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rHE JOURNAL F B I O L O G I C A LHEMISTRY Vol. 267, No. 27, Issue of September 25, pp. 19089-19094,1992B 1992 by Th e Americanociety for Biochemistry and Molecular Biology, Inc. Printed in U.S.A .

Tissue Factor-dependent Autoactivation f Human Blood CoagulationFactor VII*(Received for publication, March 13, 992)

Masakuni Yamamoto, Tomohiro NakagakiS, and Walter KisielgFrom the Blood S.y.stems Research Foundation Laboratory, Department of Pathology, Universityof New Mexico SchoolofMedicine, Albuq&rque, New Mexico87131

We recently showed that single-chain zymogen fac-tor VI1 is converted to two-chain factor VIIa in anautocatalyticmanner following complex formationwith either cell-surface or solution-phase relipidatedtissue factor apoprote in (Nakagaki, T., Foster, D. C .,Berkner, K. L., and Kisiel, W. (1991) iochemistry30,10819-10824).We have now performed a detailedkinetic analysisof the autoactivationof human plasmafactor VI1 in the presence of relipidated recombinanttissue factor apoprotein and calcium. Incubation offactor V I1 with equimolar amountsf relipidated tissuefactor apoprote in resulted in the formation of factorVIIa amidolytic activi ty coincident with the onversionof factor V I1 to factor VIIa. The time course for thegeneration of factor VIIa amidolytic activity in thissystem was sigmoidal, characterized by an initial lagphase followed by a rapid linearphase until activationwas complete. The duration of the lag phase was de-creased by the addition of exogenous recombinant fac-tor VIIa. Relipidated tissue factor apoprotein was es-sential for factor VI1 autoactivation. No factor VI1activation was observed following complex formationbetween factor VI1 and a recombinant soluble tissuefactor apoprotein construct consisting of the amino-terminal extracellular domain in the presence or ab-sence of phospholipids. Kinetic analyses revealed thatfactor VI1 activation in the presence of relipidatedtissue factor apoprotein can be defined by a second-order reaction mechanism in which factor V I1 is acti-vated by factor VIIa with an apparent second-orderra te constant of 7.2 X lo3M s. Benzamidine inhib-ited factor V I1 autoactivation with an apparent Ki of1.8 mM, which is identical to the apparent Ki or theinhibition of factor VIIa amidolytic activity by thisactive sitecompetitive inhibitor. Our da ta are onsist-entwith a factor V I1 autoactivation mechanism inwhich trace amounts of factor VIIa rapidly activatetissue factor-bound factorVI1 by limited proteolysis.

Factor VI1 is a multidomain, vitamin K-dependent glyco-protein that is synthesized in the liver and secreted into theblood as a zymogen of a serine protease, factor VIIa. In thetest tube, single-chain human factor VI1 is converted to two-chain factor VIIa by limited proteolysis of the Arg52-Ile53* This work was supported inpart by National Institu tes of HealthGrant HL 35246 and Blood Systems, Inc. The costs of publication ofthis article were defrayed in part by the payment of page charges.This article must herefore be hereby marked aduertisernent inaccordance with 18U.S.C. Section 1734 solely to indicate this fact.$Supported by agrant from the Chemo-Sero-Therapeutic Re-search Institute, Kumamoto, Japan. To whom correspondence should be addressed.

peptide bond by one of many coagulation proteases includingfactor Xa (1-5), factor IXa ( 5 , 6 ) , actor XIIa (7,8) , hrombin(9), and factor VIIa (10, 11). In addition, the proteolyticactivation of factor VI1 by factor Xa is greatly acceleratedfollowing complex formation of factor VI1 with its integralmembrane cofactor, tissue factor ( 3 , 4 ) .Following vascular injury and exposure of the subendothe-lium to the flowing blood, the formation of a bimolecularcomplex between circulating zymogen factor VI1 and theextracellular domain of cell-surface tissue factor is widelybelieved to be the initial event of the extrinsic pathway ofblood coagulation. Precisely how the presumably inactivefactor VII-tissue factor complex (10, 12-14) is converted to afunctional factor VIIa-tissue factor complex that proteolyti-cally activates factor IX or factor X in normal hemostasis isunclear. Previous studies demonstrated spontaneous activa-tion of plasma or recombinant factor VI1 following complexformation with tissue factor provided by a human bladdercarcinoma cell line, 582 (15).This spontaneous activationwas, however, not observed using an inactive mutant recom-binant factor VI1 preparation (S344A factor VII) in which theactive site serine residue was replaced with alanine, providingpresumptive evidence for the autoactivation of factor VI1 incomplex with tissue factor (11). n addition, catalytic amountsof recombinant factor VIIa, but not factor Xa, readily acti-vated factor VII-tissue factor in the presence of plasma con-centrations of antithrombin 111, the major serine proteaseinhibitor of blood coagulation proteases (11). n the presentstudy, we have performed a detailed kinetic analysis of theactivation of plasma factor VI1 in the presence of fluid-phase,relipidated tissue factor apoprotein. This analysis, togetherwith previous results ( l l ) , provides a mechanism for theinitiation of the extrinsic pathway of blood coagulation inwhich tissue factor-bound factor VI1 is rapidly converted tofunctional factor VIIa by trace amounts of circulating factorVIIa.

