fibrinogen degradation product fragment D - Europe PMC

Proc. Nati. Acad. Sci. USA

Vol. 86, pp. 8788-8792, November 1989

Cell Biology

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen degradation product fragment D FRANK M. LADUCA, LAURA A. TINSLEY, CHI V. DANG, AND WILLIAM R. BELLt Department of Medicine, Hematology Division, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205

Communicated by Victor A. McKusick, August 11, 1989

ABSTRACT The direct stimulation of fibrinogen biosynthesis by fibrinogen degradation produces (FDPs) was studied in rat hepatocyte cultures. Pure rat FDP fragment D (FDP-D) (Mr 90,000) and FDP fragment E (FDP-E) (Mr 40,000) and mixtures of the two (FDP-DE) were added to rat hepatocytes cultured in serum-free hormonally defined medium. Hydrocortisone (20 IpM) significantly increased synthesis of fibrinogen, as determined by inc( rporation of [35S]methionine. FDPD and FDP-E did not it :rease fibrinogen synthesis in the presence of hydrocortisone. However, hepatocytes cultured without hydrocortisone displayed increased fibrinogen synthesis (2.0- to 2.8-fold) with FDP-D (2.6-6.7 #M) but not with FDP-E (5.7 puM). At these FDP concentrations the synthesis of albumin, haptoglobin, and transferrin was not increased. FDP-D-induced fibrinogen synthesis was inhibited (>90%) by actinomycin D and cycloheximide, indicating that the increase in [USlmethionine incorporation was from de novo protein synthesis. The role of FDP-D was further substantiated by showing that FDP-D, but not FDP-E, bound to the hepatocytes. These data indicate that FDP-D, but not FDP-E, directly and specifically stimulates fibrinogen synthesis in rat hepatocytes; this stimulation does not require any additional serum or protein cofactors.

MATERIALS AND METHODS Reagents. Tris(hydroxymethyl)aminomethane (Tris), sodium chloride, Triton X-100, phenylmethylsulfonyl fluoride, EDTA, actinomycin D, and cycloheximide were from Sigma. Bovine serum albumin (30%) was from Miles Scientific. Cell culture reagents were cupric sulfate, sodium selenite, zinc sulfate, epidermal growth factor, hydrocortisone, and collagenase (type I, lot 94F0698) (Sigma), insulin (Eli Lilly); RPMI 1640, Hanks' buffered saline, penicillin, streptomycin, and Fungizone (GIBCO). [35S]Methionine (1132 Ci/mmol; 1 Ci = 37 GBq) was obtained from DuPont/NEN. Carrier-free "?5I (3.7 GBq of 1251 per ml) was from Amersham. Trasylol was from Mobay Chemical. Materials for SDS/PAGE and molecular weight standards were from Bio-Rad. All other chemicals were of reagent grade and obtained from Fisher Scientific. Animals. Adult male Sprague-Dawley rats (200-250 g) were used (Charles River Breeding Laboratories) for preparation of hepatocytes and rat tail collagen. Preparation of Hepatocytes. Rat hepatocytes were prepared by a modification (12) of the collagenase perfusion method of Seglen (13). The isolated cells were washed and prepared for culture by the method described (14). Hepatocyte viability was->90% in all cultures used as determined by trypan blue exclusion and lactic dehydrogenase released into the medium. Culture Conditions. Hepatocytes were cultured in serumfree hormonally defined (SFHD) medium as described (15). SFHD medium consisted of RPMI 1640 supplemented with copper (0.1 mM), selenium (3.0 nM), zinc (50 pM), and penicillin/streptomycin (100 units/ml). Fungizone (2.5 mg/ ml) was included during the first day of culture. SFHD was further supplemented with either insulin (10 pg/ml) and epidermal growth factor (50 ng/ml) and referred to as SFHDIE or with insulin, epidermal growth factor, and hydrocortisone (20 AuM) and referred to as SFHD-ICE. Cells (2 x 106 viable cells per dish) were cultured in 60-mm dishes (Coming) that were precoated with rat tail (type I) collagen (14). After 4-hr incubation (humidified 5% CO2 at 370C) the unattached cells were removed by washing with RPMI 1640, and the medium was replaced with SFHD, SFHD-IE, or SFHD-ICE medium. During the first 24 hr of culture the medium also contained preservative-free bovine lung heparin at 2 units/ml (138 units/mg; Upjohn). During the period when FDPs were added to the culture, the media contained heparin at 10 units/ml. We had previously dem-

