,CA
Received13December2005;accepted9March2006
Publishedonline30March2006inWileyInterScience(www.interscience.wiley.com).DOI:10.1002/bit.20927
Abstract: Amicroarraypresentingglycerolnanodroplets 1994; Burke, 2003;Schneppenheimetal., 2001). Precisely
offluorogenicpeptidesubstrateswasusedasabiosensor because blood is biomarker-rich (Anderson and Anderson,
for the detection of multiple enzyme activities within
2002;Andersonetal.,2004;Fujiietal.,2005;Liottaetal.,
human plasma. Using 10 different plasma proteases
2003)andcanberoutinelycollectedinclinicalsettings,rapid
(kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa,
factor Xa, thrombin, activated protein C, uPA and and thorough analysis ofblood is a keygoal for diagnostic
plasmin) and a 361-compound fluorogenic substrate andprognosticproteomics.
library(Ac-Ala-P3-P2-Arg-coumarinforP¼allaminoacids However, proteomic analysis of blood is complicated by
exceptCys),adatabasewascreatedfordeconvolutingthe
the fact that human blood plasma is a complex milieu of
relativeactivityofeachindividualenzymesignalinhuman
interactingproteinswithabundancesspanningmanyorders
plasmatreatedwithvariousactivators(calcium,kaolin,or
uPA). Three separate deconvolution protocols were ofmagnitude.Forexample,theconcentrationofendogenous
tested: searching for ‘‘optimal’’ sensing substrate seq- tissuefactormaybeinthelowfemtomolarrange(Bogdanov
uencesforasetof5enzymesandusingthesesubstrates et al., 2003; Butenas et al., 2005; Lo and Diamond, 2004),
to detect protease signals in plasma; ranking the ‘‘opti-
whereas fibrinogen concentrations are often six orders
mal’’sensingsubstratesfor10proteasesusinglocalerror
of magnitude greater, in the low micromolar range.
minimization,resultinginasetofsubstrateswhichwere
bundled via weighted averaging into a super-pixel that Furthermore, the proteins in blood plasma are dynamic, as
had biosensing properties not obtainable by any indivi- exemplified by the well-known protease cascades of
dualfluorogenicsubstrate;andtreatingeach361-element coagulation,complementactivation,andfibrinolysis.
mapmeasuredforeachplasmapreparationasaweighted
Oneofthemostimportantproteinnetworksinthebloodis
sumofthe10mapsobtainedforthe10purifiedenzymes
thetissuefactor-initiatedbloodcoagulationcascade(Fig.1).
using a global error minimization. The similarity of the
results from these latter two protocols indicated that a The kinetics of the full reaction network are fairly well
smallsubsetof<90substratescontainedthemajorityof described (Hockin et al., 2002) and the importance of this
biochemicalinformation.Theresultswereconsistentwith cascadeiswelldocumented.Problemswiththecoagulation
the state of the coagulation cascade expected when
cascadesuchasfactorVLeiden,vonWillebrand’sdisease,
treatedwiththegivenactivators.Thismethodmayallow
and hemophilia A and B demonstrate strong genotype/
development of future biosensors using minimal and
non-specificmarkers.Thesesubstratescanbeappliedto phenotype linkages in blood biology (Bertina et al., 1994;
real-time diagnostic biosensing of complex protease Burke, 2003; Schneppenheim et al., 2001). Capturing the
mixtures.(cid:1)2006WileyPeriodicals,Inc. activation states for all of the blood coagulation cascade
Keywords: protease;coagulation;microarray proteaseswouldallowdistinctactivitymapstobecreatedfor
the different transient states of a plasma sample. Ideally, a
INTRODUCTION novelkineticsignaturecouldbedetectedbycomparingsuch
activity maps of normal and diseased plasma to reveal
As a consequence of its close contact with vascularized
diagnosticinformation.
organs, it is thought that blood biochemistry reflects the
Massspectrometric(MS)methodsintandemwithliquid
physiologicalorpathologicalstateofthebody(Bertinaetal.,
chromatography and 2-dimensional gel electrophoresis
(2DGE) are currently used for the detection of biomarkers
Correspondenceto:S.L.Diamond
D.N.GosaliaandS.L.Diamondcontributedequallytothiswork. in blood diagnostics (Petricoin et al., 2004). MS- and
(cid:1)2006WileyPeriodicals,Inc.
