The role of betagamma and alphagamma complexes in the assembly of human fibrinogen.

The role of αγ and βγ dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing β and γ chains and generating βγ complexes were fused with cells expressing only the α chain. Likewise, when cells co-expressing α and γ chains and generating αγ complexes were fused with cells expressing only the β chain, fibrinogen was also formed and secreted. The relative amounts of αγ or βγ intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the β chain was limiting, the αγ dimer was the predominant intermediate; likewise, when the α chain was limiting, the βγ complex was the predominant intermediate. The incorporation of preformed αγ and βγ complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of αγ and βγ complexes as functional intermediates in the assembly of fibrinogen.

The role of ␣␥ and ␤␥ dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing ␤ and ␥ chains and generating ␤␥ complexes were fused with cells expressing only the ␣ chain. Likewise, when cells co-expressing ␣ and ␥ chains and generating ␣␥ complexes were fused with cells expressing only the ␤ chain, fibrinogen was also formed and secreted. The relative amounts of ␣␥ or ␤␥ intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the ␤ chain was limiting, the ␣␥ dimer was the predominant intermediate; likewise, when the ␣ chain was limiting, the ␤␥ complex was the predominant intermediate. The incorporation of preformed ␣␥ and ␤␥ complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of ␣␥ and ␤␥ complexes as functional intermediates in the assembly of fibrinogen.
Human fibrinogen is a large soluble plasma protein that plays a critical role in protecting the vascular network against the loss of blood following tissue injury (Hantgan, 1987). Fibrinogen (M r 340,000) is composed of two sets of three polypeptide chains including the ␣ (M r 66,000), ␤ (M r 52,000), and ␥ (M r 46,500) chains (McKee et al., 1966). The six chains, (a␤␥) 2 , contain 29 disulfide bonds and form a complex trinodular structure (Hall and Slayter, 1959;Erickson and Fowler, 1983) linked by two coiled-coil regions (Doolittle et al., 1978). The ␣, ␤, and ␥ chains are encoded by three independent genes clustered on chromosome 4 at 4q23-32 (Chung et al., 1990;Forman and Barnhardt, 1964). During the synthesis of fibrinogen, the individual chains are translated, processed, assembled, and eventually secreted into plasma as a mature fibrinogen molecule. The assembly of fibrinogen appears to follow a single sequential chain-addition pathway (Huang et al., 1993a), although other pathways have also been proposed (Hartwig and Danishefsky, 1991;Roy et al., 1991). Data from our laboratory suggest that the initial steps in fibrinogen assembly involve the formation of the ␣␥ and ␤␥ dimers linked by disulfide bonds. A third chain is then added to the each dimer to form ␣␤␥ halfmolecules. Finally, two half-molecules dimerize and become linked by five disulfide bonds to form the intact fibrinogen molecule (Huang et al., 1993b). It has not been clearly established, however, whether both ␤␥ and ␣␥ are functional intermediates in fibrinogen assembly, since no precursor-to-product relationship has been demonstrated.
Imbalances in the intracellular levels of the ␣, ␤, and ␥ chains have been observed in hepatocytes and hepatoma cells from several species. A common feature is an excess amount of ␥ chain, but limited levels of either the ␤ chain, as in human hepatocytes in culture (Yu et al., 1983 and and the rabbit (Alving et al., 1982), or the ␣ chain, as in rat (Hirose et al., 1988) and chicken (Plant and Grieninger, 1986). Unequal rates of synthesis and/or intracellular degradation may contribute to this imbalance. The effect of an imbalance of the three chains on fibrinogen synthesis and assembly is not known. In cultured chicken embryonic hepatocytes, adding serum to the culture medium restored the balance of fibrinogen chains and resulted in the salvage of those chains, which otherwise were targeted for degradation (Grieninger et al., 1984). However, the mechanism for this restoration has not been established.