Fibrinogen, the central component of the coagulation system, is synthesized in the liver. The plasma level of fibrinogen has been suggested to be an independent risk factor for coronary (1) and cerebral occlusive arterial diseases (2). Hence, it is extremely important to understand the molecular mechanisms controlling fibrinogen synthesis. Plasmin digestion of fibrinogen and fibrin results in generation of fibrinogen degradation products (FDPs) and fibrin degradation products, respectively. Our studies have previously shown that i.v. infusion of homologous FDPs into rabbits stimulates fibrinogen synthesis (3, 4). In these studies, stimulation of fibrinogen synthesis by FDP fragment D (FDP-D) was 3- to 5-fold higher than by FDP fragment E (FDP-E) (4). A similar effect of FDP-D was subsequently seen in rats (5, 6). In vitro perfusion of isolated rat liver (7) has also identified FDP-D ability to stimulate fibrinogen synthesis. Studies of FDP effects on fibrinogen synthesis in hepatocyte culture have produced discrepant results (8-11). Both direct (8) and indirect (9) effects of FDPs on fibrinogen synthesis have been reported. These discrepancies are difficult to resolve, due, in part, to a diversity of cell culture conditions. Because a sound understanding of the regulatory mechanisms of fibrinogen synthesis is of important physiologic and clinical value, we have further studied the direct role of FDPs in fibrinogen synthesis. These studies evaluate the effect of homologous rat FDP preparations of fibrinogen biosynthesis in rat hepatocytes cultured in a serum-free, hormonally defined medium.

Abbreviations: FDP, fibrinogen degradation product; FDP-D, FDP fragment D; FDP-E, FDP fragment E; HSF, hepatocyte regulatory factor; SFHD, serum-free hormonally defined medium as described; SFHD-IE, SFHD supplemented with insulin and epidermal growth factor; SFHD-ICE, SFHD supplemented with insulin, epidermal growth factor, and hydrocortisone. TTo whom reprint requests should be addressed at: Department of Medicine, Blalock 1002, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205-2101.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8788

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onstrated that heparin is required for accurate quantitation of secreted fibrinogen in serum-free medium (16). Antibody and Antigen Preparations. Sheep anti-rat fibrinogen, sheep anti-rat transferrin, rabbit anti-rat albumin, and rabbit anti-rat haptoglobin antibodies were prepared as described (14). All four antibody preparations were precipitated with 40% ammonium sulfate three times (17) and were monospecific as determined by immunoelectrophoresis (18). Rat fibrinogen was purified from pooled rat plasma as described (19). Rat albumin and rat transferrin were purchased from Cooper Biomedical. Rat haptoglobin was a gift from J. Hanley (University of Iowa). Preparation of Homologous Rat FDPs. Rat FDPs were prepared by DEAE-cellulose chromatography of plasmin digest (Thrombolysin; Merck Sharp & Dohme) as described (20). Fractions containing FDP were pooled and dialyzed against cold deionized water and lyophilized for storage at -30'C. Protein purity was determined by SDS/PAGE (21). Cell Culture Experiments with FDPs. Dilutions of stock solutions of FDP-D or FDP-E (1.056 mg/ml of SFHD-IE and heparin at 10 units/ml) were made in the same medium. An identical quantity of [35S]methionine (25 puCi per dish in Hanks' buffered saline) was added to each FDP-containing solution and two control culture media (SFHD-IE and SFHD-ICE) lacking FDPs. Hepatocytes were incubated for 24 hr in SFHD-IE (heparin at 2 units/ml). SFHD-IE was removed, and a modified Hanks' buffered saline (HBSS*) medium supplemented with the ingredients of SFHD without methionine was added to the cells. After 45 min the HBSS* medium was removed, and the respective FDP-containing medium or control medium were added to triplicate dishes (0.4 ml/dish). At the indicated times, the culture media were collected in 1 mM phenylmethylsulfonyl fluoride/Trasylol (50 kallikrein inhibitor units/ml) and frozen at -70'C. The culture dishes were washed with phosphate-buffered saline, fixed in absolute methanol for 5 min, and stored frozen for subsequent DNA assay. Immunoprecipitation Assay of Hepatocyte-Synthesized Proteins. Hepatocyte-synthesized proteins were assayed by immunoprecipitation with antibody absorbed onto protein A-bearing Staphylococcus A (Pansorbin; Calbiochem). The Pansorbin was washed three times with buffer L (40 mM Tris/200 mM NaCI/0.4% SDS, pH 7.4) and was loaded with antibody at 4°C by mixing for 5 hr. Before use the antibodyactivated Pansorbin was washed twice with buffer L. Fibrinogen Assay. Samples of spent culture media (50 ul) were mixed with buffer E (200 mM Tris, pH 7.4/100 mM NaCl/1 mM Na2EDTA/0.5% Triton/0.1% SDS/Trasylol at 50 kallikrein inhibitor units per ml/0.9% bovine serum albumin/1 mM phenylmethylsulfonyl fluoride). An excess amount (300 ,l) of activated Pansorbin containing the adsorbed sheep anti-rat fibrinogen antibody was then added. The samples were incubated overnight at 4°C, with mixing. The Pansorbin-antigen-antibody complex was recovered from the medium by centrifugation at 800 x g for 10 min at 4°C. The adsorbed antigen-antibody complex was washed three times with buffer E to eliminate nonspecific trapping. The binding capacity of the activated Pansorbin was 1 mg of rat fibrinogen per ml, as determined with 125I-labeled rat fibrinogen. The adsorbed labeled fibrinogen was solubilized in liquid scintillation fluid (Ready-Solv EP; Beckman) and counted on a Beckman LS-133 liquid scintillation counter. Albumin, Transferrin, and Haptoglobin Assays. These assays were done as described above using specific antibodyadsorbed Pansorbin. Quantitation of Secreted Fibrinogen. The amount of [35S]methionine-labeled fibrinogen measured by immunoprecipitation is an indicator of fibrinogen synthesis and secretion in the hepatocyte cultures. In an initial set of experiments, fibrinogen was quantified by immunoassay (14) in the two

