Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1886-1890, April 1987 Biochemistry Isolation of the inhibitor produced by HepG2 hepatoma cells (extrinsic pathway/plasma lipoprotein) GEORGE J. BROZE, JR.*, AND JOSEPH P. MILETICH Departments of Medicine and Laboratory Medicine, Washington University School of Medicine, The Jewish Hospital, St. Louis, MO 63110 Communicated by Philip W. Majerus, December 12, 1986 (receivedfor review November 13, 1986)

ABSTRACT Progressive inhibition of tissue factor activity have shown that not only factor VII(a) but also catalytically occurs upon its addition to human plasma (serum). This active factor Xa and an additional factor are required for the process requires the presence offactor VII(a), (a), Ca2+, generation of TF inhibition in plasma or serum. This addi- and another component in plasma that we have called the tissue tional factor, which we call the tissue factor inhibitor (TFI), factor inhibitor (TFI). A TFI secreted by HepG2 cells (human is present in barium-absorbed plasma (19) and appears to be hepatoma cell line) was isolated from serum-free conditioned associated with lipoproteins, since TFI functional activity medium in a four-step procedure including CdCl2 precipita- segregates with the lipoprotein fraction that floats when tion, diisopropylphosphoryl-factor X. affinity chromatogra- serum is centrifuged at a density of 1.21 g/cm3 (18). phy, Sephadex G-75 superfine gel filtration, and Mono Q We have shown (18) that HepG2 cells (a human hepatoma ion-exchange chromatography. The purified TFI contained a cell line) secrete an inhibitory moiety with the same charac- predominant band atMr 38,000 on NaDodS04/polyacrylamide teristics as the TFI present in plasma. This suggests that the gel electrophoresis that comigrates with inhibitory activity. may represent at least one source of this plasma Like the activity present in plasma, this TFI requires the inhibitory activity in vivo. Here we describe the purification presence of factor VII(a), factor X(a), and Ca2+ to express and preliminary characterization of the TFI produced by inhibitory activity. Its specific activity (assuming an extinction HepG2 cells. coefficient of 10 at 280 nM, for a 1-cm path length through a 1% solution) was 9800 units/mg ofprotein, where 1 unit ofTFI MATERIALS AND METHODS activity was dermed as that present in 1 ml of normal pooled serum. Materials. Affi-Gel 15 and low molecular weight standards for polyacrylamide electrophoresis were purchased from Tissue factor (TF) is a lipoprotein that enhances the Bio-Rad. Na125I (carrier-free) was purchased from New catalytic activity ofplasma coagulation factor VIIa toward its England Nuclear, and Iodo-Gen was obtained from Pierce. substrates, factor IX and factor X (1, 2). Several cell types Sephadex G-75 superfine and a Mono Q column were from that are not normally in contact with plasma (e.g., fibroblasts Pharmacia. Earle's modified essential medium (EMEM) and and smooth muscle cells) appear to synthesize TF in a fetal bovine and calf sera were obtained from KC Biological constitutive fashion (3, 4). Thus, presumably, in vivo coag- (Lenexa, KS), and liver cell growth factor was obtained from ulation may be initiated when plasma gains access to TF Miles. Bovine serum albumin, phenylmethylsulfonyl fluo- through a rent in the vascular system at a site of injury. Two ride, diisopropyl fluorophosphate (iPr2P-F), acrylamide, other cell types, however, monocytes and endothelial cells, methylenebis(acrylamide), rabbit brain cephalin, transferrin, which are in contact with plasma and which do not produce selenium, insulin, lactalbumin hydrolysate, Hepes, Mops, TF constitutively, can be induced to synthesize TF through and Trizma base were from Sigma. All other chemicals were the action of a host of stimuli (endotoxin, complement of reagent grade or better and came from Fisher or from