EXPERIMENTAL ROCEDURESMaterials-Bovine serum albumin, phosphatidylcholine, and phos-phatidylserine were obtained from Sigma. H-D-Ile-Pro-Arg-p-ni-troanilide (S-2288) was purchased from Helena Laboratories.Dansyll-Glu-Gly-Arg-chloromethyletone (DEGRck) was obtainedfrom Calbiochem. Benzamidine-HC1 was a product of Aldrich. So -dium [251]iodide wasobtained from Du Pont-New England Nuclear.All other materials were of the highest grade commercially available.Proteins-Human plasma-derived factor VII, factor Xa, andthrombin were purified essentially as described (16). Prior to use,

The abbreviations used are: dansyl, 5-dimethylaminonaphthal-ene-1-sulfonyl; BSA, bovine serum albumin; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; PS, phosphatidyl-serine; PC, phosphatidylcholine; DEGRck, dansyl-Glu-Gly-Argchlo-romethyl ketone; TF1.219, COOH-terminal truncated tissue factorapoprotein containing residues 1-219; TBS, Tris-buffered saline.

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19090 Autoactivation of Factor V IIhuman factor VI1 preparations (5 p~ in TBS: 50 mM Tris-HC1 (pH7.5), 100 mM NaCl) were routinely incubated with DEGRck (finalconcentration 5 mM) for 1 h a t 37 "C followed by exhaustive dialysisat 4 "C against TBS. Factor VI1 preparations, at 1mg/ml, containedno detectable factor X antigen as determined by a specific enzyme-linked immunosorbent assay (limit of detection = 1 ng/ml factor X) .Recombinant wild-type human factor VIIa was purified from babyhamster kidney cell culture medium as described (17). Full-lengthrecombinant human tissue factor apoprotein and a arboxyl-terminal,truncated tissue factor apoprotein consisting of the 219-amino acidextracellular domain (TF1.z19) were enerously provided by Drs. Gor-don Vehar and Lisa Paborsky, Genentech, Inc., South San Francisco.Full-length tissue factor apoprotein preparations were produced inEscherichia coli (18), whereas TF,-,,, was produced in stably trans-fected human 293 cell lines (19). Both issue factor apoprotein prep-arations were purified by immunoaffinity chromatography essentiallyas described (18).All proteins were homogeneous as judged by SDS-polyacrylamide gel electrophoresis.General Methods-SDS-polyacrylamide slab gel electrophoresis(SDS-PAGE) was performed according to Laemmli (20) using 10%polyacrylamide separating gels. Following electrophoresis, the pro-teins were visualized by staining with Coomassie Blue G-250 orautoradiography. Protein concentrations were determined accordingto Bradford (2 1 )using bovine serum albumin as the eference protein.Full-length recombinant human tissue factor apoprotein was relipi-dated as described (22) with the exception that small unilamellarphospholipid vesicles (PC:PS/75:25) were used in place of rabbitphospholipids. The effective tissue factor apoprotein concentrationin relipidated samples was assumed to be 50% of the total tissuefactor apoprotein concentration23). Phosphatidylcholine (PC):phosphatidylserine (P S) vesicles (75:25, mo1:mol) were prepared byultrasonic irradiation and ultracentrifugation as described (24). lZ5I-Labeled plasma factor VI1 was prepared to a specific radioactivity of0.5-1.0 pCi/pg using the IODO-GEN method (25) essentially asdescribed (15).Measurement of Factor VIZ Autoactivation-The autoactivation offactor VI1 was assessed in a two-stage assay as follows. In the firststage, DEGRck-treated factor VI1 was preincubated at 37 "C in a400-p1 snap-cap centrifuge tube in 91 p1 of TBS, 0.3% BSA, 5 mM CaClZ.After 5 min, 9 yl of relipidated tissue factor apoprotein or TF,-,,, wasadded to start the reaction. The final concentrations of factor VI 1and tissue factor apoprotein in the reaction mixture were each 600nM. The final concentrationof phospholipids (PC:PS/75:25) in factorVII-relipidated tissue factor apoprotein mixtures was 50 y ~ . tselected intervals, aliquots (10 pl) were removed from the incubationmixtures and transferred o a polystyrene cuvette containing 90 pl ofTBS, 0.3% BSA, 5 mM EDTA to stop the reaction. Then, 20 pl ofTF,-,,, (6 p ~ ) ,20 y1 of TBS, 0.3% BSA, 5 mM CaCl, and 60 pl of S-2288 ( 2 mM) were added to thecuvette and theabsorbance at 405 nmcontinuously recorded in a Beckman DU-65 spectrophotometer. Thefactor VIIa produced in the first stage was determined by interpola-tion from a linear standard curve relating aAlo5/min uersus knownconcentrations of recombinant factor VIIa (0-10 nM) in the presenceof 10 nM relipidated tissue factor apoprotein and 200 nM TFl.21s.Factor VIIa amidolytic assay s tandard curves (constructed with factorVIIa only) or a mixture of factor VIIa and factor VI1 (total factorVIIa and factor VI1 in the assay equals 10 nM) were superimposable,indicating that factor VI1 had no measurable effect on the tissuefactor apoprotein-dependent enhancement of factor VIIa amidolyticactivity in this system. In some experiments, the effect of benzamidineon factor VI1 autoactivation was assessed at several concentrationsof benzamidine (0-10 mM) in the first-stage incubation mixture. Inaddition, the cleavage of factor VI1 during autoactivation was exam-ined by SDS-PAGE and autoradiography in an incubation mixturecontaining 600 nM unlabeled factor VI1 and 1 nM lZ5I-labeled actorVII. In this experiment, aliquots (10 pl ) were emoved rom theincubation mixture at selected intervals, reduced with 10% merca-poethanol, 2% SDS, and subjected to SDS-PAGE. The slab gel wasthen dried and developed by autoradiography using Kodak X-Omatfilm.Th e second-order rate constant for the activation of factor VI1 inthe presence of relipidated tissue factor was obtained from the slopeof a plot of ln([VII]/ [VIIa]) versus time essentially as described byTans et al. for the activation of factor XI1 (26) and prekallikrein (27).Our model for the activation of factor VI1 in the presence of relipi-dated tissue factor apoprotein assumes that: 1) tissue factor-boundfactor VI1 is activated by solution-phase factor VIIa, and 2 ) thebinding of factor VI1 or VIIa to thephospholipid micelle is negligible