Proc. Natl. Acad. Sci. USA 86 (1989)

8789

control cultures: hepatocytes in SFHD-IE and hepatocytes in SFHD-ICE. In these cultures, because no cross-reactive FDP had been added, a proportionality was generated based on the ratio of the amount of immunoprecipitable radioactivity ([35S]methionine-cpm) and the amount of immunoassay-measurable fibrinogen (pug of fibrinogen per 2.0 x 106 cells). A linear increase of quantifiable fibrinogen in the hydrocortisone-containing cultures (SFHD-ICE) was obtained compared with the nonhydrocortisone-containing cultures (SFHD-IE). In all studies evaluating the effect of added FDPs, the ratio of [35S]methionine radioactivity between these control preparations was determined. By using this proportionality and comparing to the [35S]methionine uptake (cpm) of FDP-containing hepatocyte, a quantifiable amount of fibrinogen (,ug of fibrinogen per 2.0 x 106 cells) was estimated for each FDP-containing culture. Other Assay Methods. After completion of an experiment the cell cultures were assayed by one of two methods for the determination of hepatocyte mass. Random dishes were assayed for protein content by the method of Lowry et al. (22). The remaining dishes were assayed for DNA content as described (23). A linear correlation between total cell number and DNA content or protein content was seen. FDP Binding Assay. To identify the existence of membrane receptor sites for degradative fragments of fibrinogen, HepG2 cells (liver tumor, patent line 47; American Type Culture Collection) were grown in culture medium (Eagle's minimum essential medium supplemented with nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum; Flow Laboratories). Cells were grown to confluency, trypsinized (trypsin-EDTA; 1x; Sigma) for 10 min, and pelleted by centrifugation at 500 x g for 5 min at 250C. Cells were washed and resuspended in Tyrode's buffer (NaCl at 8 g/liter, KCI at 0.2 g/liter, NaHCO3 at 1.0 g/liter, NaH2PO4 at 0.05 g/liter, MgCl2-6H2O at 0.2 g/liter, dextrose at 1.0 g/liter, and 2% bovine serum albumin). Aliquots of 250 ,l or -0.5 x 106 cells were added to Eppendorf tubes. Ten microliters of human 125 I-labeled FDP-D or 125 I-labeled FDPE (10-200 nM; 200,000 cpm per assay; total assay volume = 260 ,l) was added to these triplicate tubes. Specimens were incubated at 4°C for 1 hr and periodically gently agitated to ensure homogenous mixing. The HepG2 cells/FDP-D or FDP-E mixture was centrifuged at 16,000 x g for 5 min. The cellular pellet was resuspended and washed three times in Tyrode's buffer. The radioactivity in the final pellet was counted (y 9000; Beckman) to determine the amounts of 1251-labeled FDP-D or 1251-labeled FDP-E bound to the hepatocytes.