In the present studies, the role of the ␣␥ and ␤␥ dimers as intermediates in the assembly of fibrinogen was examined using a cell fusion system employing stably transfected baby hamster kidney (BHK) 1 cells expressing one or two of the fibrinogen chains. The formation of the ␤␥ and ␣␥ complexes was then examined with ␣, ␤, or ␥ chains being limiting. Intracellular levels of fibrinogen chains and the ␣␥ and ␤␥ complexes in two fibrinogen-producing human hepatoma cells lines, Hep G2 (Knowles et al., 1980) and HuH-7 (Nakabayashi et al., 1982), were also measured. These data provided additional evidence for the formation of ␣␥ and ␤␥ dimers, as well as ␣␤␥ half-molecules as intermediates in the assembly of fibrinogen from its three individual chains. Furthermore, the cellular level of individual chains determines whether the ␣␥ or ␤␥ complex is the predominant intermediate.

MATERIALS AND METHODS
Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase were purchased from Promega and Boehringer Mannheim, and the Sequenase Kit from U. S. Biochemicals. Cell culture media were purchased from Life Technologies, Inc. and JRH Scientific, and fetal bovine serum from Hyclone. Polyethylene glycol 4000 (PEG-4000) was from Life Technologies, Inc. [ 35 S]Met and [ 35 S]Cys (approximately 1.1 Ci/mmol) were obtained from Amersham. Protein A-Sepharose and cycloheximide were from Sigma. Antibodies against human fibrinogen were from Accurate Chemical.
Cell Lines-The Hep G2 human hepatoma cell line (Knowles et al., 1980) was kindly provided by Dr. Mulvihill at ZymoGenetics, Inc., Seattle, WA. HuH-7 human hepatoma cell line (Nakabayashi et al., 1982) was kindly provided by Dr. Nakabayashi from the University of Calgary, Alberta, Canada. The establishment and characterization of stably transfected BHK cell lines expressing individual fibrinogen chains and intact fibrinogen have already been described (Huang et al., * This work was supported by National Institutes of Health Grant HL16919. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡  1993a). Hep G2 cells and stably transfected BHK cell lines were maintained as described earlier (Huang et al., 1993a). HuH-7 cells were maintained in RPMI 1640 medium containing 1% fetal bovine serum, antibiotics (50 mg/ml penicillin, 50 mg/ml streptomycin, and 100 mg/ml neomycin, Life Technologies, Inc.), and supplements as described (Nakabayashi et al., 1982).
Construction of Expression Vectors-The mammalian expression vector pZem229 (Mulvihill et al., 1988) was used to construct high-level expression vectors for the ␣, ␤, and ␥ chains of human fibrinogen (Huang et al., 1993a). The mammalian expression vector pZem97 was used to construct low-level expression vectors for the ␣ chain, pAZem97 (Huang et al., 1993a), and the ␥ chain, pGZem97. The expression vector pBD-1 (Farrell et al., 1991) containing the ␤ chain cDNA was used as a low-level expression vector for this chain. The cell line BG500 was established by transfecting pGZem229 (Huang et al., 1993a) into the cell line B1209-5, in which the expression level of the ␤ chain was increased by selection and amplification in 5 mM methotrexate. In BG500 cells, approximately equal amounts of ␤ and ␥ chains were synthesized as estimated by Western blotting and metabolic labeling analyses.
Metabolic Labeling and PEG-induced Cell Fusion-BHK cells (90% confluence) were labeled in Met-free and Cys-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% dialyzed fetal bovine serum, 200 mCi/ml [ 35 S]Met and [ 35 S]Cys for the indicated time. In cell fusion experiments, labeled cells were washed three times with Dulbecco's modified Eagle's medium and incubated with 2 ml of PEG-4000 (50% in phosphate-buffered saline) for 1.5 min to induce cell fusion. Cells were then washed five times with Dulbecco's modified Eagle's medium after removal of PEG-4000 and further incubated in growth medium for varying amounts of time as indicated. In some experiments, cells were incubated in growth medium containing cycloheximide (20 g/ml) for 1 h before fusion and further incubated in the same medium after fusion. Hep G2 cells (90% confluence) were labeled in Met-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.), containing 5% dialyzed serum and 200 mCi/ml [ 35 S]Met. HuH-7 cells were labeled in Met-free RPMI 1640 (BioWhittaker), containing 1% dialyzed serum, supplements, and 200 mCi/ml [ 35 S]Met. Cell lysates were prepared as described previously (Huang et al., 1993a) and stored at Ϫ70°C.