given the relatively high Kd for factor VII-phospholipid interactions(28, 29). The data were analyzed in terms of a second-order mecha-nism of autoactivation. The reaction is described in Equation 1.FVII + FVIIa + FVIIaz (1)

The rate of factor VIIa generation is given by Equation 2.d[FVIIa]/dt = kz[FVII]FVIIa] (2 )

A s described earlier by Tans et al. (26, 27), the final solution to thisdifferential equation is given by Equation 3.ln([FVII]/[FVIIa]),= -kz[FVII],,,,t + ln([FVII]/[FVIIa]),,o (3)

Here, kz is the second-order rate constant and [FVII],,, is the tota lconcentration of factor VI1 and factor VIIa present in the reactionmixture, whereas ([FVII]/[FVIIa]), and ([FVII]/[FVIIa]),=n re therespective ratios of factor VI1 and factor VIIa concentrations a t timet and time zero. A plot of ln([FVII]/[FVIIa]) uersus time should yielda straight line with a slope equal to -kz[FVII],,I, and the interceptat time zero is equal to ln([FVII]/[FVIIa]) at time zero.The inhibition of therate of factor VI1 autoactivation in thepresence of relipidated tissue factor by benzamidine was analyzedassuming competitive inhibition of factor VIIa by benzamidine ac-cording to the following equation.FVII + FVIIa + FVIIazFVIIa + I -+ FVIIa. Ii (4)

Z is benzamidine and K , is the inhibition constant. Assuming that[FVIIa. ] is an inactive species and does notcontribute to heactivation reaction, the rate is given by Equation 5.d[FVIIa]/dt = kz[FVIIa]fr,,[FVII] (5)

[FVIIaIf,, is the concentration of factor VIIa not associated with theinhibitor. Assuming rapid equilibrium of inhibitor binding to factorVIIa, Equation 5 can be rearranged to yield the following equation.d[FVIIa]/dt = (k2.Ki/(K,+ I ) ) FVIIa],,.l[FVII] (6 )