component COa, immune complexes, interleukin 1, tumor Sigma. Factor X-deficient human plasma was obtained from necrosis factor) in vitro (5-9). Therefore, the factor VII-TF George King Biomedical (Overland Park, KS). Serum sam- pathway of coagulation may also be involved in several ples from healthy blood donors were provided by the Amer- pathological conditions associated with disordered coagula- ican Red Cross (St. Louis, MO). HepG2 cells were obtained tion and in which one or more ofthese stimuli are from the American Type Culture Collection. likely to be present. . A crude preparation of TF was prepared and studies the of TF-initiated washed extensively with EDTA (18, 20). The X coagulant Early concerning regulation from Russell's viper , IIIa, factor coagulation showed that incubation of TF (in crude tissue ViIa, and factor X were purified as described (18, 21-23). thromboplastin preparations) with serum inhibited its activity Factor Xa was produced from purified factor X by incubation in vitro and prevented its lethal effect when it was infused into with insolubilized X coagulant protein and inactivated with mice (10-15). Extensive studies by Hjort (16) in 1957 con- iPr2P-F (18, 24). iPr2P-factor Xa was linked to Affi-Gel 15 at firmed and extended previous work in the area, and he a final concentration of =2 mg/ml of packed gel, using the concluded that an inhibitory moiety in serum recognized the manufacturer's instructions (Mops buffer, pH 7.5). factor VII-TF complex. Consistent with this hypothesis are Assay. A three-stage assay for TF inhibition was used the facts that the inhibition of TF that occurs in plasma during the purification procedure (18). In the first stage, 10 pl requires the presence ofCa2+ (which is also necessary for the of factor VIla (1 9ug/ml), 10 gl of factor X (10 ug/ml), 10 1.d binding of factor VII/VIIa to TF) and that inhibition can be of CaCl2 (40 mM), 10 ul of antithrombin IIIa (650 ,ug/ml), 50 prevented and/or reversed by chelation of divalent cations gl of the sample to be tested, diluted in TBSA (0.1 M with EDTA (13, 17, 18). More recent investigations (18, 19) NaCl/0.05 M Tris-HCl, pH 7.5, containing bovine serum

The publication costs ofthis article were defrayed in part by page charge Abbreviations: TF, tissue factor; TFI, TF inhibitor; iPr2P-F, payment. This article must therefore be hereby marked "advertisement" diisopropyl fluorophosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

Downloaded by guest on September 30, 2021 1886 Biochemistry: Broze and Miletich Proc. Natl. Acad. Sci. USA 84 (1987) 1887 albumin at 1 mg/ml), and 10 Al of crude, EDTA-washed TF mg/ml), and Hepes (25 mM). The conditioned medium was (10% vol/vol) were incubated at room temperature. After 30 removed and replaced with fresh serum-free medium twice min, a 10-,41 sample was diluted 100-fold into TBSA with 5 per week. The HepG2 cells could be maintained under these mM CaCl2. Fifty microliters of this diluted sample, 50 ul of conditions for >3 months. factor VIIa (1 Aug/ml), 50 Al of CaCl2 (25 mM), and 50 ;LI of factor X (10 ,ug/ml) were then incubated at 37°C. After 1 min, RESULTS 50 ,ul of a mixture containing 10 parts factor X-deficient plasma and 1 part rabbit brain cephalin stock reagent (pre- Serum-Free Culture ofHepG2 Cells. Initial studies suggest- pared as described by Sigma) was added, and the time to clot ed that TFI is present at relatively low concentrations in formation was determined with a fibrometer (Baltimore plasma. Therefore, because of the potential problems asso- Biological Laboratory, Cockeysville, MD). ciated with the isolation of lipoproteins from the large Mixtures in which TBSA rather than sample was incubated quantities ofplasma that might be required in the purification in the first stage served as controls. The concentration of procedure, alternative sources of TFI were sought. Several crude TF in the