PK K C1 sequences. We then employed various deconvolution algo-
K, plm rithmstousetheseoptimalsubstratesasnovelbiosensorsto
XII XIIa
kaolin investigate the transient state of blood during coagulation.
Xa, ATIII
TF
XI IIa XIa This approach could be clinically useful for detection of
IIa X ATIII, TFPI:Xa enzyme activity directly from plasma samples because it
IX
IXa TF allowsdirectanalysisofsmallsamplequantities.
IIa, Xa Xa, IIa
VIII VIIIa VIIa VII
TF:VIIa, IXa
PC TM:IIa APC PS MATERIALS AND METHODS
Xa, IIa
ATIII, TFPI Xa Va V
PS Proteases, Plasma,and Fluorogenic Substrates
II IIa APC
Purifiedhumanplasmakallikrein,humanfactorXa,human
fibrinogen fibrin IIa thrombinandhumanplasminwerepurchasedfromEnzyme
AP−− aannitoi−npicla psmhoinspholipid surface XIIIa XIII ResearchLaboratories(SouthBend,IN).Humanurokinase
APC− activated protein C PAI−1
ATIII − antithrombin−III plasminogen activator (uPA) and lipidated recombinant
CFD1P− C− f1ib irninh ibdietogrredation peptides Crosslinked human tissue factor were purchased from American Dia-
II− prothrombin fibrin
IIa− thrombin tPA, uPA gnostica (Stamford, CT). Whole blood was obtained by
KMG− k−a mllikarcerionglobulin plg plm AP, MG venipuncture from healthy, non-smoking volunteers in
PAI−1− plasmin activation inhibitor−1
PC− protein C plm accordance with protocols approved by the University of
PK− prekallikrein Nicked
plg− plasminogen scuPA fibrin Pennsylvania IRB and anticoagulated with 1/9 volume of
plm− plasmin
PS− protein S plm, IIa 3.8%sodiumcitrateinthesyringe(Sigma;St.Louis,MO).
TAFI − thrombin−activated fibrinolysis inhibitor tPA TAFI
TF− tissue factor Whole blood was centrifuged at 100g for 15 min to obtain
tPA − tissue plasminogen activator
uPA− urokinase plasminogen activator FDP plateletrichplasma(PRP)followedbycentrifugationofthe
supernatant at 10,000g for 5 min to generate platelet free
Figure1. Thecoagulationpathways.Thisisadiagramofthecontact(XII
plasma(PFP).
activated) and tissue factor pathways of coagulation. Note that II is
prothrombinandIIaisthrombin. Fluorogenic peptide substrates for plasma kallikrein
(Z-FR-MCA, 612.6 MW), factor Xa (Boc-IEGR-MCA,
gel-basedproteomicmethodshaveprovidedabetterunder- 730.8 MW), thrombin (Boc-VPR-MCA, 627.8 MW),
standing of plasma proteins and their roles in different urokinase plasminogen activator (glutaryl-GR-MCA,
diseases,butaredifficulttouseforroutinediseasedetection 502.5MW)andplasmin(Boc-VLK-MCA,615.8MW)were
in a clinical setting (Diamandis, 2004) because of the purchased from Bachem (King of Prussia, PA). Three
technicallydemandingandtime-consumingsampleprepara- hundred sixty-one fluorogenic substrates were synthesized
tion requirements (Diamandis, 2004; Fujii et al., 2005). in a library of the general format Ac-A-P -P -R-coumarin
3 2
2DGEuseisfurtherhinderedbyitslowthroughputnature. (where P and P were naturally occurring amino acids
3 2
These limitations are especially true in coagulation diag- except Cys) according to protocols detailed previously
nostics where point-of-care devices are often needed for (Backesetal.,2000;Gosaliaetal.,2005a,b).
emergency or home use. While MS methods are relatively
highthroughput,theydonotnecessarilyrevealthekineticsor Microarray Printing
functionalinteractionswithintheplasmaproteasecascades.
