rHE JOURNALOF BIOLOGICAL CHEMISTRY

B 1992 by The Society American

for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 27, Issue of September 25, pp. 19089-19094,1992 Printed in U.S.A .

Tissue Factor-dependent Autoactivation Human Blood Coagulation of Factor VII*

(Received for publication, March 13, 1992)

Masakuni Yamamoto, Tomohiro NakagakiS, and Walter Kisielg

From the Blood S.y.stems Research Foundation Laboratory, Department of Pathology, University of New Mexico School of Medicine, Albuq&rque, New Mexico87131

We recently showed that single-chain zymogen factor VI1 is converted to two-chain factor VIIa in an autocatalyticmanner following complex formation with either cell-surface or solution-phase relipidated tissue factor apoprotein (Nakagaki, T., Foster, D. C., Berkner, K. L., and Kisiel, W. (1991)Biochemistry 30,10819-10824). have now performed a detailed We kinetic analysisof the autoactivationof human plasma factor VI1 in the presence of relipidated recombinant tissue factor apoprotein and calcium. Incubation of of factor VI1 with equimolar amounts relipidated tissue factor apoprotein resulted in the formation of factor VIIa amidolytic activity coincident with the conversion of factor VI1 to factor VIIa. The time course for the generation of factor VIIa amidolytic activity in this system was sigmoidal, characterized by an initial lag phase followed by a rapid linearphase until activation was complete. The duration of the lag phase was decreased by the addition of exogenous recombinant factor VIIa. Relipidated tissue factor apoprotein was essential for factor VI1 autoactivation. No factor VI1 activation was observed following complex formation between factor VI1 and a recombinant soluble tissue factor apoprotein construct consisting of the aminoterminal extracellular domain in the presence or absence of phospholipids. Kinetic analyses revealed that factor VI1 activation in the presence of relipidated tissue factor apoprotein can be defined by a secondorder reaction mechanism in which factor VI1 is activated by factor VIIa with an apparent second-order rate constant of 7 2 X lo3M s. Benzamidine inhib. ited factor VI1 autoactivation with an apparent K i of 1 8 mM, which is identical to the apparent Kifor the . inhibition of factor VIIa amidolytic activity by this active sitecompetitive inhibitor. Our data are consistentwith a factor VI1 autoactivation mechanism in which trace amounts of factor VIIa rapidly activate tissue factor-bound factor by limited proteolysis. VI1

peptide bond by one of many coagulation proteases including factor Xa (1-5), factor IXa ( 5 , 6 ) ,factor XIIa (7,8), thrombin (9), and factor VIIa (10, 11). In addition, the proteolytic activation of factor VI1 by factor Xa is greatly accelerated following complex formation of factor VI1 with its integral membrane cofactor, tissue factor (3,4). Following vascular injury and exposure of the subendothelium to the flowingblood, the formation of a bimolecular complex between circulating zymogen factor VI1 and the extracellular domain of cell-surface tissue factor is widely believed to be the initial event of the extrinsic pathway of blood coagulation. Precisely how the presumably inactive factor VII-tissue factor complex (10, 12-14) is converted to a functional factor VIIa-tissue factor complex that proteolytically activates factor IX or factor X in normal hemostasis is unclear. Previous studies demonstrated spontaneous activation of plasma or recombinant factor VI1 following complex formation with tissue factor provided by a human bladder carcinoma cell line, 582 (15).This spontaneous activation was, however, not observed using an inactive mutant recombinant factor VI1 preparation (S344A factor VII) in which the active site serine residue was replaced with alanine, providing presumptive evidence for the autoactivation of factor VI1 in complex with tissue factor (11). addition, catalytic amounts In of recombinant factor VIIa, but not factor Xa, readily activated factor VII-tissue factor in the presence of plasma concentrations of antithrombin 111, the major serine protease inhibitor of blood coagulation proteases (11).In the present study, we have performed a detailed kinetic analysis of the activation of plasma factor VI1 in the presence of fluid-phase, relipidated tissue factor apoprotein. This analysis, together with previous results ( l l ) , provides a mechanism for the initiation of the extrinsic pathway of blood coagulation in which tissue factor-bound factor VI1 is rapidly converted to functional factor VIIa by trace amounts of circulating factor VIIa.
EXPERIMENTAL PROCEDURES

Factor VI1 is a multidomain, vitamin K-dependent glycoprotein that is synthesized in the liver and secreted into the blood as a zymogen of a serine protease, factor VIIa. In the test tube, single-chain human factor VI1 is converted to twochain factor VIIa by limited proteolysis of the Arg52-Ile53
* This work wassupported inpart by National Institutes of Health Grant HL 35246 and Blood Systems, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Supported by agrant from the Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan. To whom correspondence should be addressed.