[FVIIa],,,,l is the total amount of free factor VIIa plus factor VIIabound to inhibitor. Inasmuch as he inhibitor is present in greatexcess, the concentration of bound inhibitor is insignificant. Thesecond-order rate constant, kz, is now reduced by a factor of KJKi +I due to the presence of inhibitor. Thus, he second-order semi-logarithmic plots should again yield astraight line with a slopeinversely related to benzamidine concentrations.Determination of the Inhibition Constants of Benzamidine for Hu-man Factor VZZa, Factor X a , and Thrombin-Human factor VIIa(120 nM), factor Xa (10nM), and thrombin (2 nM)were addedseparately to a cuvette containing 20 p1 ofTFl.zl, (200 nM finalconcentration), 420 yI of TBS, 0.3% BSA, 5 mM CaCl,, 60 pl of s-2288 (25-200 p~ final concentration), and varying amounts of benz-amidine (0-5 mM final concentration). The change in absorbance at405 nm was determined and 1/AAlo5/min uersus enzamidine concen-tration plotted using three concentrations of S-2288. The inhibitionconstant ( K , )was determined as that concentration of benzamidineat which the three straight lines intersected.

RESULTSConstruction and Validity of the Factor VIZ AutoactivationAssay-In initial studies, we attem pted to design a n amidol-ytic assay for factor VIIa formation based on that describedby Pedersen et al. (10) or the autoactivation of recombinantfactor VI1 in th e presence of polylysine. In th at assay, factorVI1 (300 nM) was incubated withpolylysine, the eactio nstopped at various times by the add ition of 5 mM CaCl,, andthe al iquotmixed directly with a chrom ogenicubstrate (FXa-

1). Inoursystem, actor VI1 (600 nM) is ncubated withrelipidated tissue factor apopro tein (600 nM) and CaClz (5mM). In order to sto p the reaction a telected intervals, EDT Awas added to a f inal concentrat ion of 10 mM. An aliquot ofthis sample was then added to 600 ~1 of TBS, 0.3% BSA, 0.2mM S-2288 followed by measurement of theabsorbance


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Autoactivation of Factor V I I 19091change at 405 nm. Under these conditions, essentially nohydrolysis of S-2288was observed in any time point ample.In fact, incubation of 500 nM recombinant factor VIIa withTBS, 0.3% BSA, 0.2 mM S-2288produced a AA405/min f only0.002. Accordingly, we then decided to take advantage of thefact tha t calcium and tissue factor apoprotein markedly in-crease (-50-fold) the amidolytic activity of factor VIIa (14,30-32). In a second series of experiments, aliquots were re-moved from the factor VII/relipidated tissue factor apopro-tein/CaCl, incubation mixture, and made 10 mM in EDTA tostop the reaction. Each aliquot was diluted 60-fold into TBS,0.3% BSA, 5 mM CaCl,, 0.2 mM S-2288 and the U 4 0 5 re-corded. Using this system, very rapid activation of factor VI1was observed and complete activation occurred within 10min.We speculated that reassociation of factor VII/VIIa withrelipidated tissue factor apoprotein in the second stage pro-moted continued autoactivation of factor VI1 during the 5-min absorbance measurement period in the spectrophotome-ter and resulted in an anomalously high factor VIIa activityin each timed aliquot. When concurrent studies demonstratedtha t the soluble TF1-219 onstruct did not support autoacti-vation of factor VI1 in the above assay, we decided to employthis tissue factor apoprotein in the second stage at a 20-foldmolar excess over carryover relipidated tissue factor apopro-tein. We reasoned that, under these conditions, factor VIIaamidolytic activity wouldbe sufficiently enhanced for itsaccurate measurement by the TF1-219with a minimal, if any,continued autoactivation of factor VI1 in the second stage bythe residual relipidated tissue factor apoprotein. Given thatthe reported Kd or factor VII-relipidated tissue factor apo-protein association is -10-fold lower than that observed forfactor VII-TF1-P1g33), a 20-fold molar excess of TFl-219 nthe second stage in all likelihood would insure tha t the ma-jority of factor VIIa was complexed with TF,-,,,. Less certainis the issue of whether a factor VIIa-TF1-219omplex exhibitsthe same amidolytic activity as a complex of factor VIIa-relipidated tissue factor apoprotein. In this regard, it is per-haps noteworthy to point out tha t, in contrast to Ruf et al.(32) who reported comparable rates, our factor VIIa-TFl.,lgcomplex exhibited an s-2288 turnover number (AA,,,/min/nM VIIa) -50% of tha t observed for a factor VIIa-relipidatedtissue factor apoprotein complex (Fig. lA).To address theissue of continued autoactivation in the second stage of thissystem utilizing TFl-219,our mixtures of factor VI1 and factorVIIa were constructed in the presence of 5% relipidated tissuefactor apoprotein and 95% TF1-219nd incubated a t 25 C or30 min. The mixtures consisted of factor VII/VIIa at (a )lOO%/O%, ( b ) 75%/25%, ( c ) 50%/50%, and (d) 25%/75%with a final factor VII/VIIa combined concentration of 10nM. A t 0, 10, 20, and 30 min, aliquots (600 ~ 1 )ere mixed ina polystyrene cuvette containing 60 pl of S-2288 (2 mM) andthe h A 4 0 5 determined for 5 min. In each factor VII/VIIamixture, essentially no change in the AA405/min wasbservedin the first 10 min of incubation, and later incubation timesindicated only a slight increase in AA40s/min(Fig. 1B). Thisfinding demonstrated that little, if any,autoactivation isoccurring in our system in the second-stage mixture duringthe 5-min absorbance measurement. Thus, n spite of thedifferences of factor VI1 affinity and fold-enhancement offactor VIIa amidolytic activity, the above two-stage assayenabled us to freeze the reaction at selected intervals, pre-vent or minimize autoactivation n the second stage, andaccurately measure factor VIIa concentration based on astandard curve constructed with amixture of relipidatedtissue factor apoprotein and TF1-219Fig. 2) .Tissue Factor-dependent Autoactivation of Factor VII-

