The activation of factor X and prothrombin by
recombinant factor VIIa in vivo is mediated by
tissue factor.
H ten Cate, … , B L Kass, R D Rosenberg
J Clin Invest. 1993;92(3):1207-1212. https://doi.org/10.1172/JCI116691.
The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic
mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with
endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints.
Research Article
Find the latest version:
The
Activation of Factor X and Prothrombin by Recombinant
Factor
Vila In Vivo Is Mediated by Tissue Factor
HugotenCate,** KennethA.
Bauer,III
Marcel Levi,t ThomasS.Edgington,' RichardD.Sublett,** SamadBarzegar,§Bryon L.Kass,§and Robert D.Rosenberg§*
*Department ofInternal Medicine, Slotervaart Hospital, and*Centerfor Hemostasis, Thrombosis, Atherosclerosis, andInflammation Research, AcademicMedicalCenter, 1105AZAmsterdam, TheNetherlands;
IDepartment
ofMedicine, Beth IsraelHospitaland IIBrockton-WestRoxbury Department of Veterans Affairs Medical Center, Harvard Medical School, Boston,Massachusetts02215;'The Scripps Research Institute, LaJolla, California92037; **R. W. Johnson Pharmaceutical ResearchInstitute,SanDiego, California 92121; andt*DepartmentofBiologyandWhitaker College,Massachusetts InstituteofTechnology,Cambridge, Massachusetts 02139Abstract
The human coagulation system continuously generates very
smallquantities of Factor Xa and thrombin. Current evidence
suggests that basal level activation ofthe hemostatic mecha-nismoccursviaFactor VIIa-dependent activation of Factor X,
butdirectproof hasnotbeen available fortheparticipation of tissue factor in this pathway.Toexamine thisissue,weinfused relatively high concentrations of recombinant Factor VIla
(
-50ugg/
kg body wt)
into normalchimpanzees
andobservedsignificant increases in the plasma levelsofFactor IX activa-tionpeptide,Factor Xactivationpeptide,andprothrombin
acti-vationfragment
F,12.
Metabolic turnover studies withradiola-beled FactorIXactivationpeptide, FactorXactivation peptide, andF1+2 indicate that elevated levels of the activation peptides
areduetoaccelerated conversion ofthe threecoagulation sys-temzymogens into serine proteases.The administration ofa potentmonoclonalantibodytotissuefactor,whichimmediately
neutralizes function oftheFactor
VIla-tissue
factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also sup-presses basal level activation of FactorIXand FactorX.The aboveresults suggest thatrecombinant FactorVIlafunctionsas aprohemostaticagentbyinteracting with endogenous tissue factor sites,butdefinitive proofwillrequire studiesin hemo-philic animals using relevant hemostatic endpoints. (J. Clin. Invest. 1993.92:1207-1212.) Key words:blood
coagulation-FactorVII*tissuefactor*radioimmunoassay*monoclonal
an-tibody
Introduction
The human coagulation mechanism iscomposed of intrinsic and extrinsicreaction cascades, whichconverge at the levelof
Factor X and lead through a finalcommon pathway to the
generation of thrombin and formation of fibrin (1, 2).
Pre-vious in vitro investigationsdemonstrated that intrinsic
cas-cadefunction involvesthe Factor XIIa-dependentactivation of Factor XI, and the subsequentFactor XIa-dependent acti-vation ofFactorIX. Howeverithasrecently been shown that thrombin canactivateFactor XI in thepresence ofa negatively
Addresscorrespondence to Kenneth A. Bauer, M.D., Beth Israel Hospi-tal, 330 Brookline Avenue, Boston,MA02215.
Receivedfor publication 21 December1992and in revisedform 9 April 1993.
TheJournalof ClinicalInvestigation,Inc.
Volume 92,September 1993, 1207-1212
charged surface(3, 4). Earlyinvitro studiesrevealed that the FactorVIIa-tissue factor(TF)'complexcannotonlyactivate
FactorX, but also FactorIX(5, 6). Using specific immunoas-saysfor activation peptides, we have shown in humans that
Factor VII,butnot FactorXI, ismainly responsiblefor Factor
IXactivation under basal conditions (7).Indeed, Factor
VII-deficientpatients exhibit drastically reduced plasma
concen-trations of FactorIXactivationpeptide (FIXP) and FactorX
activationpeptide (FXP), whicharerestoredtonormal levels by infusion of relatively low concentrations (10-20 ,g/kg body wt) of recombinant FactorVIIa(7-9).
Although Factor VIIaatphysiological concentrations pos-sesses verylittleabilityto directly activate FactorX in vitro (10), there is currently no direct evidence that activation of thiszymogenby FactorVIa invivorequiresassociation with TF.Patients withFactor VIIIinhibitors have been infusedwith
largeamountsofrecombinant Factor VIIawithbeneficial
ef-fects on hemostasis (11-13). Considerable doubt exists
whether TFis required for the aboveresponse,andit has been demonstrated thataphospholipidsurface aloneis sufficientto promoteactivationofFactorXby Factor VIIa ( 14-16).Inthe
presentcommunication, weinfuse relatively high
concentra-tions of recombinant Factor VIIa into normal chimpanzees and demonstrate accelerated activation of FactorIX,FactorX,
and prothrombin. The administration ofa potent mAb
di-rectedagainsthumanTF totheanimals inhibits the activation of FactorXandprothrombin bytheinfused recombinant
pro-tein,and alsosuppressesbasal level activation of Factor IX and Factor X.