assay was chosen to produce control clotting tissue culture cell lines were tested, and HepG2 cell condi- times of 35-40 sec. Antithrombin IIIa was included in the tioned medium was found to contain TFI activity at the level assay to decrease the effect ofthe factor Xa formed during the present in serum, as judged by the TFI assay described in first-stage incubation upon the clotting time derived in the Materials and Methods. third stage of the assay. Relative TFI activity was calculated Further investigations led to the use of a supplemented, from a standard curve constructed by plotting (on log-log serum-free medium that would support HepG2 cells in paper) the prolongation in seconds of the clotting time culture for >3 months. Additional pilot studies suggested that beyond the control value vs. the final concentration ofnormal the level of TFI activity in conditioned medium was not pooled serum (50 donors) in the first stage of the assay. This enhanced by the inclusion of dexamethasone, ethanol, or standard curve produced a linear response from 1-10%o phorbol 12-myristate 13-acetate in the culture medium. (vol/vol) serum concentrations. One unit ofTFI activity was Purification of TMI. Except as otherwise noted, the puri- defined as that contained in 1 ml of normal pooled serum. fication procedure was performed at room temperature Results of assays of chromatography fractions are expressed (Table 1). as clotting-times and have not been converted to units/ml. Cadmium chloride precipitation. HepG2 cell serum-free NaDodSO4/PAGE was performed in 15% gels (4% stack- conditioned medium (4 liters), following the addition of ing gel) by the method of Laemmli (25). Reduced samples phenylmethylsulfonyl fluoride (final concentration 0.1 mM) were heated to 100°C for 5 min in the presence of 10% and NaN3 (final concentration 0.05% wt/vol) was centrifuged 2-mercaptoethanol prior to electrophoresis. TFI was extract- at 2500 x g for 30 min to remove particulate debris. CdCl2 (1.0 ed following NaDodSO4/PAGE by cutting the lane from the M) was then added to a final concentration of 5 mM, and the gel, allowing it to soak in 0.1 M NaCl/0.05 M Tris-HCI, pH mixture was stirred for 15 min. The precipitate, which 7.5, for 30 min, slicing it into 2-mm sections, and incubating contained the TFI activity, was collected by centrifugation each slice overnight in 100 ,ul of 1 M NaSCN/0.05 M (2500 x g for 30 min), and the supernatant was decanted. The Tris-HCl, pH 7.5/0.025% Lubrol PX containing bovine serum pellet was dissolved with 40 ml of 0.5 M EDTA/5 mM albumin at 0.5 mg/ml. A 1:50 dilution in TBSA of the iPr2P-F, pH 9.5, and then dialyzed extensively against TS extracted material was then assayed as described above for (0.1 M NaCl/0.05 M Tris-HCl/0.1 mM phenylmethylsulfonyl TF inhibition. Immunoblotting was performed as described fluoride/0.5 mM EDTA/0.02% NaN3, pH 7.5). (26), using 125I-labeled factor Xa as the probe. The amino acid iPr2P-factor Xa 1S chromatography. The dialyzed composition of purified TFI (110 pmol) was determined, Affi-Gel preparation was clarified by centrifugation (10,000 x g for 15 following 24-hr hydrolysis in 6 M HCl at 110°C, on a Beckman 6300 autoanalyzer with postcolumn ninhydrin derivatization. min) and applied to a column of iPr2P-factor Xa-Affi-Gel 15 Cell Culture. HepG2 cells were cultured in plastic or glass equilibrated in TS. The gel was washed with starting buffer, roller bottles (850 cm2). After initial seeding, the cells were and bound material was eluted with 2 M NaSCN/0.05 M grown in EMEM with nonessential amino acids and penicillin Tris-HCl, pH 7.5/0.05% Lubrol PX/0.02% NaN3/0.1 mM (103 units/ml), streptomycin (100 /kg/ml), amphotericin (250 phenylmethylsulfonyl fluoride (Fig. 1). Fractions containing ng/ml), sodium pyruvate (100 mM), L-glutamine (200 mM), TFI activity were pooled and concentrated to r1 ml, using a and 5% fetal