0.06C*E.0s 0.03

0.000 5 10

Factor Vlla (nM)

0.02

I0.00 10 10 20 30

7 TY

incubation Time (rnin)FIG. 1. Amidolytic activity of factor VIIa. A , various concen-trations of factor VIIa were added to a cuvette containing TBS, 0.3%BSA, 5 mM CaC12 and either 200 nM relipidated TF or 200 nM TF,.

219. The reaction mixture was incubated a t 25 C for 5 min. S-2288(0.2 mM final concentration) was then added to the cuvette and theamidolytic activity was determined as described under ExperimentalProcedures. B , various mixtures of factor VI1 and factor VIIa wereconstructed in a system containing TBS, 0.3%BSA, 5 mM CaC12,10nM relipidated tissue factor apoprotein, 200 nM TF,-2,9,0.2 mM S-2288, and the absorbance at 405 nm was continuously recorded at25 C for 30 min. The mixtures consisted of factor VII/VIIa a t l o o%/0% (O), 75%/25% (O), 0%/50%(V), nd 25%/75%(V) ith a finalfactor VII/VIIa combined concentration of 10 nM in each system.0.03 -

0.02 -EEa

0.01 -

0.00 cc0 5Factor Vila (nM)

10

FIG. 2. Human factor VIIa amidolytic assay standard curve.Data are presented as mean values + S.D. ( n 3).Incubation of DEGRck-treated human lasma factor VI1 withan equimolar concentration of relipidated tissue factor apo-protein resulted in the time-dependent formation of factorVIIa amidolytic activity toward the chromogenic substrate,Ile-Pro-Arg-p-nitroanilideS-2288).In the absence of relipi-dated tissue factor apoprotein, no factor VIIa amidolyticactivity was observed. The time course for the generation offactor VIIa amidolytic activity was sigmoidal, with an initiallag phase followed by a rapid linear phase until activation wascomplete (Fig. 3A). Incubation of factor VI1 with a recombi-


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19092 Autoac t iva t ion of F a c t o r V I I

0

@I

0

10 20ImuMlo n Tlme (rnln)

5 10 15 20 25 30IncubationTime (min)

FIG.3. Tissue actor-dependentautoactivation of factorVII. A , human plasma factorVI1 (600nM) was preincubate d at 37 "Cin 91 p1 of TBS, 0.3% BSA, 5 mM CaCI,. After 5 min, 9 pl ofrelipidated full-length tissue factor apoprotein 0 ) r TF1..219 0 ) asadded to s ta r t theeaction. A t selected intervals, aliquots(10pl ) wereremoved from the incubation mixture and added to 90 pl of TBS,0.3% BSA, 5 mM EDTA to stop the reaction. The amidolytic activityof factor VIIa formed was then meas ured asdescribed under "Exper-imental Procedures." B, the above experiment was repeated with theexception t ha t '251-labeled factor VI1 (1 nM fin al concen tration ) waspresent n he ncubationmixture.At he ndicated imes, 10-plaliquots weresubjected to SDS-polyacry lamide gel electrophoresisunde r reducing conditions in a 10% polyacrylamide slab gel, followedby autoradiography.nant human tissue factor apoprotein construct lacking thetransmembrane domain and cytoplasm ic tail (TF1-219)ailedto generate factor VIIa midolytic activity wheth er or not theincubationmixturecontained 50 FM mixedphospholipids(PC/PS;75/25) (Fig. 3A). Inclusion of 1nM '2sI-labeled fac torVI1 into an inc ubation m ixture conta ining00 nM unlabeledfactor VI1 and 600 nM relipidated issue factor apoproteinresulted in th e te mpo ral c onversio n f '*'I-labeled factor VI1to "'I-labeled factor VIIa as judged by SDS -PAG E and au-toradiography (Fig. 3B). In addition, the produc tion of two-chain ""I-labeled factorVIIa n hissystemqualitativelyparallele d the forma tion f factor V IIa amidolytic activity.The sigmoidal nature of factor VIIaamidolytic activitygeneration in the presencef relipidated tissue factor a popro-tei n and calcium ions provided presum ptive evidence for th eautoac tivation of factor VI1 by trace am ount sof contaminat-ing factor VIIa not inhibited by pretre atm ent of the factorVI1 preparation with DEG Rck. This pattern is s imilar to thatreported for the autoactivationf factor XI1 and prekallik reinin the presence f sulfatides and dextran sulfate,espectively(26, 27), as well as the autoactiva tion f recombinant humanfactor VI1 in the pres enc e of polylysine (10).When the t imecourse of factor VI1 activation was analyzedy a second-ordersemi-logarithmic plot, a straight line was obtained in whichth e slope is equal to the prod uct of the second-order ratecons tan t (-k2) nd the to ta l concentra t ion f factor VI1 andfactor VIIa in the systemFig. 4). The addition f recombinantfactor VIIa (1-10% of the tot al facto r VI1 conc entra tion ) tothe incubation mixture shortened theag phase in proportion