Methods
Chimpanzees.Adult malechimpanzees (Pan troglodytes)werehoused
atthe NewYorkUniversity MedicalCenterLaboratoryfor Experimen-tal Medicine andSurgeryin Primates(Tuxedo, NY) and the New MexicoRegionalPrimate Research Laboratories (Alamagordo,NM). Theanimals selected for study weighed between 52 and 62 kg, exhib-ited normalkidneyaswellasliverfunction,andhadnotparticipatedin infectiousdisease investigations duringthetimeperiodofthe Factor VIIa infusions. The general anesthetics used in these investigations
wereeitherketamineorhalothane, and the length of the experiments did notexceed 6 h. In separate controlexperiments, nosignificant differenceswereobserved between thetwoanesthetics withregardto
the levels ofactivationpeptidesgenerated (datanotshown).Thestudy
protocols wereapproved bythe animal health and welfare committee of the primate centers andwereconductedaccording to the guidelines oftheAmericanPhysiological Society.
Blood collectionandprocessing. Venous blood was obtained from theantecubital veins of chimpanzeesby twosyringe technique using 21-gaugebutterflyneedles.Blood samplesforthe FIXPand FXP RIAs were drawnintoplastic syringes preloaded with the following anticoag-ulant: 38 mmol/liter citric acid, 75mmol/litersodium citrate, 136 mmol/literdextrose, 6mmol/literEDTA,6mmol/literadenosine, and 25 U/mlheparin. The ratio of anticoagulanttoblood used was 0.2:1.0(vol/vol).For theF,12andfibrinopeptideA(FPA)assays, an anticoagulantcontainingathrombininhibitor, EDTA,andaprotinin was purchasedfrom Byk-Sangtec (Dietzenbach, Germany); the ratio ofanticoagulanttoblood usedwas0.1:0.9(vol/vol). The measure-mentsofcoagulation factors,FactorVII/VIIa antigenlevels, and cell counts werecarriedoutonblood collected insodium citrateat afinal concentration of3.8% (wt/vol). Allplasma specimenswere centri-fugedat4VCfor20 minat1,600g,and storedat-70'Cuntilassayed. Assays. FactorVII/VIIaantigen levels were determined with an enzyme-linked immunosorbent assay (Asserachrom VII; American BioproductsCo.,Parsippany,NJ). TheFIXPs,FXPs,and were quan-tified withspecific immunoassaysasdescribedpreviously (7, 8, 17, 18). The FPAmeasurementswere establishedbyradioimmunoassay with a kit provided by Byk-Sangtec.
Chimpanzeeproteinpurification.ChimpanzeeFactorIX,Factor X, and prothrombin were isolated from barium chloride-adsorbed plasma by affinity chromatography usingspecificmAbs forhuman plasma proteins, which weregenerously provided by Drs. D. Bing (Center forBloodResearch, Boston, MA),D.S. Fair(University of Texas,Tyler,TX), and B.Furie(NewEngland Medical Center, Bos-ton,MA),respectively. Briefly, chimpanzee plasma (I liter)was sub-jectedtobarium chlorideadsorption (final concentration 70 mmol/ liter), elution with 0.17 mol/liter trisodium citrate, and dialysis againstaTris-buffered salinesolution(0.05MTris-HCl, 0.5MNaCl, pH 7.4)containing 0.02% (vol/vol)Tween 20(19).Theanti-human Factor IX mAbmatrix (6.6mgofmagnesium-dependent antibody coupledto2mlofSepharose[Pharmacia Fine Chemicals, Piscataway, NJ]) wasequilibrated with dialysis buffer adjustedto7.5mmol/liter
MgCl2.Theplasmaeluate wascirculated throughtheaffinitycolumn for90 min, thematrixwaswashedwith 1 literof equilibration buffer, and Factor IXwaselutedwith thesamebuffertowhichwasadded10 mmol/literEDTA, butnoMgCl2.Thepurification of Factor Xwas
accomplished inasimilar manner to thatdescribed for FactorIX, except thatadsorptionof plasmaeluatetotheanti-humanFactor X mAb (5 mg ofantibody coupledto5 mlofAgarose[ PharmaciaFine
Chemicals])wascarriedoutwithTBScontaining5mmol/literCaC12, and boundproteinwaseluted withTris-buffered saline containing20 mmol/liter ofEDTA.Theisolation ofprothrombinwasundertakenas
outlined above except thatadsorption of plasmaeluatetothe
anti-hu-manprothrombinmAb(6mgofantibody coupledto2mlof Sepha-rose)wasconductedwithTBScontaining 10mmol/literCaCl2and boundproteinwaselutedwithTBScontaining3mmol/ literEDTA.