bovine serum and 5% calf serum, with twice- YM5 membrane (Amicon). weekly medium changes. After 2 weeks, the medium con- Gelfiltration and acetone delipidation. The concentrated taining serum was removed, and the cells were washed gently sample was applied to a column of Sephadex G-75 superfine with EMEM and thereafter cultured in serum-free medium equilibrated in 1 M NaSCN/0.05 M Tris-HCl, pH 7.5/0.05% consisting of the same ingredients listed above plus transfer- Lubrol PX/0.02% NaN3/0.1 mM phenylmethylsulfonyl flu- rin (5 ,.g/ml), selenium (5 ng/ml), insulin (5 yg/ml), liver cell oride (Fig. 2). Fractions containing TFI activity were pooled growth factor (20 pg/ml), lactoalbumin hydrolysate (5 and mixed with 8 volumes of acetone. After 30 min, the Table 1. Purification of TFI Protein, Activity, Specific activity, Yield, Purification, Step mg* units units/mg % -fold Serum-free medium 23,400 4400 0.188 (1.55)t 100 1 (2840)t CdCl2 precipitation, elution, and dialysis 480 4090 8.52 93 45 (5.50)t iPr2P-factor Xa-Affi-Gel 7.0 1240 180 28 957 (116)t Sephadex G-75 0.83 870 1050 20 5,590 (677)t Mono Q 0.06 590 9800 13 52,100 (6320)$ *Calculated assuming an extinction coefficient at 280 nm of EPm = 10. tMedium extensively dialyzed against TS buffer. WValues based upon dialyzed medium. Downloaded by guest on September 30, 2021 1888 Biochemistry: Broze and Miletich Proc. Natl. Acad Sci. USA 84 (1987)

1120

10010-

1.0 2 0.7; 1001 806

U 0.5 -60

0.1~~~~~~t40I 40 0.3 6_i }~90 0 20 40 60 80 Fraction FiG. 1. iPr2P-factor Xa-Affi-Gel affinity chromatography. The dialyzed TFI preparation ("100 ml) following CdC12 precipitation and extraction was applied to a 1.5 x 40-cm column of iPr2,P'factor X,-Affi-Gel 15 equilibrated in 0.1 M NaCl/0.05 M Tris*HCl, pH 0 20 40 60 7.5/0.5 mM EDTA/0.02% NaN3/0.1 mM phenylmethylsulfonyl Fraction fluoride. After washing with start buffer, the TFI was eluted with 2 M NaSCN/0.05 M TrisHCl, pH 7.5/0.05% Lubrol PX/0.1 mM FIG. 2. Sephadex G-75 superfine gel filtration. The concentrated phenylmethylsulfonyl fluoride. The flow rate was 3 ml/hr, and sample (-1 ml) from iWr2P-factor Xr-Affi-Gel chromatography was fraction size was 4 ml. Samples were diluted 500-fold for TFI assay. applied to a 1 x 120 cm column ofSephadex G-75 superfine. The flow Fractions 66-70 were pooled. rate was 1.5 ml/hr, and fraction size was 1 ml. Samples were diluted 2000 fold for TF assay. Fractions 34-41 were pooled. precipitate was collected by centrifugation (10,000 X g, 20 min, 100C). tifig (data not shown). The amino acid composition of the Mono Q ion-exchange chromatography. The acetone pre- purified TFI is shown in Table 2. The purified TFI appeared cipitate was solubilized with 1.0 ml of 6 M urea/0.02 M to be similar to the inhibitor present in serum in that it Tris'HCl/0.05% Lubrol PX, pH 8.3, and applied to a Mono required factor VII., factor X, and Call for the expression of Q column equilibrated in the same buffer. The column was activity (18) (Table 3) and dilutions of the purified TF1 developed with a 30-ml linear gradient from starting buffer to produced a line parallel to that of the normal human serum 6 M urea/0.5 M Tris HCl/0.05% Lubrol PX, pH 8.3 (Fig. 3). standard curve (Fig. 5). Fractions containing TFI activity were pooled as indicated in Fig. 3, concentrated to ;1 ml (YM5, Amicon), dialyzed DISCUSSION against 1 M NaSCN/0.05 M Tris HCl/0.05% Lubrol PX, pH The purification scheme is summarized in Table 1. Since 7.5, and stored at 4TC. much of the absorbance at 280 0mpresent in the starting Properties of the TFI. NaDodSO4/PAGE of the purified medium wits due to low molecular weight substances, the TFI showed a predominant bafd at Mr 38,000 (unreduced) or eventual fold purification is somewhat misleading. Thus, 39,000 (reduced) (Fig. 4). Extraction of gel slices following alternative values are presented in the table based upon the NaDodSO4/PAGE of an unreduced sample of the purified protein content of the medium following extensive dialysis preparation showed that inhibitory activity comigrated with (dialysis membrane with Mr 10,000 cut-off). the Coomassie-gtained band. As expected, this same band The serum-free conditioned medium from HepG2 cells was recognized by '25I-labeled factor Xa upon immUnoblot- proved to be a convenient starting material for the putifca- 100 0.10k 75 90 Q FIG. 3. Mono Q ion-exchange chromatography. 