Isolation of the Tissue Factor Inhibitor Produced by Hepg2 Hepatoma Cells (Extrinsic Coagulation Pathway/Plasma Lipoprotein) GEORGE J
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Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1886-1890, April 1987 Biochemistry Isolation of the tissue factor inhibitor produced by HepG2 hepatoma cells (extrinsic coagulation 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), factor X(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. liver 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 cofactor 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 thrombosis in which one or more ofthese stimuli are from the American Type Culture Collection. likely to be present. Proteins. 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 protein from Russell's viper venom, antithrombin 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.