Materials-Bovine serum albumin, phosphatidylcholine, and phosphatidylserine were obtained from Sigma. H-D-Ile-Pro-Arg-p-nitroanilide (S-2288) was purchased from Helena Laboratories. Dansyll-Glu-Gly-Arg-chloromethyl ketone (DEGRck) was obtained from Calbiochem. Benzamidine-HC1 was a product of Aldrich. Sodium [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, and thrombin were purified essentially as described (16). Prior to use, The abbreviations used are: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PS, phosphatidylserine; PC, phosphatidylcholine; DEGRck, dansyl-Glu-Gly-Argchloromethyl ketone; TF1.219, COOH-terminal truncated tissue factor apoprotein containing residues 1-219; TBS, Tris-buffered saline.

19089

19090

Autoactivation of Factor VII

given the relatively high Kd for factor VII-phospholipid interactions (28, 29). The data were analyzed in terms of a second-order mechanism of autoactivation. The reaction is described in Equation 1. FVII kz + FVIIa + 2 FVIIa
(1)

human factor VI1 preparations (5 p~ in TBS: 50 m Tris-HC1 (pH M 7.5), 100 m NaCl) were routinely incubated with DEGRck (final M concentration 5 mM) for 1 h at 37 "C followed by exhaustive dialysis at 4 "C against TBS. Factor VI1 preparations, at 1 mg/ml, contained no detectable factor X antigen as determined by a specific enzymelinked immunosorbent assay (limit of detection = 1 ng/ml factor X). Recombinant wild-type human factor VIIa was purified from baby hamster kidney cell culture medium as described (17). Full-length recombinant human tissue factor apoprotein and acarboxyl-terminal, truncated tissue factor apoprotein consisting of the 219-amino acid extracellular domain (TF1.z19) were generously provided by Drs. Gordon Vehar and Lisa Paborsky, Genentech, Inc., South San Francisco. Full-length tissue factor apoprotein preparations were produced in Escherichia coli (18), whereas TF,-,,, was produced in stably transfected human 293 cell lines (19). Both tissue factor apoprotein preparations were purified by immunoaffinity chromatography essentially as described (18).All proteins were homogeneous as judged by SDSpolyacrylamide 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 proteins were visualized by staining with Coomassie Blue G-250 or autoradiography. Protein concentrations were determined according to Bradford (21) using bovine serum albumin as thereference protein. Full-length recombinant human tissue factor apoprotein was relipidated as described (22) with the exception that small unilamellar phospholipid vesicles (PC:PS/75:25) were used in place of rabbit phospholipids. The effective tissue factor apoprotein concentration in relipidated samples was assumed to be 50% of the total tissue factor apoprotein concentration (23). Phosphatidylcholine (PC): phosphatidylserine (PS) vesicles (75:25, mo1:mol) were prepared by ultrasonic irradiation and ultracentrifugation as described (24). lZ5ILabeled plasma factor VI1 was prepared to a specific radioactivity of 0.5-1.0 pCi/pg using the IODO-GEN method (25) essentially as described (15). Measurement of Factor VIZ Autoactivation-The autoactivation of factor VI1 was assessed in a two-stage assay as follows. In the first stage, DEGRck-treated factor VI1 was preincubated at 37 "C in a400p1 snap-cap centrifuge tube in 91 p1 of TBS, 0.3% BSA, 5 m CaClZ. M After 5 min, 9 yl of relipidated tissue factor apoprotein or TF,-,,, was added to start the reaction. The final concentrations of factor VI1 and tissue factor apoprotein in the reaction mixture were each 600 nM. The final concentrationof phospholipids (PC:PS/75:25) in factor . VII-relipidated tissue factor apoprotein mixtures was 50 y ~ At selected intervals, aliquots (10 pl) were removed from the incubation mixtures and transferredto a polystyrene cuvette containing 90 pl of TBS, 0.3% BSA, 5 m EDTA to stop the reaction. Then, 20plof M , M TF,-,,, (6 p ~ )420 y1 of TBS, 0.3% BSA, 5 m CaCl, and 60 pl of S2288 (2 mM) were added to thecuvette and theabsorbance at 405 nm continuously recorded in a Beckman DU-65 spectrophotometer. The factor VIIa produced in the first stage was determined by interpolation from a linear standard curve relating aAlo5/min uersus known concentrations of recombinant factor VIIa (0-10 nM) in the presence of 10 nM relipidated tissue factor apoprotein and 200 nM TFl.21s. Factor VIIa amidolytic assay standard curves (constructed with factor VIIa only) or a mixture of factor VIIa and factor VI1 (total factor VIIa and factor VI1 in the assay equals 10 nM) were superimposable, indicating that factor VI1 had no measurable effect on the tissue factor apoprotein-dependent enhancement of factor VIIa amidolytic activity in this system. In some experiments, the effect of benzamidine on factor VI1 autoactivation was assessed at several concentrations of benzamidine (0-10 mM) in the first-stage incubation mixture. In addition, the cleavage of factor VI1 during autoactivation was examined by SDS-PAGE and autoradiography in an incubation mixture factor containing 600 nM unlabeled factor VI1 and 1 nM lZ5I-labeled VII. In this experiment, aliquots (10 pl) wereremovedfrom the incubation mixture at selected intervals, reduced with 10% mercapoethanol, 2% SDS, and subjected to SDS-PAGE. The slab gel was then dried and developed by autoradiography using Kodak X-Omat film. The second-order rate constant for the activation of factor VI1 in the presence of relipidated tissue factor was obtained from the slope of a plot of ln([VII]/[VIIa]) versus time essentially as described by Tans et al. for the activation of factor XI1 (26) and prekallikrein (27). Our model for the activation of factor VI1 in the presence of relipidated tissue factor apoprotein assumes that: 1) tissue factor-bound factor VI1 is activated by solution-phase factor VIIa, and 2) the binding of factor VI1 or VIIa to thephospholipid micelle is negligible