Thefinalconcentrations ofthechimpanzeeFactorIX,FactorX, andprothrombinpreparationswere 100nmol/liter,and 617nmol/ liter, and 1,473nmol/liter, respectively,asdeterminedby immunoas-says directed againstthe various humanproteins(7, 18).The above products were also examined bynondenaturing gel electrophoresis, andwereshowntobehomogeneous with respecttosizebytheSDSgel electrophoreticsystemof Laemmli (20).Theindividualvitamin K-dependent proteinspurifiedfromchimpanzee plasmamigratedatthe
same rate ashumanFactorIX, human FactorX,andprothrombin, respectively. The levels ofcontaminating activation peptidesin the FactorIX, Factor X, andprothrombin preparationsasjudgedby im-munoassaysagainst the human activation fragment counterparts were undetectable,1%,atd0.1%,respectively, on a molar basis as compared
tothezymogens.Human FactorIX,human FactorX,and prothrom-binwereobtained from Enzyme Research Laboratories, South Bend, IN, andwerephysicallyhomogeneousasjudgedbynondenaturating gelelectrophoresis. The human coagulation proteinswerediluted in
0.05 MTris-HCl, 0.10MNaCl,pH 7.5,containing4%(wt/vol) oval-bumintoconcentrations comparabletothechimpanzee preparations. Thechimpanzee and human proteins were then activated in vitro, and the levels of activationpeptidesdetermined. The Factor IX and Factor Xpreparations were incubated for 3 h at370Cwith final concentra-tions of 5.7 nmol/liter to 50 nnmol/liter recombinant Factor VIla, 4.9 nmol/liter relipidated human brain tissue factor (Drs. Ronald Bach and YaleNemerson, Mount Sinai Medical Center, New York), and 4.9mmol/liter CaCl2. Similarly, prothrombin preparations were incu-bated for 60 minat240Cin I mlreaction mixturescontaining10 mM CaCl2with 10Mghuman Factor V, 10gghumanFactor Xa, 500Mg prothrombin, and 70Migrabbitcephalin.
Determination ofthe metabolic behavior ofactivation peptides. The radiolabelingofFIXP, FXP, andF,12wascarried out by the chlora-mine-T method of Greenwood et al. (21 ) using 3Mgof human activa-tion peptideand0.5mCiofcarrier-freeNa'251 orNa 31I (New England Nuclear,Billerica, MA). The activation fragments were separated from free iodide by Sephadex G-10 gel filtration (FIXP and FXP), or Sepha-dexG-25 gel filtration(F,1+2)(Pharmacia FineChemicals). Before ad-ministration of radiolabeled peptide, animals were given 1 ml of Lu-gol'ssolution intheir drinking water, and the above treatment was continued forthreedays after infusion. Approximately50MCi of la-beledpeptide was infused as a bolus into a peripheral vein of chimpan-zees, andserial blood samplesweredrawn bytwo-syringe technique into theanticoagulant usedfor theF,+2and FPA assays through an indwellingvenous catheterplaced in theoppositearmvein.After cen-trifugingthebloodsamplesat1,600gfor1O min,the1251/'3'I counts in 0.5 mlofplasma werequantifiedin a Gamma 8000 Counting System (Beckman InstrumentsInc.,Irvine,CA). The plasma protein radioac-tivitydeterminationsaredescribedas afunction of timeby a two-ex-ponentialcurve,
CIe'rI
+C2er2t(22).Thefractionalbreakdown rate, kB,wascalculatedfrom (C,/r, +C2/r2)-'
(22).Recombinant proteins and monoclonal antibodies. Human recom-binant Factor VIIa (23)wasobtained asalyophilizedpowderfrom NovoNordisk (Gentofte, Denmark) and was reconstituted in sterile waterbefore use. The anti-TF mAb TF8-5G9, which blocks Factor X activation by the Factor VIIa-TF complex in vitro, was provided at a proteinconcentration of 5.5 mg/ml in PBS (24, 25).
Statisticalanalysis.The withinandbetween group differences were analyzed by ANOVA, and P values were calculated with Newman-Keul's test to correctfor multiple comparisons (26). P values below 0.05wereconsideredstatistically significant.
Results
The immunoreactivities of chimpanzee FIXP, chimpanzee
FXP,andchimpanzee
F,+2
werecomparedwith their respec-tive humanfragment homologues.This wasaccomplished by activating purified chimpanzee and humanzymogens,which had beenquantified by immunoassays using human proteins, and thenmeasuringthe levels of activationpeptides using im-munoassaysdirectedagainst the human activation fragments. Ona molarbasis,the resultsobtained show thatchimpanzeeFactorIX, chimpanzee Factor X, and chimpanzee prothrom-binwere converted to their activatedproductsat a levelof100, 61, and 100%, respectively, whereashuman FactorIX, human
FactorX, and human factorprothrombinwereconvertedto
their activated productsatalevelof 87, 75, and 91%,
respec-tively,under identical reactionconditions.Thesedataindicate
thattheimmunoreactivities ofchimpanzeeand human zymo-gensand therespectiveactivationpeptidesarevirtually iden-tical.
Given these observations, we used immunoassays
previ-ously developedtoquantifyhumancoagulationzymogen
acti-vation to measurethe effects of recombinanthuman Factor
Table . Temporal Changes in Plasma FIXP Levels after Infusion ofRecombinant Factor VIIa (3 mg) Alone,
or in Combination with mAb TF8-5G9 (600
jig/kg
Body Wt) in Chimpanzeesmin
Animal I=0 1 =5 t= 15 t=30 t=60 t=120 t =180 i=240 t=300
pmol/liter
Factor VIIa
1 103 160 199 167 161 132 154 101 109
2 165 151 222 192 224 179 132 121 143
3 116 111 137 141 179 162 118 116 122
4 123 207 ND 223 180 166 126 134 116
5 196 199 280 393 345 294 252 203 197
Mean 141 166 210* 223$ 218§ 187 156 135 137
SEM 17 17 30 44 33 28 25 18 16
FactorVIa+TF8-5G9
6 234 ND ND 227 259 237 188 143 120
7 153 178 230 292 305 296 230 209 168
8 280 364 375 499 470 409 314 337 260
Mean 222 271 302 33911 34511 314 244 230 183
SEM 37 93 72 82 64 50 37 57 41
*P<0.05, P<0.01, §P< 0.02, ascompared with FIXPat t=
0.