4) 50 After acetone precipitation, TFI was solubilized E , with 6 M M 0.05h 70 urea/0.02 Tris-HCl, pH 8.3/0.05% 25 'll Lubrol PX and applied to a 1-ml Mono Q column . equilibrated in the same buffer. After washing with 50 start buffer, the column was developed with a 30-ml a 0 gradient from start buffer to end buffer (6 M urea/0.5 M Tris HCl, pH 8.3, 0.05% Lubrol PX). The flow rate was 20 ml/hr, and fraction size was 1 20 30 40 50 60 ml. Samples were diluted 2000-fold for TOl assay. Fraction Horizontal bar indicates fractions that were pooled. Downloaded by guest on September 30, 2021 Biochemistry: Broze and Miletich Prof. Natl. Acad. Sci. USA 84 (1987) 1889 Slice Table 3. Requirement for factor VII, factor X, and Ca2' for the expression of activity by purified TFI Clotting time,t Deletion from first stage* sec None 95.3 Factor X 40.2 Factor VIIa 41.3 Ca2+ (5 mM EDTA) 40.8 u) Control 37.7 4) *See description of TR assay in Materials and Methods. tResults are the means of duplicate assays. The final concentration of TFI was 10 ng/ml in the first stage of the assay.

-0 0 u medium was found to be a rapid and effective means of concentrating the preparation. We suspect that Cd2+ forms an insoluble salt with phosphate present in the culture medium to which TFI binds (TFI also binds to hydroxy- apatite; data not shown), but we have not investigated the mechanism of the precipitation step further. A final CdCl2 concentration >3 mM was sufficient to precipitate all of the TFI activity. Extraction of TFI from the pellet could be accomplished with sodium phosphate (0.5 M) or EDTA (0.5 M), though the latter appeared to produce slightly greater U yields in activity. Since factor X was required for the generation of TF I inhibition in serum (19) and our earlier studies had suggested R that stoichiometric concentrations of factor VIIa, TF, factor Xa, and TFI might be involved in the inhibitory complex (18), the purification of TFI using factor Xa-Affi-Gel 15 was FIG. 4. NaDodSO4/PAGE of purified TFI. Purified TFI (3.75 pug attempted. TFI activity bound to iPr2P-factor Xa-Affi-Gel 15, per lane) was subjected to NaDodSO4/PAGE either unreduced (2 lanes) or following reduction with 10%o 2-mercaptoethanol (1 lane). and this binding did not require the presence of Ca2+. This (Upper) One ofthe lanes containing unreduced TFI was cut from the suggests that the requirement for catalytically active factor gel and sliced, and TFI activity was extracted for assay. (Lower) Xa for TF inhibition in serum may be related to activation of Coomassie blue stainipg of reduced (R) and unreduced (U) TFI. an inactive precursor form of TFI or to proteolytic modifi- Origin is at left. cation ofeither (or both) constituents ofthe VIIa-TF complex leading to enhanced binding by TFI, or that the of tion ofthe TFI. The cells themselves could be maintained for prolonged periods under serum-free culture conditions, with 100 the limit to their viability being microbial contamination related to twice-per-week manipulation. Precipitation ofTFI 0 activity following the addition of CdCl2 to the conditioned Table 2. Amino acid composition of TFI c) Mol per 38,000 g u) Amino acid of protein 4) 4) Aspartic acid 40 20 (A Serine 1 19 u Glutamic acid 44 Qc) Proline 15 Glycine 33 Alanine 19 Cysteine 13* Valine 12 Methionine t 15 Leucine 27 Tyrosine 10 III I I, I I, Phenylalanine 20 1 10 Histidine 5 Inhibitor concentration