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

As described earlier by Tans et al. (26, 27), the final solution to this differential 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 total concentration of factor VI1 and factor VIIa present in the reaction mixture, whereas ([FVII]/[FVIIa]), and ([FVII]/[FVIIa]),=nare the respective ratios of factor VI1 and factor VIIa concentrations at time t and time zero. A plot of ln([FVII]/[FVIIa]) uersus time should yield a straight line with a slope equal to -kz[FVII],,I, and the intercept at time zero is equal to ln([FVII]/[FVIIa]) at time zero. The inhibition of the rate of factor VI1 autoactivation in the presence of relipidated tissue factor by benzamidine was analyzed assuming competitive inhibition of factor VIIa by benzamidine according to the following equation. FVII
kz + FVIIa + 2 FVIIa

FVIIa

Ki + I -+ FVIIa. I

(4)

Z is benzamidine and K, is the inhibition constant. Assuming that [FVIIa.I] is an inactive species and does notcontribute tothe activation 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 the inhibitor. Assuming rapid equilibrium of inhibitor binding to factor VIIa, 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 VIIa bound to inhibitor. Inasmuch as the inhibitor is present in great excess, the concentration of bound inhibitor is insignificant. The second-order rate constant, kz, is now reduced by a factor of KJKi + I due to the presence of inhibitor. Thus,the second-order semilogarithmic plots should again yield astraight line with a slope inversely related to benzamidine concentrations. Determination of the Inhibition Constants of Benzamidine for Human Factor VZZa, Factor Xa, and Thrombin-Human factor VIIa were added (120 nM), factor Xa (10nM), and thrombin (2 nM) separately to a cuvette containing 20 p1 ofTFl.zl, (200 nM final concentration), 420 yI of TBS, 0.3% BSA, 5 m CaCl,, 60 plof sM 2288 (25-200 p~ final concentration), and varying amounts of benzamidine (0-5 m final concentration). The change in absorbance at M 405 nm wasdetermined and 1/AAlo5/min uersus benzamidine concentration plotted using three concentrations of S-2288. The inhibition constant (K,)was determined as that concentration of benzamidine at which the three straight lines intersected.
RESULTS

Construction and Validity of the Factor VIZ Autoactivation Assay-In initial studies, we attempted to design an amidolytic assay for factor VIIa formation based on that described by Pedersen e t al. (10)for the autoactivation of recombinant factor VI1 in the presence of polylysine. In that assay, factor VI1 (300 nM) was incubated withpolylysine, thereaction stopped at various times by the addition of 5 mM CaCl,, and the aliquot mixed directly with a chromogenic substrate (FXa1). Inoursystem,factor VI1 (600 nM) isincubated with relipidated tissue factor apoprotein (600 nM) and CaClz (5 mM). In order to stop the reaction at selected intervals, EDTA was added to a final concentration of 10 mM. An aliquot of this sample was then added to 600 ~1 of TBS, 0.3% BSA, 0.2 mM S-2288 followed by measurement of theabsorbance