Immunoprecipitation and Electrophoresis-Immunoprecipitation was carried out with a rabbit antibody (IgG fraction) to human fibrinogen and Protein A-Sepharose as described previously (Huang et al., 1993a).
Electrophoresis including one-and two-dimensional non-reduced/ reduced SDS-PAGE were performed according to Laemmli (1970) and Davidson et al. (1977), respectively. Gels for autoradiography were impregnated with Amplify (Amersham) prior to drying. The radioactivity in the gel bands was quantitated by a PhosphorImager (Molecular Dynamics). The specific activities for each of the three labeled chains were calculated by normalizing the radioactivity to the number of methionine and/or cysteine residues in each chain Lottspeich and Henschen, 1977;Doolittle et al., 1979;Henschen et al., 1979;Watt et al., 1979).

Formation and Secretion of Fibrinogen in Fused BHK Cells
Expressing Fibrinogen Chains-To determine whether ␣␥ and ␤␥ complexes were essential intermediates in the assembly of fibrinogen, experiments were designed to introduce one or more of the three chains of fibrinogen into individual stable BHK cells followed by cell fusion in the presence of PEG (Davidson et al., 1976). These experiments were undertaken to test whether fused cells could synthesize ␣␥, ␤␥, and a␤␥ complexes as well as mature fibrinogen. The formation of ␤␥ and ␣␥ complexes was initially examined by the fusion of stably transfected cells expressing the ␤ chain with cells expressing the ␥ chain (beta cell/gamma cell fusion). By this approach, each pair of BHK cells were co-cultured in a dish, labeled with [ 35 S]Met and [ 35 S]Cys, and then fused in the presence of PEG. The intracellular complexes present in the fused cells were analyzed by immunoprecipitation, using a human fibrinogen-specific antibody and Protein A-Sepharose, followed by two-dimensional SDS-PAGE. The ␤␥ complex was readily formed in the beta cell/gamma cell fusion (data not shown). Likewise, the ␣␥ com-plex was rapidly formed after the fusion of alpha cells and gamma cells. However, no ␣␤ complex was formed after fusion of alpha cells with beta cells. These preliminary results demonstrated that cells synthesizing individual fibrinogen chains remain competent to assemble potential fibrinogen intermediates after fusion and can be used to follow the precursor-toproduct relationship in the assembly process. Since the fusion of cell mixtures is random, various combinations are likely to occur. Consequently, not all the labeled precursors are expected to be converted to assembled products in the fused cells.
Experiments were then carried out in which the ␣ chain was introduced into BHK cells synthesizing and accumulating the ␤␥ complex, and in which the ␤ chain was introduced into cells synthesizing the ␣␥ complex. In these experiments, two pairs of stably transfected BHK cell lines were used. The first pair included a cell line (BG500) which expressed ␤ and ␥ chains generating ␤␥ complexes, and a cell line (A104) that expressed only the ␣ chain (Huang et al., 1993a). The second pair included a cell line (AG1302) that expressed ␣ and ␥ chains generating ␣␥ complexes and a cell line (B1209) that expressed only the ␤ chain (Huang et al., 1993a). When the cells expressing the ␤ and ␥ chains were fused with the cells expressing only the ␣ chain (beta,gamma cell/alpha cell fusion), fully assembled intracellular fibrinogen was observed (Fig. 1A, lane 4). Very little or no fibrinogen was identified in the control or non-fused cells (lane 3). The assembled fibrinogen was also secreted into the culture medium (lane 2) in contrast to the control (lane 1). Two-dimensional, non-reduced/reduced SDS-PAGE showed that fibrinogen formed in the fused cells contained ␣, ␤, and ␥ chains as expected (Fig. 1B). Complexes of ␤␥ and ␣ 2 chains were also observed. Similarly, when cells expressing ␣ and ␥ chains were fused with cells expressing only the ␤ chain (alpha,gamma cell/beta cell fusion), fully assembled intracellular fibrinogen was observed ( Fig. 2A, lane 4), but not in the control or non-fused cells (lane 3). Fibrinogen was also secreted into the culture medium (lane 2) in contrast to the control (lane 1). Furthermore, two-dimensional, non-reduced/reduced SDS-PAGE showed that the fibrinogen formed in the fused cells contained ␣, ␤, and ␥ chains (Fig. 2B). Complexes of ␣␥, ␤␥, and a␤␥ were also observed (Fig. 2B). These results demonstrated that fibrinogen could be assembled and secreted by the fusion of cells containing preformed ␣␥ or ␤␥ complexes with cells containing the third fibrinogen chain.