Lysine 26 Arginme 18 FIG. 5. Comparison of purified TFl and normal human serum in Tryptophan ND TFI assay. Dilutions of purified TFI were assayed in the TFI functional assay and compared to a standard curve constructed using ND, not determined. normal human serum. The y axis is prolongation of the clotting *Cysteine value from 24-hr hydrolysis; performic acid oxidation was beyond the control value (39 sec), and the x axis is the final not performed. concentration of TFI (e, in ng/ml) or normal human serum (o, in % tMethionine was oxidized during hydrolysis; none was detected. vol/vol) in the first stage of the assay (see Materials and Methods). Downloaded by guest on September 30, 2021 1890 Biochemistry: Broze and Miletich Proc. Natl. Acad. Sci. USA 84 (1987) factor Xa is required for the binding of a Xa TFI complex to functional activity (18, 19). Assignment ofa more appropriate the VIIa-TF complex. Since the capacity of the iPr2P-factor name than TFI will await further studies concerning its mode Xa-Affi-Gel 15 column appeared to be much lower'than of action and a recommendation by the International Com- predicted, an additional possibility is that catalytically active mittee on Thrombosis and Haemostasis. Xa is required for the complex formation between Xa and TFI, and that the iPr2P-Xa-Affi-Gel 15 contained small We thank Dr. T. C. Wun (Monsanto, St. Louis, MO), for sharing amounts of active Xa. his expertise concerning the serum-free culture of HepG2 cells, and Frequently, an additional protein band, constituting as Dr. Thomas Rucinsky, Cynthia Kunz, and Lyle Hutsell ofthe Tissue much as of the total protein, has been visualized after Culture Support Center (Washington University, St. Louis, MO), 20%6 who provided much of the HepG2 cell conditioned medium used in NaDodSO4/PAGE of the final TFI preparation. It has a these studies. The amino acid analysis of TFI was performed by molecular weight of 56,000 unreduced, and 63,000 following Michael Jennings (Monsanto). Excellent technical support was also reduction. This protein elutes slightly later than, though provided by Darryl Higuchi, Melissa Pigg, Louise Warren, Robert overlapping with, the Mr 38,000 protein from the Mono Q Finney, and James Girard. We thank Betty Greene for preparing this column but, within the experimental error associated with manuscript. This work was supported by the Monsanto Company low protein concentrations, appears to have the same TFI and by National Institutes of Health Grant HL34462. specific activity as the Mr 38,000 protein. Thus, we believe it may represent a precursor or dimeric form of the Mr 38,000 1. Silverburg, S. A., Nemerson, Y. & Zur, M. (1977) J. Biol. TFI rather than an unrelated protein contaminant. Chem. 252, 8481-8488. Dilutions of the purified TFI from HepG2'cells produced a 2. Zur, M. & Nemerson, Y. (1980) J. Biol. Chem. 255, 5703-5707. 3. Maynard, J. R., Heckman, C. A., Pitlick, E. A. & Nemerson, curve parallel to that ofnormal human'serum in the TFI assay Y. (1975) J. Clin. Invest. 55, 814-824. (Fig. 5), with purified TFI at a concentration of 100 ng/ml 4. Maynard, J. R., Drayer, B. E., Stemerman, M. B. & Pitlick, apparently equivalent to the inhibitory activity in serum. In F. A. (1977) Blood 50, 387-3%. addition, mixing experiments showed that incubation with 5. Lerner, R. G., Goldstein, R. & Cummings, G. (1971) Proc. serum did not abrogate the inhibitory effect of the purified Soc. Exp. Biol. Med. 138, 145-148. TFI. It seems likely that the TFI purified from HepG2 cells 6. Muhlfelder, T. W., Niemetz, J., Kreutzer, D., Beebe, D., is the same as that present in plasma, but further studies will Ward, P. A. & Rosenfeld, S. I. (1979) J. Clin. Invest. 63, be required to prove identity. It remains possible that the TFI 147-150. secreted by 7. Rothberger, H., Zimmerman, T. S., Spiegelberg, H. L. & these neoplastic HepG2 cells is dissimilar to that Vaughan, J. H. (1977) J. Clin. Invest. 