Autoactivation of Factor VII

change at 405 nm. Under these conditions, essentially no hydrolysis of S-2288 was observed in any time pointsample. In fact, incubation of 500 nM recombinant factor VIIa with M of TBS, 0.3% BSA, 0.2 m S-2288produced a AA405/min only 0.002. Accordingly, we then decided to take advantage of the fact that calcium and tissue factor apoprotein markedly increase (-50-fold) the amidolytic activity of factor VIIa (14, 30-32). In a second series of experiments, aliquots were removed from the factor VII/relipidated tissue factor apoprotein/CaCl, incubation mixture, and made 10 m in EDTA to M stop the reaction. Each aliquot was diluted 60-fold into TBS, 0.3% BSA, 5 m CaCl,, 0.2 m S-2288 and the U 4 0 5 reM M corded. Using this system, very rapid activation of factor VI1 was observed and complete activation occurred within 10 min. We speculated that reassociation of factor VII/VIIa with relipidated tissue factor apoprotein in the second stage promoted continued autoactivation of factor VI1 during the 5min absorbance measurement period in the spectrophotometer and resulted in an anomalously high factor VIIa activity in each timed aliquot. When concurrent studies demonstrated that the soluble TF1-219 construct did not support autoactivation of factor VI1 in the above assay, we decided to employ this tissue factor apoprotein in the second stage at a 20-fold molar excess over carryover relipidated tissue factor apoprotein. We reasoned that, under these conditions, factor VIIa amidolytic activity wouldbe sufficiently enhanced for its accurate measurement by the TF1-219 with a minimal, if any, continued autoactivation of factor VI1 in the second stage by the residual relipidated tissue factor apoprotein. Given that the reported Kd for factor VII-relipidated tissue factor apoprotein association is -10-fold lower than that observed for factor VII-TF1-P1g (33), a 20-fold molar excess of TFl-219 in the second stage in all likelihood would insure that the majority of factor VIIa was complexed with TF,-,,,. Less certain is the issue of whether a factor VIIa-TF1-219 complex exhibits the same amidolytic activity as a complex of factor VIIarelipidated tissue factor apoprotein. In this regard, it is perhaps noteworthy to point out that, in contrast to Ruf et al. (32) who reported comparable rates, our factor VIIa-TFl.,lg complex exhibited an s-2288 turnover number (AA,,,/min/ nM VIIa) -50% of that observed for a factor VIIa-relipidated tissue factor apoprotein complex (Fig. lA).To address the issue of continued autoactivation in the second stage of this system utilizing TFl-219, four mixtures of factor VI1 and factor VIIa were constructed in the presence of 5% relipidated tissue and factor apoprotein and 95% TF1-219 incubated at 25 Cfor 30 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 10 nM. At 0, 10, 20, and 30 min, aliquots (600 ~ 1 were mixed in ) a polystyrene cuvette containing 60 pl of S-2288 (2 mM) and the h A 4 0 5 determined for 5 min. In each factor VII/VIIa mixture, essentially no change in the AA405/min was observed in the first 10 min of incubation, and later incubation times indicated only a slight increase in AA40s/min(Fig. 1B). This finding demonstrated that little, if any,autoactivation is occurring in our system in the second-stage mixture during the 5-min absorbance measurement. Thus, in spite of the differences of factor VI1 affinity and fold-enhancement of factor VIIa amidolytic activity, the above two-stage assay enabled us to freeze the reaction at selected intervals, prevent or minimize autoactivationin the second stage, and accurately measure factor VIIa concentration based on a standard curve constructed with amixture of relipidated (Fig. tissue factor apoprotein and TF1-219 2). Tissue Factor-dependent Autoactivation of Factor VII0.06

19091

*E

. s
C
10

0.03

0.00
0

10

Factor Vlla (nM)

0.02

0.00 1 0

10

20

30

incubation Time (rnin)

FIG. 1. Amidolytic activity of factor VIIa. A , various concentrations of factor VIIa were added to a cuvette containing TBS, 0.3% BSA, 5 m CaC12 and either 200 nM relipidated T F or 200 n TF,. M M 219. The reaction mixture was incubated at 25 C for 5 min. S-2288 (0.2 m final concentration) was then added to the cuvette and the M amidolytic activity was determined as described under Experimental Procedures. B, various mixtures of factor VI1 and factor VIIa were M constructed in a system containing TBS, 0.3% BSA, 5 m CaC12,10 nM relipidated tissue factor apoprotein, 200 nM TF,-2,9, 0.2 m SM 2288, and the absorbance at 405nmwas continuously recorded at 25 C for 30 min. The mixtures consisted of factor VII/VIIa at loo%/ 0% (O), 75%/25% (O),50%/50% (V), and 25%/75% (V)with a final factor VII/VIIa combined concentration of 10 nM in each system.
0.03

0.02

2 a
0.01

0.00

cc
0

10

Factor Vila (nM)

FIG. 2. Human factor VIIa amidolytic assay standard curve. Data are presented as mean values + S.D. ( n = 3).

Incubation of DEGRck-treated human plasma factor VI1 with an equimolar concentration of relipidated tissue factor apoprotein resulted in the time-dependent formation of factor VIIa amidolytic activity toward the chromogenic substrate, Ile-Pro-Arg-p-nitroanilide (S-2288).In the absence of relipidated tissue factor apoprotein, no factor VIIa amidolytic activity was observed. The time course for the generation of factor VIIa amidolytic activity was sigmoidal, with an initial lag phase followed bya rapid linear phase until activation was complete (Fig. 3A). Incubation of factor VI1 with a recombi-

19092

Autoactivation of Factor VII

10

20

ImuMlon Tlme (rnln)

@
I

10

20

30

Incubation Time (min)

FIG. 4. Tissuefactor-dependentautoactivation of factor of VII: effect of varying the initial concentration factor VIIa.
The experimental conditionswere identical to those described in Fig. 2A with the exception that recombinant factor VIIa was added to incubation mixtures a t time zero to a final concentration of either 6 nM ( 0 )or 60 nM (V), addition to an incubation mixture without in exogenous factor VIIa (0).Thefactor VIIa concentratein each reaction mixture was determined temporally as described under "Experimental Procedures." The ratioof [factor VII] to [factor VIIa] was calculated a t eachtimepoint,andthenatural logarithm of this quotient was plotted versus incubation time.