"0.05<
P<
0.10 ascomparedwith FIXPat t=0.
VIla (-=
50,gg/kg
body wt) to five chimpanzees produced at 5 plasma FactorVIlaconcentrations also was associated with an min animmediateincrease in mean FactorVII/VIIa
antigen increase in meanF1+2
levels of 2.2-fold at 2 h, which remainedlevelsfrom 53% ofnormal±6.4 (SD) to 191%±21. The levels high at the termination of the experiments (Table III). The
only graduallyreturned towardsbaselinelevels, and remained time-dependent changes in plasma FPA levels were virtually
elevated at 88%±9.0 (SD) at S h. The augmentation of the identical to those of
F,+2,
rising from 8.8 nmol/liter to a peakplasmaFactor VIIa measurements was associated with an aver- of 68.2 nmol/liter at 1 h, and remained elevated at 20.8 nmol/ ageincreasein FIXP and FXPof 1.6-foldat30min(Table
I)
literattheendof theexperiments (datanotshown).Itshouldand3-foldat 15min(TableII),respectively. These elevations be noted that the highest concentrations of
F1+2
as well asFPAgradually returned to baseline values at 4 h. The elevation in in chimpanzees were observed at 1-2 h, which were delayed as
TableII. Temporal ChangesinPlasmaFXPLevelsafter Infusion ofRecombinantFactor VIIa(3mg)Alone,
orinCombination with mAbTF8-5G9(600,ug/kg) inChimpanzees
min
Animal t=0 t=5 t= 15 t=30 t=60 t=120 t=180 t=240 t=300
pmol/liter
FactorVIla
1 73 211 ND 184 124 93 64 38 31
2 157 207 383 365 336 268 203 142 157
3 108 214 317 316 328 174 162 116 113
4 82 244 ND 329 214 116 93 86 93
5 178 220 366 371 372 279 236 206 201
Mean 120 219* 355$ 313$ 275$ 186§ 152 118 119
SEM 21 7 20 34 46 38 32 28 29
FactorVIla+TF8-5G9
6 177 ND ND 162 178 138 121 109 114
7 72 83 97 110 94 95 65 60 50
8 108 88 92 142 121 129 92 ND 77
Mean 119 85.5 94.5 138 131 121 92.7 84.5 80.3
SEM 31 2.5 2.5 15 1 13 16 24 18
Table III. Temporal Changesin PlasmaF1,2LevelsafterIntravenousInfusionofRecombinant Factor VIla (3 mg) Alone, orinCombination with mAbTF8-5G9(600,ug/kgBody Wt)inChimpanzees
min
Animal t =0 t=5 1= 15 =30 t=60 t=120 t=180 t=240 t = 300
pmol/liter
Factor VIa
1 1.01 0.99 1.16 1.32 1.68 1.31 1.43 1.41 1.46
2 1.55 1.71 2.03 2.04 2.40 2.71 2.93 3.27 ND
3 0.83 0.89 1.10 1.42 2.70 2.76 1.98 1.77 2.14
4 1.23 0.96 ND 1.18 1.98 1.50 1.06 1.04 1.01
5 0.68 1.59 1.80 2.18 2.59 3.34 3.89 3.03 2.72
Mean 1.06 1.23 1.52 1.63 2.27* 2.32t 2.26t 2.10§ 1.83
SEM 0.15 0.17 0.23 0.20 0.19 0.39 0.51 0.44 0.38
FactorVIla+TF8-5G9
6 0.79 ND ND 0.74 0.78 0.81 0.80 0.76 0.72
7 0.87 0.82 0.78 0.83 0.78 0.71 0.59 0.54 0.58
8 1.53 1.42 1.63 1.55 1.56 1.71 1.52 1.21 1.09
Mean 1.06 1.12 1.20 1.04 1.04 1.08 0.97 0.84 0.80
SEM 0.23 0.30 0.42 0.26 0.26 0.32 0.28 0.20 0.15
*P<0.02. tP<0.01,
§P<0.05,ascomparedwithF,+2att=O.
compared with the peak FXP levels at 15-30 min and
re-mained elevated fora greaterperiod oftime (TablesII and III).
The elevated plasma concentrations ofFIXP, FXP, and
F,12
areattributabletoincreasedzymogenactivation andfrag-mentgeneration rather than decreased clearance of the activa-tionpeptides. Either '31I-FIXP and '251-FXP or
'25I-F,12
wereinfused into chimpanzees, withandwithoutrecombinant Fac-tor VIIa; the fractional breakdown constants of the markers
wereestimated asoutlined in Methods. The calculated frac-tional breakdownconstantsofFIXP, FXP, andF1+2are2.34,
2.59,and0.34, respectively, in the absence of Factor
VIla,
and2.78,3.13, and 0.43, respectively, in thepresenceof the serine
protease.