The time course of intracellular fibrinogen formation in the fused cells was determined by Western blot analysis of cell lysates using an affinity purified antibody to human fibrinogen. As shown in Fig. 3, fully assembled fibrinogen was detected in both types of fused cells, including the beta,gamma cell/alpha cell fusion and the alpha,gamma cell/beta cell fusion. Assembly was detectable within 30 min after PEG-induced fusion. As the post-fusion incubation time increased, the amount of fibrinogen increased.
In some experiments, (beta,gamma cell/alpha cell fusion), the ␤␥ complex, fibrinogen, as well as ␣ chain dimers were formed in the fused cells (Fig. 1B). In these experiments, ␣ dimers were generated in the ␣ chain cell line. In other fusion experiments (alpha,gamma cell/beta cell fusion), the ␣␥ and ␣␤␥ complexes, as well as some ␤␥ complex, were formed in the fused cells (Fig. 2B). The ␤␥ complex was generated by the interaction of ␤ chains in the ␤ chain cell line with the free excess ␥ chains in the alpha,gamma cell line. The presence of the ␤␥ complex in these fused cells suggested the possibility that fibrinogen may have been synthesized via the newly formed ␤␥ complex, as well as the pre-formed ␣␥ complex.
Effect of Cycloheximide on Fibrinogen Formation in Fused BHK Cells-The effect of cycloheximide on the synthesis of fibrinogen was tested by measuring the incorporation of [ 35 S]Met and [ 35 S]Cys into fibrinogen chains after exposure to 20 g/ml cycloheximide for 1 h. The intracellular fibrinogen chains were then immunoprecipitated and analyzed by reduced SDS-PAGE. The results indicated that the synthesis of fibrinogen was completely inhibited after incubation for 1 h with cycloheximide, as shown by the absence of any radioactive fibrinogen bands on the autoradiogram (Fig. 4).
To test whether pre-existing ␣␥ and ␤␥ complexes were functional intermediates in the assembly process, cells were cocultured and prelabeled with [ 35 S]Met and [ 35 S]Cys, followed by incubation for 1 h with media containing cycloheximide to stop further protein synthesis. The cells were then fused with PEG and examined for intracellular fibrinogen. In order to avoid the complications of an excess of ␥ chains in the assembly process, BHK cell lines BG1205 and AG105 were used in these fusion experiments. In the BG1205 cells, the ␤ chain was in considerable excess of the ␥ chain and no detectable free ␥ chain was present, as shown by the absence of a monomeric ␥ chain on the autoradiogram (Fig. 5, lane 1). Similarly, in AG105 cells, the ␣ chain was in considerable excess of the ␥ chain. Consequently, there was no detectable amount of free intracellular ␥ chain present as indicated by the absence of monomeric ␥ chain on the autoradiogram (Fig. 5, lane 4).