0 5 10 15 20 25 30
IncubationTime (min)

FIG. 3. Tissuefactor-dependentautoactivation of factor VII. A , human plasma factorVI1 (600 nM) was preincubated at 37 "C in 91 p1 of TBS, 0.3% BSA, 5 mM CaCI,. After 5 min, 9 pl of to added factor VIIa (data not shown) and resulted in a series or (0) relipidated full-length tissue factor apoprotein(0) TF1..219 was of parallel lines in the second-order plot (Fig. 4). In this plot, added to start the reaction. At selected intervals, aliquots(10 p l ) were they intercept reflects the concentrationof either endogenous removed from the incubation mixture and added to 90 pl of TBS, or 0.3% BSA, 5 mM EDTA to stop the reaction. The amidolytic activity endogenous plus exogenous factor VIIa at timezero. Effect of Benzarnidine on the Tissue Factor-dependent Auof factor VIIa formed was then measured asdescribed under "Experimental Procedures." B, the above experiment was repeated with the toactivation of Factor VU-Previous studiesdemonstrated exception that '251-labeled factor VI1 (1 nM final concentration) was that the activation of factor VI1 in complex with either cell10-pl surface tissue factor (15) or polylysine (10) was inhibited by presentintheincubationmixture.Attheindicatedtimes, aliquots weresubjected to SDS-polyacrylamide gel electrophoresis the reversible serine protease inhibitorbenzamidine. Accordunder reducing conditions in a 10% polyacrylamide slab gel, followed ingly, we investigated the effect of several concentrations of by autoradiography.

benzamidine on the rate of factor VI1 autoactivation in the presence of relipidated tissue factor apoprotein and calcium nant human tissue factor apoprotein construct lacking the ions. Fig. 5A illustrates thatbenzamidine inhibited factor VI1 transmembrane domain and cytoplasmic tail (TF1-219) failed autoactivation in a concentration-dependent manner as to generate factor VIIaamidolytic activity whether or not the judged by the decreasingslope of the second-ordersemiincubationmixturecontained 50 FM mixedphospholipids logarithmicplotas a function of increasing benzamidine (PC/PS;75/25) (Fig. 3A). Inclusion of 1 nM '2sI-labeled factor concentration. Fig. 5B is a plot of the apparent second-order VI1 into an incubation mixture containing nM unlabeled rate constant obtained asa function of benzamidine concen600 factor VI1 and 600 nM relipidated tissue factor apoprotein tration. The solid line represents a simulated curve based on resulted in the temporal conversion '*'I-labeled factor VI1 an apparent Ki of 1.8 mM and employing the equation, k: = of to "'I-labeled factor VIIa as judged by SDS-PAGE and au- (K;/Ki+ I)(k2)where k; is the second-order rate constant in toradiography (Fig. 3B). In addition, the production of two- the presence of inhibitor, I t 2 is thesecond-order rate constant chain ""I-labeled factorVIIainthissystemqualitatively in the absence inhibitor, K; is the inhibition constant, and of paralleled the formation factor VIIa amidolytic activity. of I is the concentration of inhibitor. As shown in Fig. 5B, the The sigmoidal nature of factor VIIaamidolytic activity simulated curve closely matches one drawn through the data generation in the presence relipidated tissue factor apopro- points and supports Ki value of 1.8 mM benzamidine for the of a tein and calcium ions provided presumptive evidence for the inhibition of factor VI1 autoactivation. autoactivation of factor VI1 by trace amountsof contaminatIn order to substantiate that factor VIIa, and not other ing factor VIIa not inhibited by pretreatment of the factor proteases, was responsible for the activationof factor VII, we VI1 preparation with DEGRck. This pattern is similar to that examined the effect of benzamidine on the amidolytic next reported for the autoactivationfactor XI1 and prekallikrein activities of factor VIIa, human factor Xa, and human thromof in the presenceof sulfatides and dextran sulfate, respectively bin toward S-2288. The results for the inhibition of factor (26, 27), as well as the autoactivation recombinant human VIIa amidolytic activity by benzamidine is shown in Fig. 6A of factor VI1 in the presence of polylysine (10). When the time as a Dixon plot. The type of inhibition was competitive, and course of factor VI1 activation was analyzed a second-order an apparent Ki of 1.8 mM benzamidine was obtained at the by semi-logarithmic plot, a straight line was obtained in which point where the three lines intersected.Benzamidine also the slope is equal to the product of the second-order rate competitively inhibited the amidolytic activities of factor Xa constant (-k2) and the total concentration factor VI1 and (Fig. 6B) and thrombin (Fig. 6C) with apparent K; values of of factor VIIa in the system 4). The addition recombinant 0.2 and 5 mM benzamidine, respectively. Thus, the resultsof (Fig. of factor VIIa (1-10% of the total factor VI1 concentration) to these studies indicate that the inhibition of factor VI1 autoacthe incubation mixture shortened the phase in proportion tivation by benzamidine (K;= 1.8 mM) is entirely consistent lag