Wethendetermined the involvement ofTFinFactor
VIIa-induced alterations of coagulationsystemactivation by
admin-isteringtothree chimpanzeesasingle intravenousdoseof 600
,ug/kg
ofmAbTF8-5G9just before infusing 3mgofrecombi-nantenzyme.Theelevations of theplasmaconcentrations of
FXPand
F,12
induced byFactor VIIa wereabolished(Tables IIandIII), butsuppressionofthe augmentedFIXPlevelswas not
observed intwoof the three animals (TableI). Wealso
exam-ined theeffectoftheanti-TF mAbonthe basalactivity ofthe
coagulationsystem. TwochimpanzeeswereadministeredmAb TF8-5G9at adoseof4mg/kg bodywtandtwochimpanzees
wereinfused with buffer without mAb. The plasma levels of
FIXP, FXP, and F1,2weredetermined initiallyand atvarying intervalsup to 24 hafter mAbadministration. The mean
con-centrations ofFIXP and FXPdecreased from 86.8 and 108
pmol/liter initially,to41.9 and 66.4 pmol/literat 6 h, and to 22.7 and 26.5 pmol/liter at 24 h, respectively, in the mAb-treated animals. In the control animalsreceiving buffer, the mean concentrations of FIXP and FXP were 202 and 158
pmol/liter initially,200 and 177 pmol/liter at 6 h,and 170 and 121 pmol/literat 24 h,respectively.These resultssuggest that
neutralization ofFactor VIIa-TF by the mAbsuppresses basal levelactivation ofFactor IX and Factor X, although a
pro-longedperiodof time isrequired for maximal effect.
Interest-ingly,nodownward trendwasnoted inplasmaF,12 measure-ments(data not shown).
Discussion
Underphysiologic conditions,thehumancoagulation system continuously generates small amounts of Factor Xa and thrombin(8, 18).The current evidence suggests that this basal level activation ofthe hemostatic mechanism occursviaFactor VIIa,and theparticipation ofTF, thoughimplied, remainsto be substantiated(7-9).The present setof studies in
chimpan-zeesattempts toresolve this issue. Firstly,wedemonstrate that recombinant Factor VIla infusions (
50,ug/kgbody
wt)pro-ducesignificant increasesin the levels of FIXP, FXP, and
F,1+2*
Secondly,weprovideevidencethat recombinant Factor
VIIa-inducedelevations in the levels ofFXPand
F,12
are duetoaccelerated conversion ofthe coagulation system zymogens
into theirrespectiveserineproteasesrather than altered
meta-bolicbehavior of the activation fragments. Thirdly,we show that neutralization ofTFand Factor VIIa-TFcomplex by ad-ministration ofaspecificmAb(TF8-5G9)abolishes the activa-tion ofFactor X and prothrombin by recombinant Factor VIIa.Fourthly,wedemonstrate thatinfusionsof this anti-TF mAbleadto atime-dependentreduction in the basal levelsof
FIXPand FXP.
Theexperimental resultsoutlinedabove can beexplained byhypothesizingthatTFmoleculeson cellsurfaces in
chim-panzees aresaturatedwithvarying proportionsofFactor VIIor Factor VIIa. The Factor VIIa-TF complex results from the
proteolytic activation ofFactor VII-TF by Factor Xa (27),
Factor IXa(28),orFactor VIIa-TF(29). The resultant
com-plexes initiatelowlevelconversion ofFactor IXandFactor X toactiveenzymes, the latterofwhich is responsible foramajor
portionofthebasalactivityofthecoagulation mechanism(9). Upon infusion of recombinantFactorVIIa, increasedamounts
ofFactor VIIavisavisFactor VIIassociate withTF.Giventhat the Factor VIIa-TFcomplex,ascompared withFactor VII-TF
interaction product, exhibits 120-foldor greaterprocoagulant
potency, the activation of Factor IX and Factor X is
aug-mented(30, 31 ).
The FIXPandFXPlevels in chimpanzees reachapogees at
15-30 min, somewhat delayed in comparison to the almost immediateincreases in plasma concentrations of recombinant
FactorVIa. Itis of interestto note thatsimilar investigations conducted with lower doses of recombinant FactorVIIa (10-20
jig/kg
bodywt) in aseverelyFactorVII-deficient humanwith essentially no coagulant protein showed thatproduction ofFIXPand FXPtakes place almost immediately, whereas
virtuallyidentical studies carriedoutwith a Factor VII-defi-cientpatient withareasonable level of immunoreactive
pro-tein(17% ofnormal) revealed that generation ofFIXP and
FXP occurs with some time delay as described above (9).
Thus, itappearsthatplasma FactorVII mustbecompetitively
displacedfromsome TFsites by recombinant Factor VIIa
be-fore activation of Factor IX and Factor X is observed. The
elevationsofFXPand FIXP levels inchimpanzees persist for
- 2 h, which cannot be explained by Factor VIIa-induced
alterations in the metabolicbehaviorofthe activation markers. Indeed, in theFactorVII-deficientpatients whowereinfused withtherecombinantprotein, increasedFXPand FIXP
con-centrationssimilarlyoccur overrelatively prolonged periods of time (9). Thesepersistentelevations probablyresultfrom
on-going activation of thezymogensbyFactorVIIa-TF complex which isonly slowlyneutralizedbytissuefactorpathway
inhibi-tor(TFPI)(32).Theambientplasmaconcentrations ofTFPI average2.5nmol/liter
(33),
whichareaboutonefourth of the peak plasma levelsof recombinantfactor VIa achieved inourchimpanzee studies. Thus, normal plasma concentrations of
TFPI maybeinsufficienttoinhibitthe levelsofFactor
VIIa-TFcomplexgenerated inourinvestigations.Alternatively,the requirement forTFPIto initially interact withFactor Xa
fol-lowed byassemblywithFactor VIIa-TF maydiminish therate
ofinhibitiontopermitthe observed FIXP andFXPactivation
rates(34).TheelevationsofF,12aswellas FPAin
chimpan-zeesreach maximal valuesat 1-2h, whicharedelayedas
com-paredtothepeak levels ofFXP at15-30minandremain raised for alonger period oftime. The delayed generation of
F,12
versus FXPand FIXP has also been observedin humans in-fused with recombinant FactorVIla (9),andagaincannot
re-sultfrom altered metabolic behavior of this activation marker in the animals. We hypothesize that the above phenomena
may bedueto agradual time-dependent loss ofTFPI
inhibi-toryactionor atime dependent activation of cell surfaces such
as platelets resulting in enhanced prothrombinase assembly
andfunction (35).