0 10 20 30Incubation Time (min)

FIG.4. Tissue actor-dependentautoactivation of factorVII: effect of varying the initial concentration f factor VIIa.Th e experimental conditionswere identical t o those described in Fig.2A with the exception th at recom binant factor VIIa was added toincubation mixtures a t time zero to a final concentr ation of either 6nM ( 0 ) r 60 nM (V),n addition to an incubation mixture withoutexogenous factor VIIa (0) . he actor VIIa concentrate n eachreaction m ixture was determ ined temporally as described under "Ex-perimental Procedures." The ratioof [facto r VII] to [factor VIIa] wascalculated at each imepoint,and henatur al logarithm of thisquotient was plotted versus incubation time.to ad ded factor IIa (data notshown) an d resulted in a seriesof parallel lines in theecond-order plot (Fig. 4). In this plot,they in tercepteflects the conc entratio n f eith er endogenousor endogenous plus exogenous factor VIIa at tim ezero.Effect of Benzarnidine on the Tissue Factor-dependent Au-toactivation of Fa cto r VU-Previous stud iesdemonstratedthat the activationof factor VI1 in complex wit h eith er cell-surface tissue factor (15) or polylysine (10) was inhibited byth e reversible serine protea se inh ibitor enzamidine. Accord-ingly, we investigated the effect of several concentrations ofbenzamidine on the rate of factor VI1 autoactivation in thepresence of relipidated tissue factor apoprotein and calciumions. Fig. 5A illustrates thatbenzamidine inhibite d factor VI1autoactivation in concentration-dependentmannersjudged by the decreasingslope of th e second-ordersemi-logarithmicplotas a functi on of increa sing benzamidineconcentration. Fig. 5B isa plot of the appa rent second-orderrate constan t ob tained as function of benzamidine concen-tratio n. The solid line represents a simulated curve based onan appa ren tK i of 1.8 mM and employing the e quation , k: =( K ; / K i+ I)(k2)where k; is the second-order rate constan t inthe presence of inhibitor, I t 2 is the econd-order rate constantin the abse nce f inhibitor, K; is the inhibition constant, andI is the conce ntration of inhibitor. As shown in Fig. 5B , thesimulated curve closely matches one drawn through the datapoin ts and suppor ts Ki value of 1.8mM benzam idine for th einhibition of factor VI1 autoactivation.In order to substantiate that factor VIIa, and not otherproteases, was responsible for the acti vat ionof factor VII, wenex t exa mi ned the effect of benzamidine on the amidolyticactivities of factor VIIa, human factor Xa, and human throm-bin toward S-2288. The resu lts for the inhibition of factorVIIa amidolytic activity by benzamidine is shown in Fig. 6Aas a Dixon plot. Th e typ e of inhib ition was competitive, andan apparent Ki of 1.8 mM benzamidine was obtained at thepoint where the hree ines intersected.Benzamidine alsocompetitively inhi bite d the amidolytic activities of factor Xa(Fig. 6B) and throm bin (Fig. 6C) with apparent K; values of0.2 and 5 mM benzamidine, respectively. Thus, the resultsofthese s tudies indicate that the inhibitionf factor VI1 autoac-tivation by benzamidine ( K ;= 1.8 mM) is entirely consistent


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Autoactivation of Factor V I I 19093I 1 I 1

0 50 100lncubatlonllme (min)

I I I 1

I \ I

0 5 10[8enzamldlm] (mM)