Autoactivation of Factor VII

I
1 I

19093

50

100

lncubatlonllme (min)

[Bcnramidine] (mM)

I
0

O \

I
5 10

[8enzamldlm] (mM)

FIG. 5. Inhibition of tissue factor-dependent factor VI1 autoactivation 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 and benzamidine (O), no D 5 0.25 mM benzamidine (O),1mM benzamidine (V), 5 mM benzamidine [Bsnurnldim] (mu) (V), and 10 m benzamidine (0). M The apparent second-order rate constants for each benzamidine concentration were calculated from FIG.6 . Dixon plot for the inhibition of factor VIIa, factor the slope of these lines and plotted as a function of benzamidine Xa, and thrombin amidolytic activity by benzamidine. The concentration ( B ) .The curve generated in B represents a simulated final enzyme concentrations were 120 nM factor VIIa, 10 nM factor curve assuming competitive inhibitionof autoactivation by benzam- Xa, and 2 n thrombin. A , factor VIIa; S-2288 concentrations used M idine with a I(, = 1.8 mM. were: 0.2 mM (O),0.1 mM (O),0.05 mM (V).B, factor Xa; S-2288 concentrations used were: 0.2 mM (o), mM (e), 0.1 0.05 mM (7). c, 0.05 mM with the proteolytic activation of factor VI1 by factor VIIa thrombin; S-2288 concentrations used were: 0.075 mM (V), and not trace amounts contaminating factor Xa or throm- (v),0.025 mM (0). of bin. cent (14) substrates 50-100-fold. Presumably, as argued by DISCUSSION factor VIIa undergoes an active Lawson and co-workers(14), In this report,we describe the kinetics for the spontaneous site alterationfollowing complex formation with tissue factor activation of human factor VI1 in the presenceof relipidated that enables or markedly enhances thehydrolysis rate for the tissue factor apoprotein and calcium ions. On the basis of substrate or increases the rate of active site modification by previous studies (10, 11) andinhibitionstudiesreported covalent inhibitors. In this report, provide evidence that a we herein, we concludethatthe enzymeresponsible forthe membrane-associated tissue factor apoprotein was essential proteolytic activation of factor VI1 in this system is trace for the activationof factor VI1 by factor VIIa. A recombinant amounts of factor VIIa generated during theisolation proce- tissue factor apoprotein construct consistingof the extraceldure and resistant to inhibition by the active site inhibitor, lular domain and lacking both the transmembrane and cytodansyl-Glu-Gly-Arg-chloromethyl ketone.Inaddition,the plasmic domains (TF1-219) to support factor autoacfailed VI1 sigmoidal nature of the time course for the appearance of tivation in the presence or absenceexogenous mixed phosof factor VIIa amidolytic activity in this system would tend to pholipid micelles (PC/PS;75/25). Assuming that factor VI1 rule out catalysis by a contaminating serine protease other does not interact with the phospholipid surface following its than factor VIIa. A second-order rate constantfor the tissue interaction with tissue factor apoprotein (33-35), this finding factor-dependent autoactivation of factor VI1 of 7.2 X lo3M" suggest that zymogen factor VI1 undergoes a conformation s" was obtained from linear second-order plots, and is con- change upon binding to relipidated tissue factor apoprotein sistent with rates autoactivation reported earlier human that renders itexquisitely sensitive to proteolytic cleavage a t of for factor XI1 (26), human prekallikrein (27), and recombinant the Arg'52-Ile153 peptide bond. Furthermore, our data indicates human factor VI1 (10). that the interaction factor VI1 with relipidated tissue factor of Previous studies have shown that the interaction factor apoprotein incomparison to TF1-2L9uniquely different with of is VIIa with tissue factor increased the catalytic efficiency of respect to not only autoactivation,but alsoexpression of factor VIIa toward small chromogenic (10, 30-32) or fluores- factor VIIaamidolytic activity following activation. In our