Intwoof the three chimpanzees that received anti-TF mAb before recombinant FactorVIa,suppression of the increased
FIXPlevelswas notobserved, but increasedFactor Xa
genera-tion as wellas prothrombin activation did not occur. Ithas recently been demonstrated that an antibody to TF
(TF8-11 D12), which is undoubtedlyidenticaltoTF8-5G9, also in-hibits Factor IX activationand Factor VII autoactivation in
additionto Factor X activationby the Factor VIIa-TF
com-plex in vitro (36). Theseantibodiesdo notinterfere with Fac-torVIIabindingtoTF, but rather appear toblockthe accessof the macomolecularsubstrates tothe enzyme's active site by sterichindrance (25, 36).We areuncertain ofthe reasonfor the discordance between the HXPand FXP results in these
animals.It couldbe duetothe presenceofanintermediatein the FactorIXactivation pathway,suchasFactorIXa, thatis
resistanttoinhibition bytheanti-TFmAb uponconversionto
FactorIXa by Factor VIIa-TF (37). Alternatively, it is conceiv-able that TF exists inadifferent conformation in vivoas
com-pared to in vitro and the mAb is unable to suppress Factor VIIa-TF-dependent conversion ofFactor IXbecause of the alteredstructure.
In the studies in which anti-TF mAb was administered alone, a marked decrease was notedinthe basal levelsofFIXP and FXP,whichwasmaximalat 24 h. At present, we cannot
explainthe absenceofaconcordantdecline in
F1.2
concentra-tions.However,inFactorVII-deficient patients,our
recombi-nant Factor VIIastudies indicate that the
F1,2
levelis not assensitive as FIXPand FXPto alterations in Factor VIIa-TF
activity.Inthesepatients, infusion oftherecombinant Factor
VIlaonly induced two- tofourfold increases inF1,2levels while
the othertwoactivation markers increased 10-20-fold. RecombinantFactorVIla preparationsare currentlybeing evaluated for treating bleeding episodes in hemophiliac
pa-tients with circulating Factor VIII inhibitors (12, 13). The aboveinvestigationsdemonstrate that the procoagulanteffect ofthe recombinant protein requires interaction ofenzymewith endogenousTFandthatmodestbut prolongedburstsof
coagu-lationsystemactivityoccurafter infusion.Theseobservations
suggest that recombinantFactor VIIapreparationsshould be
cautiouslyusedinsituationswherehighlevelsofTFexpression
are suspected toavoid excessive activation ofthehemostatic mechanismthat could lead tothrombotic complications.
The cellularsiteatwhichFactorVIIainteracts withTFhas
not been determined. The infused recombinant Factor VIa
could havecomplexedwith endothelial cellTF. However, histo-chemical and in situhybridization studies of human blood
ves-sels fail to detect TF protein or TF mRNA in unperturbed endothelial cells, although TFprotein has beenobserved on
foam cells of atherosclerotic plaques (38, 39).Itis possible that
TF is constituitively orepisodically expressed by endothelial cells andmonocytesinquantities sufficienttomaintain basal levelcoagulation activity,but too low to bereadilydetected. It seemsunlikelythattheoccurrenceofatheroscleroticplaques in
chimpanzees
could beresponsible
for basalactivity
of thecoag-ulation mechanism since such lesionsareinfrequent in animals used inourstudies that receive vegetarian diets (40). The
in-fusedrecombinantFactor VIIa cannotreadilybeimplicatedin
triggeringsynthesis of endothelial cellormonocyte TF. From
in vitro studies, it isknown thatendotoxin aswell as tumor
necrosis factor stimulates biosynthesis ofTF(41,42), butthis is unlikely with recombinant Factor VIIa thatis free ofthese
mediators. Theinfused recombinant Factor VIIa could have reachedsubendothelial sites whichhas been documentedwith otherlarge moleculesunderinvivoconditions (43). Perivascu-larandintramural fibroblastsexpress TF (39) and extremely
smallquantitiescanbefunctionallyidentifiedonvascular sub-endothelium (44).
Insummary, wehave demonstrated that the procoagulant responseelicited by infusion of highdoserecombinantFactor VIIa isdependent upon TF. The data suggest that the
prohe-mostatic effect ofthe recombinant protein, observed in
pa-tients withFactorVIIIinhibitors, necessitatesthe same interac-tion. However, ourexperimentswereperformedin
chimpan-zees,withouta bleeding diathesis,that were notsubjectedto vascular trauma. At suchsites, there could be excessive expo-sureof activated phospholipidmembranes aswellas TF. Thus
definitive proof ofthe mechanism of action of recombinant
Factor VIla will require comparisons of relevant hemostatic
presenceandabsenceofTFinhibition. Thepresentdataalso indicate that basal level coagulationsystemactivity isatleast
partially dependentupontheamountsofTFonbiologic
sur-faces in contactwithbloodcomponents. We havepreviously
documented age dependent elevations in activation peptide markers, andspeculate thatthistypeofbiochemical
hypercoag-ulability could, inpart,be duetoincreased biosynthesis of TF
in vivo (45).