Lysinecoatedglassslides(ErieScientific,NewHampshire)
Currently,activityinformationisaccessedinclinicalsettings
for arraying were cleaned in 100% ethanol and dried. The
through activated partial thromboplastin time (aPTT) or
specificfluorogenicsubstratesweredilutedin50%glycerol/
activated clotting time (ACT, also known as prothrombin
DMSO to 1 mM prior to arraying and printed in quad-
time, PT), which crudely measure activity through blood
ruplicates per sub-array with two sub-arrays per slide. The
gelation rates. Capturing activity information more effi-
361 compound fluorogenic substrate library, also in 50%
cientlyandspecificallyshouldprovidedramaticincreasesin
glycerol/DMSO, was printed at 50 mM in a 16(cid:1)24 format
diagnosticpower.
equivalent to a 384-well plate. All slides had internal
One way to rapidly and selectively capture protease
calibrationstandardstoenableintra-andinter-slidequanti-
activityinformationistousefluorogeniccoumarinsubstrates
ficationandnormalization.Thefluorogenicsubstrateswere
(Backes et al., 2000). These substrates become fluorescent
printedaccordingtoprotocolspreviouslydescribed(Gosalia
upon proteolytic cleavage, thereby allowing for ready
et al., 2005a,b). After printing, the slides were refrigerated
detection of protease activity. The challenge is that the
andstoreddesiccatedandinthedarkuntiluse.
proteasesinthebloodcoagulationcascadehaveoverlapping
specificities, and no exclusive one-to-one linkage exists
Reactions and Data Analysis
between each protease and an individual fluorogenic
substrate.Assuch,wehaveusedsolutionphasemicroarrays The proteases were reconstituted and diluted in buffers
of a larger set of substrates to profile the individual blood recommended by the manufacturers. The proteases and
proteases to help define groups of optimal substrate plasmamixtureswere individuallydeliveredtothespecific
1100 BiotechnologyandBioengineering,Vol.94,No.6,August20,2006
DOI10.1002/bit
substratearraysandthe361compoundlibraryaspreviously Determinationof Optimal Substrates
described (Gosalia and Diamond, 2003; Gosalia et al.,
Todeterminetheoptimalrelativesubstrate,thecleavageofeach
2005a,b) and underwent (cid:2)30-fold dilution from the final
substrate(s)byasingleenzyme(subscripte)wascomparedto
deliveryconcentrationaftermixinginthemicrospot(Gosalia i
thecleavageofthatsubstratebyallenzymestodeterminethe
andDiamond,2003;Gosaliaetal.,2005a,b).
specificity (S) in Eqn. 2. At the same time, the cleavage
The final delivery concentrations were: 1 mM plasma
percentageforallsubstrateswasnormalizedandscaled(C)on
kallikrein in 4 mM NaOAc-HCl, 150 mM NaCl (pH 5.3);
therange0–1asshowninEqn.3.Theoptimalsubstrate(Q)
20 mM factor Xa in 20 mM Tris, 700 mM NaCl (pH 7.4); e
wasdeterminedtobethesubstratewithamaximumweighted
10mMhumanthrombinin50mMsodiumcitrate,200mM
combination of specificity (weighted by w) and cleavage
NaCl,0.1%PEG-8000(pH6.5);10mMuPAin,150mMPBS s
percentage(weightedbyw )asshowninEqn.4.