19094

Autoactivation of Factor VI1

hands, the specific amidolytic activity of the factor Vila- circulating factorVIIa (10-100 PM) plays a significant role in relipidated tissue factor apoprotein complex toward 5-2288 the conversion of the firstcomplex of factor VII-tissue factor was approximately twice that observed for an equimolar mix- to factor VIIa-tissue factor. The newly formed factor VIIature of factor VIIa-TF,-,lg. Whether thisreflects a significant tissue factorcomplex can then generate factor Xa, which also difference in the affinity factor VIIa for TF,_z19 reported catalyzes continued proteolytic activation of tissuefactorof as by Ruf et al. (33) or an inability of TF1_,,,to generate the bound factor VI1 in addition to that catalyzed by fluid-phase optimal catalytic conformation for substrate hydrolysis will factor VIIa. Alternatively, circulating factor VIIa may effecrequire additional studies. Our data do not support that of tively competewith zymogen factor VI1 foravailable cellRuf et al. (32), who reported equivalent enhancement factor surface tissue factor and this factor VIIa-tissue factor can of VIIa amidolytic activity by membrane-associated full-length initiate the extrinsic pathway and activate factor X, which tissue factor apoprotein and TF,-219 toward the peptidyl sub- then reciprocally activates factorVII-tissue factor. In relation strate, methoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine- to theautoactivation of factor VII, the contribution of factor p-nitroanilide. The reason for this difference is not readily Xa in this feedback reaction is, however, difficult t o assess apparent,butmayrelatetosignificant differences in the given its affinity for factor Va, the presence of its substrate, TF,.,,, preparations used. In any event, our datadefinitively prothrombin, and its sensitivity to plasma inhibitors such as demonstrate that the interaction of factor VI1 with TF1.219 antithrombin I11 and tissue factor pathway inhibitor. does not apparently increase its proteolytic susceptibility to Acknowledgments-We are grateful to Drs. Gordon Vehar and Lisa factor VIIa. Evidence that this might also be the case for other proteases, including factor Xa, was obtained by Pabor- Paborsky (Genentech, South San Francisco) for providing us with exhibited preparations of recombinant human tissue factor apoprotein. We also sky et al. (19),whoshowed that soluble TFl.219 thank Anders Pedersen (Novo Nordisk, Copenhagen) for recombi-0.16% activity relative to full-length relipidated tissue factor factor VIIa and Nancy Basore for excellent technical assistance. nant apoprotein in a chromogenic assay consisting of detergentREFERENCES solubilized recombinant tissue factor apoprotein, factor VII, 1. Radcliffe, R., and Nemerson, Y. (1976) J. Biol. Chem. 261,4797-4802 observed in this and factor X. The weak activity of TF1-219 2. Bajaj, S. P., Rapaport, S. I., and Brown, S. F. (1981) J. Biol. Chem. 2 5 6 , system may be due, in large part, to its inability to support 253-259 3. Nemerson, Y., and Repke, D. (1985) Thromb. Res. 40,351-358 either the factor VIIa- or factor Xa-mediated activation of 4. Rao, L. V. M., and Rapaport, S. I. (1988) Proc. Natl. Acad. Sei. U. S. A. 86, the factor VII-TF1.219complex. In addition, the recently de6687-6691 5. Wildgoose, P., and Kisiel, W. (1989) Blood 73,1888-1895 scribed coagulation assay specific for factor VIIa using 6. Masys, D. R., Bajaj, S. P., and Rapaport, S. I. (1982) Blood 6 0 , 1143-1150 TF1-2,9 (36, 37) presumably is based upon theability of 7. Kisiel, W., Fujikawa, K., and Davle, E. W. (1977) Biochemistry 1 6 , 41894194 TF1.,,g to bind factor VIIa and support factor X activation 8. Broze, G. J., and Majerus, P. W. (1981) Methods Enzymol. 