RESULTS Purification of Rat FDPs. Plasmin digestion of rat fibrinogen produced four distinct protein bands on SDS/PAGE electrophoresis corresponding to FDP fragments D1, D2, D3, and E. This FDP mixture eluted from DEAE-cellulose demonstrated three distinct peaks of protein identifiable as FDPD and FDP-E (Fig. 1). The purified FDP fragments were added to hepatocyte cultures as a pure fragment D preparation (Dl), a pure fragment E preparation, or a mixture of fragments D and E. Characteristics of Hepatocyte Cultures. Hepatocytes cultured in SFHD-IE secreted fibrinogen at a rate of 11.2 ± 0.5 ,ug per 2 x 106 cells per 24 hr for up to 72 hr in culture. With hydrocortisone (hepatocytes cultured in SFHD-ICE), fibrinogen production was significantly increased (16.8 ± 3.0 ,ug). The increase of secreted fibrinogen was due to de novo synthesis as measured by [35S]methionine incorporation into

newly synthesized protein. Exposure of Hepatocytes to FDPs in SFHD-ICE. A mixture of FDP-D (2.6 uM) and FDP-E (5.7 /xM) was added to

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FIG. 1. Purification of rat FDPs. Purified rat fibrinogen was digested with plasmin and subsequently eluted on a DEAE-cellulose column using a linear ionic double gradient (19). The constituents of the three major protein peaks were identifiable as D1, D2-D3, and E on SDS/PAGE analysis; fraction size was 6 ml. (Insets) Coomassie blue-stained polyacrylamide gels of fragments D1 (94 kDa) and E (52 kDa).

hepatocytes cultured in steroid-containing medium (SFHDICE). The effects of FDP on fibrinogen synthesis were assessed by two approaches: (i) The FDPs were added to the culture dishes, and aliquots of medium were removed at selected periods of incubation. In these samples, secreted newly synthesized fibrinogen was measured by [35S]methionine incorporation. (ii) FDPs were added to the cultures, and at various times the medium was removed, and the cells were harvested for determination of intracellular and extracellular fibrinogen synthesis, as measured by [35S]methionine incorporation. With both conditions extracellular fibrinogen was not increased with FDPs compared to the control cultures. There were no differences in intracellular fibrinogen levels at various incubation times (Fig. 2). Characteristics of Corticosteroid-Containing (SFHD-ICE) Culture. Because FDPs did not increase fibrinogen biosynthesis in hydrocortisone, we evaluated the capacity of the hepatocyte cultures to produce fibrinogen in the absence of 26

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FIG. 3. Steroid stimulation of fibrinogen biosynthesis. Hepatocytes were cultured in either SFHD-IE (o) or SFHD-ICE (x). Dexamethasone (1 ,uM) was added to both culture preparations, and the amount of secreted fibrinogen was quantified by immunoassay (14). In SFHD-ICE, dexamethasone addition did not elicit increased fibrinogen synthesis. In SFHD-IE, dexamethasone significantly increased secreted fibrinogen. Data represent percent change of fibrinogen synthesis with dexamethasone.

corticosteroids. Hepatocytes were cultured either in SFHDIE or in SFHD-ICE for 24 hr; then the media were replaced with the corresponding fresh media. Triplicate culture dishes were further supplemented with 1 nm to 10 ,.M dexamethasone. In SFHD-IE cultures, the addition of dexamethasone caused a dose-responsive stimulation of fibrinogen synthesis. In SFHD-ICE cultures, however, dexamethasone did not induce fibrinogen synthesis (Fig. 3). In fact, there was a steady decrease in fibrinogen secretion with additional steroid. Cultures in SFHD-ICE was designated as "maximally" stimulated. Exposure of Hepatocytes to FDPs Cultured in SFHD-IE. A mixture of FDP-D and FDP-E was added to submaximally stimulated hepatocytes cultured without corticosteroid (SFHD-IE). Fibrinogen synthesis was not measured during the first 16 hr. After 24 hr of culture extracellular fibrinogen increased significantly, whereas the amount of albumin, transferrin, and haptoglobin biosynthesis was identical in FDP-containing and control cultures (Fig. 4). Specificity of FDP-D Stimulatory Effect. Further experiments were conducted to determine the stimulatory effect of