FIG.5. Inhib ition of tissue facto r-depend ent factor VI1 au-toactivation by benzamidine. A , the autoactivation of factor VI1(600 nM) at 37 "C in the presence of TBS, 0.3% BSA, 5 mM CaC12,600 nM relipidated tissue factor apoprotein ando benzamidine (O),0.25 mM benzamidine (O),1mM benzamidine (V), 5 mM benzamidine(V), nd 10 mM benzamidine (0). he apparent second-order rateconstants for each benzamidine concentration were calculated fromthe slope of these lines and plotted as a function of benzamidineconcentration ( B ) .The curve generated in B represents a simulatedcurve assuming competitive inhibitionof autoactivation by benzam-idine with a I(,= 1.8mM.with the prote olytic activa tion of factor VI1 by factor VIIaand not t r ace am ounts f contamina t ing fac tor Xa or throm-bin.

DISCUSSIONIn th is r epor t ,we describe the kine tics for the spon taneo usactivation of hum an factor VI1 in the presenceof relipidatedtissue factor apoprotein and calcium ons. On the basis ofprevious tudies (10, 11) and nhibit ion tudies eported

herein, we con clud e hat he enzyme esponsible for heproteolytic activation of factor VI1 in his syste m is traceamounts of factor VIIa generated during the solation proce-dure and resistant to inhibit ion by the active si te inhibitor ,dansyl-Glu-Gly-Arg-chloromethyletone. n d dit ion, hesigmoidal nature of the ime course for the appearance offactor VIIa amidolytic activity in this system would te nd torule out catalysis by a contaminating ser ine protease othertha n f a c to rVIIa. A second-order rate constant or the t issuefactor-depend ent autoactivation of factor VI1 of 7.2 X lo3M"s" was obtained from linear second-orde r plots, and is con-s is tent wi th ra tesf autoactivation reported ear l ieror hum anfactor XI1 (26), hum an prekall ikrein (27) , and recombin anthum an factor VI1 (10).Previous studies hav e sho wn that the in teraction f factorVIIa with tissue factor increa sed the cataly tic efficiency offactor V IIa tow ard sm all chromogenic (10, 30-32) or fluores-

0 5[Bcnramidine] (mM)

D 5[Bsnurnldim] (mu)

FIG.6. Dixon plot for th e inhibition of factor VIIa, factorXa, and thrombin am idolyt ic activity by benzamidine. Thefinal enzyme concen trations were 120 nM factor VIIa, 10 nM factorXa, and 2 nM throm bin . A , factor VIIa; S-2288 concentrations usedwere: 0.2 mM (O),0.1 mM (O),0.05 mM (V). , actor Xa; S-2288concen trations used were: 0.2 mM (o),.1 mM (e), .05 mM (7). ,thrombin; S-2288 concen trations used were: 0.075 mM (V), .05 mM(v), .025 mM (0) .cent (14) subs trates 50-100-fold. Presu mab ly, as argued byLawson and co-workers(14), actor VIIa undergoes an activesite alteratio n ollowing complex formation with tissue factorthat enab les or arkedly enhances thehydrolysis rate for thesubstrate or increases the rate of active site modification bycovalent inhibitors. In this report, e provide evidence th at amembrane-associated t issue factor apoprotein was essentialfor the activa tionof factor VI1 by factor VIIa. A recomb inanttissue factor apoprotein construct consistingof the extracel-lular domain and lacking both the transm embrane and cyto-plasmic domains (TF1-219)ailed to sup port factor I1 autoac-t ivation in the presence or absencef exogenous mixed ph os-pholipid micelles (PC/P S;75/2 5). Assuming that fact or VI1does not interac t with th e phospholipid surface following itsinteraction with tissue factor apoprotein33-35), this f indingsuggest that zymogen factor VI1 undergoes a conformationchange upon binding to relipidated tissue factor apoproteintha t r enders i texquisitely sen sitiv e to proteolytic cleavage atth e Arg'52-Ile153eptide bond. Furthermore, our data indicatestha t the in te rac t ionf factor VI1 with relipidated tis sue factorapoprotein incomparison to TF1-2L9s uniquely d ifferent withrespect to no t only autoactivation,but alsoexpression offactor VIIa amidolytic activity following activation. In our