When cell fusion experiments in these cells were carried out after inhibition of protein synthesis by cycloheximide, fibrinogen was formed in both the beta,gamma cell/alpha cell fusion (Fig. 5, lane 2), as well as the alpha,gamma cell/beta cell fusion (Fig. 5, lane 4). Fibrinogen assembly did not occur in the control experiments (lanes 1 and 3). These data strongly suggest that fibrinogen was assembled from the pre-existing ␤␥ complex and the ␣ chain in the beta,gamma cell/alpha cell fusion or from the pre-existing ␣␥ complex and ␤ chain in the alpha,gamma cell/ beta cell fusion. These results support the conclusion that pre-formed ␤␥ and ␣␥ complexes as well as individual ␣ and ␤ chains were intermediates in fibrinogen assembly. Moreover, fibrinogen assembly did not require de novo protein synthesis.
Effect of Imbalance of Fibrinogen Chains on Formation of ␤␥ and ␣␥ Complexes in Transfected BHK Cells-To examine further the effect of imbalance of fibrinogen chains on the formation of ␤␥ and ␣␥ complexes, BHK cell lines expressing all three chains of fibrinogen, but only limiting amounts of the ␣ chain (cell line FAi), the ␤ chain (cell line FBi), or the ␥ chain (cell line FGi), were established. Two promoters were used to express fibrinogen chains in these cell lines, including the wild-type metallothionein-1 promoter and a modified version. The expression level of the modified metallothionein-1 promoter was 10 -20-fold higher than the wild-type metallothionein-1 promoter (Mulvihill et al., 1988). The latter promoter was used to express the fibrinogen chain which was designed to be limiting.
In the cell line FAi, the ␣ chain was present in limiting amounts (Fig. 6A, lane 1). In this cell line, the major intracellular intermediate was the ␤␥ complex, while only a trace amount of the ␣␥ complex was observed (Fig. 6B, lane 1). In the cell line FBi, the ␤ chain was limited relative to the ␣ and ␥ chains (Fig. 6A, lane 2). In this cell line, the major form of intermediate was the ␣␥ complex and only a trace amount of the ␤␥ complex was generated (Fig. 6B, lane 2). In cell line FGi, the ␥ chain was limited relative to the ␣ and ␤ chains (Fig. 6A,  lane 3). In these experiments, ␣ and ␤ chains were present as ␣ chain oligomers and ␤ chain oligomers, respectively. Both the ␤␥ and ␣␥ complexes were present in trace amounts as determined both by one-dimensional (Fig. 6B, lane 3) and twodimensional SDS-PAGE (data not shown). Fully assembled fibrinogen was produced by all three of these cell lines (Fig. 7B) and secreted into the culture media (data not shown). The chain compositions of fibrinogen and the ␤␥ and ␣␥ intermediates in these cells were confirmed by two-dimensional SDS-PAGE analyses (data not shown).
These results demonstrated that the amounts of ␤␥ and ␣␥ intermediates were determined by the relative levels of the ␤ and ␣ chains. When the ␣ chain was limiting, the ␤␥ complex was formed preferentially, whereas in the case of limited ␤ chain, the ␣␥ complex was formed preferentially. However, when the ␥ chain was limited relative to the ␤ and ␥ chains, formation of both ␤␥ and ␣␥ complexes were greatly reduced due to the limited availability of the ␥ chain.
Relative Rate of Synthesis of Fibrinogen Chains and Formation of ␤␥ and ␣␥ Complexes in Hep G2 and HuH-7 Cells-The rate of synthesis of fibrinogen chains in both Hep G2 cells and HuH-7 cells were determined by measuring the rate of appearances of [ 35 S]Met in fibrinogen chains during the course of continuous labeling. In Hep G2 cells, there were approximately equal rates of synthesis for the ␣ and ␤ chains and a much faster rate of synthesis for the ␥ chain (Fig. 7A). In HuH-7 cells, approximately equal amounts of the ␤ and ␥ chains were observed and a much smaller amount of the ␣ chain (Fig. 7B). These results indicated that the ␥ chain was in excess compared to the other two chains in Hep G2 cells, whereas the ␣ chain was limited compared to the other two chains in HuH-7 cells.