Acknowledgments
WearegratefultoDr.W. W.Sochaand thetechnical staff of
Labora-toryfor Experimental Medicine and Surgery in Primates ofNewYork
University (Tuxedo, NY) for help with the chimpanzee experiments.
We thank Drs.Ulla Hedner and StevenGlazer(Novo Nordisk,
Gen-tofte, Denmark) for providing the recombinant Factor VIla used inour investigations.
ThesestudiesweresupportedbyNational Institutes ofHealthgrant
PO1 HL-33014, and byagrantfromtheDutchOrganization for Scien-tific ResearchtoDr.tenCate. Dr. Bauer isanEstablished Investigator oftheAmerican Heart Association.
References
1.MacFarlane, R.G. 1964.Anenzymecascadeinthe blood clotting
mecha-nism,andits functionasabiochemical amplifier. Nature (Lond.). 202:498-499.
2. Davie,E. W., and0.D. Ratnoff. 1964.Waterfallsequencefor intrinsic blood clotting. Science (Wash. DC). 145:1310-1312.
3. Naito,K.,and K.Fujikawa. 1991. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces. J. Biol. Chem. 266:7353-7358.
4.Gailani, D., andG. J. Broze, Jr. 1991. Factor XI activationinarevised modelof bloodcoagulation. Science (Wash. DC). 253:909-912.
5. Josso, F., and0.Prou-Wartelle. 1965. Interaction of tissue factorand factor
VIIattheearliestphaseof coagulation. Thromb.Diath. Haemorrh. 17:35-44. 6. Osterud, B., and S. I. Rapaport. 1977. Activation offactor IXby the
reac-tion product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc. Natl. Acad. Sci. USA. 74:5260-5264.
7. Bauer, K. A.,B. L. Kass, H.tenCate, J. J.Hawiger, andR. D.Rosenberg. 1990. Factor IX isactivated in vivo by the tissue factor mechanism. Blood. 76:731-736.
8. Bauer, K.A.,B. L.Kass, H.tenCate, M.A.Bednarek, J. J. Hawiger, and R. D. Rosenberg. 1989. Detection of Factor X activationin humans.Blood. 74:2007-2015.
9. Bauer, K.A.,P.M.Mannucci,A.Gringeri, F.Tradati, S. Barzegar, B. L. Kass,H.tenCate, A. S. Kestin, D. B. Brettler, and R.D.Rosenberg. 1992. Factor IXa-factor VIlla-cellsurface complex doesnotcontributetothebasal activation of the coagulation mechanism in vivo. Blood. 79:2039-2047.
10.Ruf, W.,A.Rehemtulla, and T. S. Edgington. 1991. Phospholipid-inde-pendentand -dependent interactions requiredfor tissue factor receptor and
co-factor function. J. Biol. Chem. 266:2158-2166.
1 1.Hedner, U., and W. Kisiel. 1983. Use of humanFactorVIIainthe treat-mentoftwohemophiliaApatients with high titerinhibitors. J. Clin. Invest.
71:1836-1841.
12. Hedner, U.,S.Glazer, K. Pingel, K.A.Albert,M.Blomback,S.
Schul-man,and H. Johnson. 1988. Successful useofrecombinant factorVIIaina
patient withseverehemophilia A duringsynovectomy. Lancet. ii: 1 193. 13.Macik,B.G.,J.Hohneker,H. R.Roberts, andA. M.Griffin.1989. Useof
recombinant activatedfactor VII fortreatmentofaretropharyngeal hemorrhage inahemophilic patient withahigh titer inhibitor.Am.J. Hematol.32:232-234.
14. Osterud, B.1983. Howtomeasurefactor VII and factorVIIactivation.
Haemostasis. 13:161-168.
15. Telgt, D. S., B.G. Macik, D. M. McCord,D. M.Monroe, andH. R.
Roberts. 1989.Mechanism by which recombinant factor VIa shortens the aPTT: activation of factor X in the absence of tissue factor. Thromb. Res. 56:603-609. 16. Rao, L. V. M., and S. I. Rapaport. 1990. Factor VIIa-catalyzed activation of factorXindependent of tissue factor: its possible significance for controlof
hemophilic bleedingbyinfused factor VIIa. Blood. 75:1069-1073.
17. Lau, H. K., J.S.Rosenberg,D.L.Beeler, andR.D.Rosenberg. 1979. The isolationandcharacterization ofaspecific antibody population directed against
the prothrombin activation fragments F2 and F+2. J.Biol. Chem.
254:8751-8761.
18. Teitel, J. M.,K. A.Bauer, H.K.Lau,and R. D.Rosenberg.1982. Studies of theprothrombinactivationpathway utilizingradioimmunoassaysfor theF2/
F,+2fragment and thethrombin-antithrombin complex. Blood. 59:1086-1097. 19.Liebman, H. A., S. A.Limentani,B.C.Furie,and B. Furie. 1985.
Immu-noaffinity purificationof factor IX(Christmas factor) byusing
conformation-specificantibodies directedagainstthe factor IX-metal complex.Proc. Nail. Acad. Sci. USA. 82:3879-3883.
20. Laemmli, U. K. 1970.Cleavage of structural proteinsduring the assembly of the head ofbacteriophage T4. Nature(Lond.).227:680-685.
21.Greenwood,F.C., W. M. Hunter, and J. S. Glover. 1963. The preparation of '311-labelled growth hormone ofhigh specific radioactivity. Biochem. J. 89:114-123.
22.Nosslin, B. 1973.Analysis of disappearance time-curves after single injec-tion of labelledproteins. CIBA Found. Symp. 9:113-130.