59, 549-557. presumably synthesized by normal liver in vivo, or even that 8. Bevilacqua, M. P., Pober, J. S., Majeau, G. R., Cotran, R. S. the predominant TFI in plasma is not produced by the liver. & Gimbrone, M. A. (1984) J. Exp. Med. 160, 618-623. Other investigators have found that the activation offactor 9. Bevilacqua, M. P., Pober, J. S., Majeau, G. R., Fiers, W., X by factor VIIa-TF is inhibited by plasma lipoproteins. Cotran, R. S. & Gimbrone, M. A. (1986) Proc. Natl. Acad. Carson (27, 28) initially demonstrated an inhibitory effect of Sci. USA 83, 4533-4537. high density lipoprotein and, later, of purified apolipoprotein 10. Schneider, C. L. (1946) Am. J. Physiol. 146, 140-145. AII upon this reaction. He found that apolipoprotein AII at 11. Schneider, C. L. (1947) Am. J. Physiol. 149, 123-129. a concentration of 0.775 (13 pug/ml) produced a 1.5-fold 12. Thomas, L. (1947) Bull. Johns Hopkins Hosp. 81, 26-42. juM 13. Mann, F. D. & Hum, M. (1949) Fed. Proc. Fed. Am. Soc. increase in the apparent K1/2 for factor VIla, with a concom- Exp. Biol. 8, 105 (abstr.). itant 43% decrease in Vma,, and suggested that apolipoprotein 14 McClaughry, R. I. (1950) J. Mich. St. Med. Soc. 49, 685 AII caused a loss of -activator (factor VII0-TF) (abstr.). complex. Kondo and Kisiel (29) isolated a lipoprotein that 15. Lanchantin, G. F. & Ware, A. G. (1953) J. Clin. Invest. 32, comigrated with factor X through several chromatographic 381-389. steps and also inhibited the activation of factor X by factor 16. Hjort, P. F. (1957) Scand. J. Clin. Lab. Invest. 9, Suppl. 27, VIIa-TF. Their studies showed this lipoprotein to be a 76-97. noncompetitive inhibitor (Ki 22 nM) of the reaction, suggest- 17. Hubbard, A. R. & Jennings, C. A. (1986) Thromb. Res. 42, ing to them that its mechanism of action involves the 489-498. 18. Broze, G. J., Jr., & Miletich, J. P. (1987) Blood 69, 150-155. sequestration of either factor VIIa or factor X. Whether the 19. Sanders, N. L., Bajaj, S. P., Zivelin, A. & Rapaport, S. I. inhibitory'properties ofthe lipoproteins or apolipoprotein AII (1985) Blood 66, 204-212. noted by these investigators can be ascribed to the presence 20. Broze, G. J., Jr., & Majerus, P. W. (1980) J. Biol. Chem. 255, of TFI in the purified preparations is difficult to determine 1242-1247. due to the markedly different assay methods employed. 21. Esmon, C. (1974) Dissertation (Washington Univ., St. Louis, Further, it is quite conceivable that additional mechanisms MO). unrelated to TFI exist in plasma for the inhibition ofthe factor 22. Peterson, C. B. & Blackburn, M. N. (1985) J.-iol. Chem. 260, VIIa7TF complex. 610-615. We have called this inhibitor the tissue factor inhibitor 23. Miletich, J. P., Broze, G. J., Jr., & Majerus, P. W. (1981) Methods Enzymol. 80, 221-228. (TFI) based on historical terminology and the fact that our 24. Bajaj, S. P., Rapaport, S. I. & Prodanos, C. (1981) Prep. assay system measures the loss of TF activity. It should be Biochem. 11, 397-412. reemphasized, however, that TFI appears to recognize the 25. Laemmli, M. K. (1970) Nature (London) 227, 680-685. VIIa-Ca2+-TF (or possibly the VII-Ca2+-TF-X,) complex 26. Broze, G. J., Jr., Hickman, S. & Miletich, J. P. (1985) J. Clin. rather than TF alone. Hjort (16) suggested the name anti- Invest. 76, 937-946. convertin (VII-TF) for this entity, but given the newer 27. Carson, S. D. (1981) FEBS Lett. 132, 37-40. nomenclature, this term would seem unfeasible. Additional 28. Carson, S. D. (1986) Fed. Proc. Fed. Am. Soc. Exp. Biol. 45, attributes that might be incorporated into its name would be 1640 (abstr.). its apparent association with lipoproteins and the require- 29. Kondo, S. & Kisiel, W. (1986) Fed. Proc. Fed. Am. Soc. Exp. ment 'for the presence of factor Xa for the expression of Biol. 45, 1073 (abstr.). Downloaded by guest on September 30, 2021