Intracellular fibrinogen and fibrinogen intermediates in Hep FIG. 4. Effect of cycloheximide (CHX) on synthesis of fibrinogen chains in transfected BHK cells. Pairs of transfected BHK cells as indicated were co-plated onto 35-mm dishes, and 24 h later, the cells were exposed to cycloheximide-containing growth medium (20 g/ml) for 1 h. The cells were then labeled with 200 Ci/ml [ 35 S]Met and [ 35 S]Cys in the presence of 5% dialyzed fetal bovine serum for 30 min. After removal of the labeling medium, the cells were washed with phosphate-buffered saline and lysed. The cell lysates were immunoprecipitated and analyzed by reduced SDS-PAGE as described under "Materials and Methods." Panel A, cells treated with cycloheximide; panel B, cells treated the same as in panel A except without cycloheximide.
FIG. 5. Effect of cycloheximide on fibrinogen formation in fused BHK cells expressing fibrinogen chains. Pairs of transfected BHK cells as indicated were co-plated onto 35-mm dishes and radiolabeled for 15 min as in Figs. 1 and 2. The cells were then incubated with 20 g/ml cycloheximide-containing medium for 1 h. The control cells were then lysed, while the fusion cells were treated with PEG as in Fig.  1 and further incubated in 20 g/ml cycloheximide-containing medium for 1 h before lysis. The cell lysates were immunoprecipitated with anti-fibrinogen antibody and the immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. G2 and HuH-7 cells were analyzed using the same approach as employed for the transfected BHK cells (Fig. 8). Both fully assembled fibrinogen and various intermediates including a␤␥ half-molecules, ␣␥, and ␤␥ complexes were present in both cell lines. However, the relative amounts of ␣␥ and ␤␥ complexes differed in these two cell lines. In Hep G2 cells, the amount of the ␣␥ complex was greater then that of the ␤␥ complex (Fig. 8,  top). In contrast, in HuH-7 cells, the amount of the ␣␥ complex was less then that of the ␤␥ complex (Fig. 8, bottom). These results clearly showed that the relative amount of ␣␥ and ␤␥ complexes in human hepatoma cells was determined by the relative amounts of the component fibrinogen chains. In HuH-7 cells, the ␣ chain was limiting and the ␤␥ complex was preferentially formed. This was similar to the BHK cell line FAi, which was limited in the ␣ chain (Fig. 6, lane 1). In Hep G2 cells, there were approximately equal amounts of the ␣ and ␤ chains, and a large excess of the ␥ chain. Under these conditions, more of the ␣␥ complex was formed than the ␤␥ complex. This suggested that the rate of formation and/or rate of degradation of the ␣␥ and ␤␥ complexes also affects the relative amount of the two ␣␥ and ␤␥ intermediates. Two-dimensional gel electrophoresis also showed that in both the Hep G2 and HuH-7 cells, the extended ␣ chain variant, ␣ E (Fu et al., 1992) was expressed and incorporated into fibrinogen (Fig. 8, top and  bottom). FIG. 7. Relative rates of synthesis of fibrinogen chains in Hep G2 and HuH-7 cells. Hep G2 and HuH-7 cells, about 90% confluence, were labeled with 200 Ci/ml [ 35 S]Met in Met-free Dulbecco's modified Eagle's medium containing 5% dialyzed fetal bovine serum and Metfree RPMI 1640 containing 1% dialyzed serum and the supplements, respectively. At indicated time points, labeling media were removed, and the cells were washed and lysed. The cell lysates were then immunoprecipitated with anti-fibrinogen antibody and the immunoprecipitates were run on reduced SDS-PAGE. The gels were dried after fixing and scanned in a PhosphorImager. The specific radioactivity of gel bands was quantified as described under "Materials and Methods." The data shown here are representative of three independent labeling experiments.

FIG. 8. Formation of fibrinogen intermediates in Hep G2
and HuH-7 cells. Hep G2 and HuH-7 cells were labeled for 18 h and the cell lysates were immunoprecipitated as in Fig. 7. The immunoprecipitates were analyzed by two-dimensional SDS-PAGE as described in the legend to Fig. 1. Only the autoradiograms for the second dimensional gel are shown.