80,228-237 (32) without supporting incipient activation of zymogen factor 9. Broze, G. J., and Majerus, P. W. (1980) J. Biol. Chem. 2 6 6 , 1242-1247 10. Pedersen, A. H., Lund-Hansen, T., Bisgaard-Frantzen, H., Olsen, F., and VI1 during the time required for clot formation tooccur. Petersen, L. C. (1989) Biochemistry 28,9331-9336 The second-order rate constant for the autoactivation of 11. Nakaeaki. T.. Foster. D. C.. Berkner. K. L.. and Kisiel.. W. (1991)Biochem. . is& 30,10819-10824 factor VI1 ( k 2 = 7.2 X IO3 M" s-') obtained in this study is 12. Williams, E. B., Krishnaswamy, S., and Mann, K. G. (1989) J. Biol. Chem. relatively small and raises the question as to whether basal 2 6 4 , 7536-7545 levels of circulating factor VIIa or factor Xa catalyzes the 13. Wildgoose, P., Berkner, K. L., and Kisiel, W. (1990) Biochemistry 2 9 , 3413-3420 conversion of factor VII-tissue factor to factor VIIa-tissue 14. Lawson, J. H., Butenas, S., and Mann, K. G. (1992) J. Biol. Chem. 2 6 7 , 4834-4843 factor following vascular injury. While the kinetic constants 15. Sakai, T., Lund-Hansen, T., Paborsky,L., Pedersen, A. H., and Kisiel, W. for the activationof factor VII-tissue factor factor Xa have by (1989) J. Biol. Chem. 264,9980-9988 Kondo, S., and Kisiel, W. (1987) 70,1947-1954 not, to our knowledge, been reported, our previous data (11) 16. Thim, L., Bjoern, S., Christensen,BloodNicolaisen, E. M., Lund-Hansen, T., 17. M., indicated that, qualitatively, factor Xa is amoreefficient Pedersen, A. H., and Hedner, U. (1988) Biochemistry 2 7 , 7785-7793 Harris, L., catalyst of this reaction than factor VIIa. However, in the 18. Paborsky, L. R., Tate, K.M., O'Brien, R. J., Yansura, D. G., Band, R., D. P., Chang, J. Y., Swartz, J. McCray, G., Gorman, C. M., Fung, V. P., Thomas, J. N., and Vehar, G. A. (1989) Biochemistry 2 8 , presence of plasma levels of antithrombin 111, no factor VII8072-8077 tissue factor activation by factor Xa was observed while the 19. Paborsk L.R. Caras I. W. Fisher, K. L., and Gorman, C.M. (1991) J. Biol. 8hm. 266,21811-21'916 autoactivation of factor VII-tissue factor proceeded unabated Laemmli, 227,680-685 (11).Furthermore, incubation of 582 bladder carcinoma cell 20. Bradford, U. K. (1970) NatureBiochem. 72,248-254 21. M. M. (1976)A d . 22. Broze, G. J., Leykam, J. E., Schwartz, B. D., and Miletich, J. P. (1985) J . monolayers (a source of tissue factor) withlZ5I-labeled-S344A Biol. Chem. 260,10917-10920 factor VI1 followed by a 10-fold dilution of normal pooled 23. Bach, R., Gentry, R., and Nemerson, Y. (1986) Biochemistry 2 6 , 40074020 plasma in the presence of hirudin resulted in the time-deY . pendent conversion of '251-labeled-S344A factor VI1 t o 1251- 24. Barenholz, F. ,Gibbes, D., Litman, B. J., Goll, J., Thompson, T. E., and Carlson, D. (1977) Biochemistry 16,2806-2810 labeled S344A factor VIIa. Factor XI, factor VIII, factor IX, 25. Fracker, P. J., and Speck, J. C. (1978) BLochern. Biophys. Res. Commun. 00,849-857 factor V, and prothrombin congenitally deficient plasmas, 26. Tans, G., Rosing, J., and Griffin, J. H. (1983) J. Biol. Chem. 2 5 8 , 82158222 when substituted for normal pooled plasma, produced quali- 27. Tans, G., Rosing, J., Berrettini, M., , B., and Griffin, J. H. (1987) J. Biol. tatively similar S344A factor VI1 activation rates, whereas Chem. 262,11308-11314 factor X-deficient plasma (-3 ng/ml factor X antigen) pro- 28. Nelsestuen, G. L., Kisiel, W., and DiScipio, R. G. (1978) Biochemistry 1 7 , 2134-2138 duced a markedly reduced rate of S344A factor VI1 activation. 29. Bom, V. J. J., and Bertina, R. M. (1990) Biochem. J. 266,327-336 Wiber F.C., Christensen, P. Insharpcontrast,factor VII-deficient plasma ( 4 ng/ml 30. Pedersen A. HK.Nordfang, O., Norris, F., Beck, 'f! C., Norris, K., Hedner, M., Mdeller, B., Meidahl-Pedersen,J., factor VI1 antigen) produced no detectable S344A factor VI1 U., and Kisiel, W. (1990) J. Biol. Chem. 2 6 5 , 16786-16793 H., Lund-Hansen, T.,Komlyama, Y., Petersen, L. C., Oesteractivation in this system.' When assayed for factor VIIa using 31. Pedersen, A.B., and Kisiel, W. (1991) Thromb. Haemostasis 66,528-534 gaard, P. the newly described TFl-219 assay (36,37), all deficient plasma Ruf, W., Reherntulla, A,, and Edgington, T. S. (1991) J. Bcol. Chem 2 6 6 , 32. 2158-2166 samples, with the exception of factor VII-deficient plasma, 33. Ruf, W., Kalnik, M. W., Lund-Hansen, T., and Edgington, T. S. (1991) J. contained factor VIIa in variable concentrations (0.3-4 ng/ Bid. Chem. 2 6 6 , 15719-15725 ml). From these types studies, we tentatively conclude that 34. Rao, L. V. M., Rapaport, S. I., and Tait, J. F. (1990) Circulotion 8 2 , IIIof 132
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M. Yamamoto, D. Foster, and W. Kisiel, manuscript in preparation.

35. Wild oose, P , Jorgensen, T., Komiyama, Y., Nakagaki, T., Pedersen, A., and Kisie1,'W. (1992) Thrombos. Haemostasis,67, 679-685 36. Morrissey, J. H., and Macik, B. G. (1991) Arterroscler. Thromb. 1 1 , 1544a 37. Macik, B. G., and Morrissey, J. H. (1991) Blood 78, 61a