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0 Time in cLiture, hr FIG. 2. Exposure of hepatocytes to FDPs in hydrocortisonecontaining medium. Rat hepatocytes were purified and cultured in SFHD-ICE (hydrocortisone-containing medium). For each experiment triplicate culture dishes were prepared. After 24-hr incubation the media was removed, the cells were washed with methionine-free buffer, and fresh SFHD-ICE, containing [35S]methionine, was added. In one set of cultures FDP-D (2.6 ,uM) and FDP-E (5.7 ,uM) were added. At the indicated times (0, 4, 8, 18, and 24 hr) the media from triplicate dishes were collected and assayed for extracellular secreted fibrinogen [control (o); FDP-DE (A)], and the cells were assayed for intracellular fibrinogen [control (o); FDP-DE (x)]. No difference in fibrinogen synthesis was seen between the FDPcontaining cultures and the control cultures.

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FIG. 4. FDP-stimulated fibrinogen biosynthesis: hepatocytes cultured in SFHD-IE. Hepatocytes (triplicate dishes) were cultured as described in Fig. 2 in SFHD-IE (control media) or SFHD-IE containing a mixture of FDP-D (2.6 ,uM) and FDP-E (5.7 ,uM). Extracellular protein was quantified by [35S]methionine incorporation. After 24 hr a significantly increased amount of fibrinogen was detected in FDP-containing cultures (P < 0.005). Albumin, haptoglobin, and transferrin syntheses were not altered. The degree of [35S]methionine uptake in FDP-containing cultures is expressed as percent control.

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Proc. Natl. Acad. Sci. USA 86 (1989)

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FIG. 5. Specificity of the FDP-D stimulatory effect. Hepatocytes (triplicate dishes) were cultured as described in Fig. 2 in either SFHD-IE (control media), SFHD-IE containing FDP-D (5.2 AM) (filled bars), or SFHD-IE containing FDP-E (5.7 ,uM) (hatched bars). Protein synthesis was measured by [35S]methionine incorporation. After 24 hr increased fibrinogen was detected in media from the FDP-D cultures (P < 0.005); FDP-E had no effect. The other hepatocyte marker proteins (albumin, haptoglobin, and transferrin) were not altered. Data are shown as percent control.

the FDP-D and FDP-E mixture. Pure FDP-D (5.2 ,uM) or pure FDP-E (5.7 ,uM) was added to triplicate culture dishes. The culture media were collected after 24 hr and assayed for fibrinogen, albumin, haptoglobin, and transferrin. Fibrinogen biosynthesis assayed by [35S]methionine incorporation was significantly increased in cells cultured with FDP-D, but not with FDP-E. Levels of several hepatocyte marker proteins were not increased with FDP-D (Fig. 5). The variability of response to FDP-D among hepatocytes obtained from different rats was studied. In each instance, the fibrinogen biosynthesis was significantly increased with FDP-D (Table 1). In individual hepatocyte cultures from different animals, the degree of FDP-D increased synthesis and the concentration of FDP-D required were variable. Fibrinogen biosynthesis was stimulated 2.0- to 2.8-fold with FDP-D. The stimulatory effect of FDP-D was inhibited (90%) when the hepatocytes were cultured with the protein synthesis inhibitors actinomycin D (3 tug/ml) and cycloheximide (3 ,tg/ml). Quantification of Fibrinogen Synthesis. The amounts of fibrinogen synthesized in control cultures (11.2 ± 0.5 ,tg per 2 x 106 cells per 24 hr) and hydrocortisone-containing cultures (16.8 ± 3.0,ug) were determined from nine experiments in which [35S]methionine metabolic labeling was used. The fibrinogen produced was measured from the radioactivities seen in FDP-containing cultures compared with those of control cultures. With FDP-D, fibrinogen synthesis was 22.1 ± 2.0 ,ug per 2 x 106 cells per 24 hr, and in the FDP-D/FDP-E mixture, synthesis was 21.5 ± 1.0 ,ug; in cultures containing FDP-E, fibrinogen synthesis was 12.5 ± 1.3 Ag. Table 1. FDP-D-stimulated fibrinogen response from different animal hepatocyte donors Animal FDP-D, ,uM Increase, % P 1 5.2 250