6/6

19094 Autoactivation of Factor VI1han ds, he specific amidolytic activity of the factor Vila-relipidated tissue factor apopro tein complex toward 5-2288was approx imately twice tha t observed for an equimolar m ix-ture of factor VIIa-TF,-,lg. Whe the r this eflects a significantdifference in the affinity f factor VIIa for TF,_z19s reportedby Ruf et al. (33) or an inabil i ty of TF1_,,, o generate theoptim al catalytic conform ation for subs trate hydrolysis willrequire add itional studies. Our data do not suppo rt that ofRuf et al. (32) ,who reported equivalent enh ancement f factorVIIa am idolytic activity by m embran e-associated full-lengtht i ssue fac tor apoprote in and F,-219oward the peptidyl sub-strate , methoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine-p-nitroanilide. T h e reason for this difference is not readilyapparent ,butmay e la te osign ificant differences in heTF,.,,, preparations used. In any event, our data efinitivelydem onstra te that the interactio n of factor VI1 with TF1.219does not apparently increase its proteolytic susceptibility tofactor VIIa. Evid ence that this mig ht also be the case forother proteases, ncluding factor Xa, was obtained by Pabor-sky et al. (19),whoshowed th at soluble TFl.219 xhibited-0.16% activity relative to full-len gth relipid ated tissue factorapopro tein in a chromogenic assay consisting of detergent-solubilized recombin ant tissue factor apopro tein, factor VII,and factor X. T he weak activity of TF1-219bserved in thissystem may be due, in large par t , to i ts inabil i ty to sup portei ther the factor VIIa- or factor Xa-med iated activation ofthe facto r VII-TF1.219 omplex. In a dd itio n, th e recently de-scribed coagulation assay specificor facto r VIIa singTF1-2,936, 37) presumably is basedupon theability ofTF1., ,g to bind factor VIIa and support factor X activation(32) without supporting incipient activationf zymogen factorVI1 during the t ime equired for clot formation to ccur.Th e second-orde r rate constant for the autoactivation offactor VI1 ( k 2= 7.2 X IO3 M" s-') obtained in this study isrelat ively small and raises the question as to w hether basallevels of circulating factor VIIa or factor Xa catalyzes th econversion of factor VII-tiss ue factor o factor VIIa-tis suefactor following vascular injury. While the kinetic constantsfor the activationof factor VII-tissue factory factor X a havenot, to our knowledge, been reported, our previous data (11)indicated hat ,quali tat ively,factorXa s amoreefficientcataly st of this reactio n than factor VIIa. However, in thepresence of plasma levels of antithrombin 111, no factor VII-tissue factor activation by factor Xa was observed while theauto activa tion of factor VII-tissue factor roceeded unabated(11).Furthermo re, incubation of 582 b ladder carcinoma cellmonolayers (a source of tissu e facto r) with Z5I-labeled-S344Afact or VI1 followed by a 10-fold dilu tion of norm al pooledplasma in the presence of hirudin resulted in the t ime-d e-pende nt conve rsion of '251-labeled-S344A factor VI1 to 1251-labeled S344A factor VIIa. Factor XI, factor VIII, factor IX,factor V, and proth rom bin congenital ly def icient plasmas,when substi tuted for n ormal pooled plasma, produced quali-tatively similar S344A factor VI1 activation rates, whereasfactor X-deficient plasma (-3 ng/ml factor X antigen) pro-duced a marked ly reduced ra te of S344A factor VI1 activation.In harpcon trast, actor VII-deficient plasm a ( 4 ng/mlfactor VI1 antig en) produced no detectable S344A factor VI1activation in thisystem.' W hen assayed for factor VIIa usingth e newly described TFl-219ssay (36,37) , a l l def icient plasmasam ples, with the exception of factor VII-deficient plasm a,contained factor VIIa in var iable concentrat ions (0.3-4 ng/ml). From these types f studies, we tentatively conclude th at

M. Yamamoto, D. Foster, and W. Kisiel, manuscript in prepara-tion.

circulating factorVIIa (10-100 PM ) plays a significant role inth e conversion of the firstcomplex of factor VII-tissue factorto factor VIIa-tis sue factor. The newly formed factor VIIa-tissue factorcomplex can then generate factor X a,hich alsocatalyzes continued proteolytic activation of tissue factor-boun d factor VI1 in add ition to tha t atalyzed by fluid-phasefacto r VIIa. Alternatively, circu lating fac tor VIIa may effec-tively competewith zymogen facto r VI1 foravailable cell-surface tissue factor and his factor VIIa-tissue factor caninit iate the extr insic pathway and activate factor X, whichthen reciprocally activates factorVII-tissue factor. In relationto th e utoactivation of factor VII, the contr ibution of factorXa in this feedback reaction is, however, difficult to assessgiven its a ffinity for factor Va, the presen ce of its sub strate,prothrombin, and i ts sensit ivi ty to plasma inhibitors such asantith rom bin I11 and tissue factor path way inhibitor.

Acknowledgments-We are grateful to Drs. Gordon Vehar and LisaPaborsky (Genentech, South San Francisco) for providing us withpreparations of recombinant human tissue factor apoprotein. We alsot ha nk Anders Pedersen (Novo Nordisk, Copenhagen) for recombi-nant factor VIIa and Nancy Basore for excellent technical assistance.REFERENCES

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