(pH7.2);10mMplasminin100mMHEPES,100mMNaOAc c
s
(pH8.5);10mMfactoraXIIain4mMNaOAc-HCl,150mM S ¼ i;e ð2Þ
NaCl (pH 5.3); 100 nM factor XIa in 4 mM NaOAc-HCl, i;e Psi;e
150mMNaCl(pH5.3);10mMfactorIXabin20mMTris- e
HCl,100mMNaCl(pH7.4);5mMfactorVIIain20mMTris-
s (cid:3)minðs Þ
HCl,100mMNaCl,1mMCaCl2(pH7.4);and10mMAPCin i;e i;e
C ¼ i ð3Þ
20mMTris-HCl,100mMNaCl(pH7.4).CitratedPFPwas i;e maxðs Þ(cid:3)minðs Þ
i;e i;e
diluted threefold before being delivered to the specific i i
substrate array and the 361 compound microarray. The
Q ¼maxðw S þw C Þ ð4Þ
mixtures consisted of 1 part citrated plasma, 1 part 30 mM e s i;e c i;e
i
calciumchlorideinHank’sbalancedsaltsolution(HBSS)and Severalvaluesofw andw between0.3and0.7weretested
s c
1partactivators[kaolin(1.5mg/mL),tissuefactor(81pM), andtherewasnotasignificantdifferencenotedintheselected
plasmin (1.5 mM) or uPA (1 mM)]. HBSS buffer was substrates.Also,modificationsofEqn.4weretestedwherethe
substitutedinabsenceofcalciumchlorideoractivators.Each maxfunctionwaschosenoftheproductofS andC .The
i,e i,e
mixturewasincubatedforexactly1or10minat378Cpriorto product maximization produced poorer results in the diag-
beingusedwiththeenzymeassaymicroarray,thusrepresent- onalizationofthesubstrateandsubstratecocktailmatrices.As
ingatransientstateofplasma.Intheeventofaclotformation, aresult,forallreportedresults,w ¼w ¼0.5(Eqn.4).
s c
themixturewascentrifugedandtheserumwasused.
Theslides(n¼4forspecificsubstrates,n¼6forsubstrate
Deconvolution of Array Data
library) were incubated at 378C for 30 min to 6 h. This
incubationtimeresultedin(cid:2)5to25%cleavage ofthebest Thedatafromthemicroarraysweredeconvolutedusingthe
substrate on each array, assuring that the assays were run lsqnonnegfunctionfromMatlabR14servicepack2basedon
within the linear range. The activated slides were scanned Lawson and Hanson’s nnls function (Lawson and Hanson,
using a cooled CCD-based imager (NovaRay, Alpha 1974). This function works by computing the value of
Innotech,SanLeandro,CA),atExl:405/40nmandEml: theregressedweights(W)forarepresentationoftheprotease
475/40 nm with integration times of 250 msec and 15 mm maps as vectors (E) that minimizes the Euclidean distance
pixelresolution.Imageswereacquiredina16-bitformatand betweentheactualplasmamaps(A)andtheregressedvalues
the analysis was performed using Array Vision (Imaging (R) (Eqns. 5 and 6). The non-negativity constraint was
Research,Ontario,Canada). maintainedbyselectingthemostpositiveelementfromthe
The percent cleavage for every substrate in the 361 regression where all other elements are maintained at 0,
compoundlibrarywasdeterminedforeachproteaseusingthe holding that element as required, and then repeating the
followingformula regressionwiththatelementnon-zeroandchoosingthenext
f (cid:3)f mostpositiveelement.Ittheniterateduntiltheallremaining
PercentCleavage ¼1(cid:3) max;e i;e ð1Þ
i;e f (cid:3)f zeroweightelementsresultedinnegativeorzeroweights.
max;e min;e
minkA(cid:3)EWk ð5Þ
where fmax is the mean fluorescence intensity of the W(cid:4)0 2
calibration standard (n¼8 spots per slide of 50 mM
unacylated coumarin), f represents mean blank (with no X
min R ¼ WE ð6Þ
substrate)intensity(n¼15spots/slide)andf standsforthe i i i
i,e i
fluorescenceofindividual substrates (i)after cleavage by a
particular enzyme (e). The final cleavage percentages for
RESULTS
each substrate in the library were averaged (n¼6) and
combinedtogivecleavagepercentreferencemapsforevery
FluorogenicSubstrate Microarraysfor
protease. The maps were represented using Cluster and
Individual Proteases and Plasma
Treeview (Ewald and Eisenberg, 1995). For the most
preferred substrates the signal/background ratio was typi- ThefluorogenicpeptidesubstratesZ-FR-MCA,Boc-IEGR-
cally>60with CV<5%. MCA, Boc-VPR-MCA, glutaryl-GR-MCA, and Boc-VLK-
Gosaliaetal.:FunctionalBloodMicroarrays 1101
BiotechnologyandBioengineering.DOI10.1002/bit