23. Thim, L., S.Bjoern,M.Christensen,E. M.Nicolaisen,T. Lund-Hansen, A.H.Pedersen, and U. Hedner. 1988. Amino acid sequence and
posttransla-tional modifications of humanfactor VIla fromplasmaandtransfectedbaby
hamsterkidneycells.Biochemistry. 27:7785-7793.
24. Morrissey,J.H., D. S.Fair,and T.S. Edgington. 1988. Monoclonal
antibody analysis of purified and cell-associated tissue factor. Thromb. Res.
52:247-261.
25.Ruf, W., and T. S.Edgington. 1991. An anti-tissue factor monoclonal
antibody which inhibitsTF-VIIacomplexis a potentanticoagulantinplasma. Thromb.Haemostas.66:529-533.
26. Zar, J. H. 1974. BiostatisticalAnalysis. Prentice-Hall, Inc.,Englewood
Cliffs,NJ.620 pp.
27. Radcliffe,R., and Y. Nemerson. 1975. Theactivationand control of
factorVIIby activated factorXandthrombin. Isolationandcharacterizationofa
single chain form of factorVII.J.Biol. Chem. 250:388-395.
28.Seligsohn, U.,B.Osterud,S. F.Brown,J. H.Griffin,and S. I.Rapaport. 1979.Activation ofhuman factor VII inplasmaand inpurifiedsystems. Roles of
activated Factor IX, kallikrein, and activated Factor XII. J. Clin. Invest. 64:1056-1065.
29.Nakagaki,T., D. C. Foster, K. L. Berkner, and W. Kisiel. 1991. Initiation
oftheextrinsic pathway of blood coagulation: evidence forthetissue factor
de-pendent autoactivation of human coagulation factor VII. Biochemistry.
30:10819-10824.
30.Zur, M., R. D.Radcliffe,J.Oberdick,and Y.Nemerson. 1982. The dual roleof factor VII in blood coagulation. Initiation and inhibition of a proteolytic systemby a zymogen. J.Biol. Chem.257:5623-563 1.
31.Williams,E. B.,S.Krishnaswamy, and K. G. Mann. 1989. Zymogen/en-zyme discrimination using peptide chloromethyl ketones. J. Biol. Chem.
264:7536-7545.
32.Rapaport,S.I. 1991. Theextrinsicpathwayinhibitor: a regulator of tissue
factor dependentbloodcoagulation. ThrombHaemostas.66:6-13.
33. Novotny, W. F., M. Palmier,T. C. Wun, G.J. Broze, Jr., and J. P.
Miletich. 1991.Purificationandproperties ofheparin-releasable
lipoprotein-as-sociatedcoagulation inhibitor. Blood. 78:394-400.
34. Broze, G. J., Jr., and J. P.Miletich.1987.Characterization of the
inhibi-tionof tissue factor inserum.Blood. 69:150-155.
35. Mann, K. G., M. E.Nesheim,W. R. Church, P. Haley, and S. Krishna-swamy. 1990.Surface-dependent reactionsof the vitamin K-dependent enzyme
complex. Blood.76:1-16.
36.Fiore,M.M.,P. F.,Neuenschwander,and J. H.Morrissey. 1992. An unusualantibodythat blockstissuefactor/factor VIla functionbyinhibiting cleavageonly ofmacromolecularsubstrates.Blood.80:3127-3134.36.
37.Lawson, J. H., and K.G.Mann.1991. Cooperative activation ofhuman
factorIXbythehumanextrinsic pathway ofbloodcoagulation.J.Biol.Chem.
266:11317-11327.
38.Wilcox,J.N.,K. M.Smith,S. M.Schwartz,and D.Gordon. 1989.
Local-izationoftissue factorin the normal vessel wall and in theatheroscleroticplaque. Proc.Natl.Acad.Sci. USA.86:2839-2843.
39. Drake,T. A., J. H.Morrissey,and T. S.Edgington.1989. Selective cellular
expression oftissue factorin humantissues. Implications for disorders ofhemosta-sisandthrombosis.Am.J.Pathol. 134:1087-1097.
40.Wissler,R.W.,and D.Vesselinovitch. 1977. Atherosclerosis in nonhu-manprimates.Adv.Vet.Sci. Comp.Med.21:351-420.
41.Moore, K. L., S. P.Andreoli,N. L.Esmon,C. T. Esmon, and N. U. Bang. 1987.Endotoxin enhances tissuefactorand suppressesthrombomodulin expres-sionofhumanvascular endothelium in vitro. J.Clin.Invest.79:124-130.
42.Conway, E. M., R. Bach, R. D. Rosenberg, and W. H. Konigsberg. 1989. Tumornecrosisfactorenhancesexpression oftissuefactormRNA inendothelial cells.Thromb.Res.53:231-241.
43.Dvorak,H.F., D. R.Senger,A.M.Dvorak, V. S. Harvey, and J. McDon-agh. 1985.Regulationof extravascularcoagulationbymicrovascular
permeabil-ity.Science(Wash. DC).227:1059-1061.
44.Weiss, H. J., V. T. Turitto, H. R. Baumgartner, Y. Nemerson, and T. Hoffmann. 1989. Evidence for the presence of tissue factor activity on suben-dothelium.Blood.73:968-975.
45. Bauer, K. A., L. M. Weiss, D. Sparrow, P. S. Vokonas, and R. D. Rosen-berg. 1987.Aging-associated changesinindices of thrombingenerationand pro-tein Cactivation in humans. Normativeagingstudy. J.Clin.Invest. 80:1527-1534.
