The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets.

The extended (E) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (EC). A recombinant form of EC (rEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography. Purified rEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in E. Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs E613/644 and E780/793. In the presence of 5 mmol/L EDTA, rEC is highly susceptible to plasmic degradation, but Ca2+ (5 mmol/L) renders rEC resistant. No protective effect from plasmic degradation was conferred to rEC by the peptides GPRPamide or GHRP, nor did rEC bind to a GPR peptide column. These results suggest that the EC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in the gammaC domain, we predict that the EC calcium binding site involves residues E772-778: DADQWEE.

Formation of the human fibrinogen subclass fib420: disulfide bonds and glycosylation in its unique (alphaE chain) domains.

COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel alphaE chain and those of the more abundant subclass whose alpha chains lack alphaE's globular C-terminus. That region, referred to as the alphaEC domain, is closely related to the ends of beta and gamma chains of fibrinogen (betaC and gammaC). Transfection of COS cells with alphaE, beta, and gamma cDNAs alone results in secretion of the symmetrical molecule (alphaEbetagamma)2, also known as Fib420. Cotransfection with cDNA for the shorter alpha chain yielded secretion of both (alphabetagamma)2 and (alphaEbetagamma)2 but no mixed molecules of the structure alphaalphaE(betagamma)2. Exploiting the COS cells' fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the alphaE chain. No evidence from Cys --> Ser replacements was found for interchain disulfide bridges involving the four cysteines of the alphaEC domain. However, for fibrinogen secretion, the alphaE, beta, and gamma subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.

Fibrinogen assembly: insights from chicken hepatocytes.

In all vertebrate species studied, the complex, disulfide-linked structure of fibrinogen is essentially the same: a hexamer assembled from three different subunits (A alpha, B beta, gamma)2. This study utilized species differences in fibrinogen subunit monomer pools to address the question of how these surplus subunit pools may affect the assembly process. We used a chicken model system in which B beta and gamma-subunits are present in excess, in contrast to the A alpha and gamma-subunit surplus found in human model systems. Analysis was based on pulse-chase experiments with electrophoretic separation of intracellular forms and secreted fibrinogen on reducing and nonreducing gels. The chicken liver-derived cells employed for this purpose, primary hepatocytes and a hepatoma cell line with a fortuitous defect in fibrinogen synthesis, together offer advantages over human systems for resolving the complexes formed in the early stages of assembly. The results demonstrate that in chicken hepatocytes there is an initial binding of gamma to A alpha subunits rather than to B beta subunits, as occurs in human hepatoma cells. Nevertheless, the presence of similar intracellular fibrinogen-related forms in both chicken- and human-derived cells, in the context of their differing subunit monomer pools, suggests an assembly pathway common to both species, with the versatility to be regulated by limitation of A alpha or B beta subunit production.

Characterization of a chicken hepatoma cell line with a specific defect in fibrinogen secretion.

This study characterizes plasma protein synthesis and its hormonal regulation in a chicken hepatoma cell line, with particular emphasis on fibrinogen. Whereas virtually all aspects of hemopexin, transferrin and albumin production in these cells corresponded to those of cultured primary hepatocytes, fibrinogen was not secreted. Analysis of fibrinogen subunit synthesis revealed a specific defect in synthesis of one subunit, gamma, correlating with a lack of its mRNA. Pulse-chase and electron microscopic studies demonstrate that, despite the inability of these cells to secrete the A alpha and B beta subunits produced, there is no long-term accumulation of unsecreted fibrinogen. The B beta fibrinogen subunits are largely degraded 2 hr after synthesis. During this time, approximately half of the A alpha subunits are degraded; the rest are converted to the glycosylated form. The implications of this type of defect with respect to the pathogenesis of fibrinogen storage disease are discussed.

Fib420: a normal human variant of fibrinogen with two extended alpha chains.

In fibrinogen, alpha E chains form a subpopulation of alpha subunits that are distinguished by a carboxyl extension homologous to the C termini of the other two constituent chains: beta and gamma. The molecular mass of alpha E is > 50% greater than that of the common alpha subunit, due in part to an extra 236 amino acids. These residues are encoded by exon VI, a recently discovered extension of the fibrinogen alpha gene. Additional mass is contributed by posttranslational processing, including N-glycosylation, which, based on experiments with the inhibitor tunicamycin, was found to account in large measure for alpha E migration on SDS/PAGE at approximately 110 kDa rather than at its calculated mass of 92,843 Da. An antibody specific for the exon VI-encoded domain of alpha E (anti-VI) and capable of recognizing alpha E-containing fibrinogen in both native and denatured form was generated using a recombinant protein as immunogen. Its use in Western blot analysis of fractions of normal human blood (plasma and preparations of fibrinogen) revealed a single, sharp, alpha E-containing band migrating behind the position of the broad, predominant fibrinogen band, (alpha beta gamma)2. Designation of the upper band as Fib420, an approximately 420-kDa homodimer of the formula (alpha E beta gamma)2, is based on the overwhelming proportion of alpha E subunits (> 80% of the total alpha chains) found in anti-VI-immunoprecipitable material from hepatoma cell medium. Several lines of evidence suggest that the alpha E subunit, alone or incorporated into fibrinogen, is more stable than the common alpha chain, a feature of potential clinical importance.

Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits.

Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology.

Assembly and secretion of fibrinogen. Degradation of individual chains.

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.

The beta chain of chicken fibrinogen contains an atypical thrombin cleavage site.

A cDNA corresponding to almost the entire coding region of the mRNA for the beta chain of chicken fibrinogen was sequenced. At the protein level, significant homology to the beta subunits of other vertebrate fibrinogens was found, with the highest degree of amino acid identity localized in the C-terminal region. In general, features conserved in the fibrinogens from other species also characterize the chicken sequence, including the cysteine motifs bordering an alpha-helical permissive region of fixed length and a single glycosylation site in the C-terminal region. However, the site of thrombin-catalyzed cleavage, which in other species consists of an Arg-Gly peptide bond, is instead an Arg-Ala bond in the chicken beta chain. The Ala was confirmed directly from a sequencing analysis of the purified beta chain of chicken fibrin. This finding may explain the observed slow clotting time of chicken fibrinogen relative to that of other species.

Synthesis and secretion of multiple forms of beta 2-interferon/B-cell differentiation factor 2/hepatocyte-stimulating factor by human fibroblasts and monocytes.

The cDNA for human beta 2-interferon (IFN-beta 2)/B-cell differentiation factor 2/hepatocyte-stimulating factor was expressed in Escherichia coli to yield a fusion protein which contains the 182 carboxyl-terminal amino acids of IFN-beta 2 fused to a 34-amino acid prokaryotic leader peptide (rIFN-beta 2). When added to cultures of human hepatoma cell line Hep3B2, rIFN-beta 2 as well as preparations of natural IFN-beta 2 enhance secretion of positive acute phase reactants such as alpha 1-antichymotrypsin, complement C3, fibrinogen, and alpha 1-acid glycoprotein and inhibit secretion of albumin, confirming that a protein derived from the IFN-beta 2 gene can have hepatocyte-stimulating factor activity. We have prepared a rabbit polyclonal antiserum to the E. coli-derived human IFN-beta 2 fusion protein. This polyclonal antiserum inhibits the hepatocyte-stimulating and B-cell differentiation activities of appropriate IFN-beta 2 preparations. The anti-rIFN-beta 2 antiserum has been used in immunoprecipitation experiments and in Western blots to help define the secretory proteins derived from the IFN-beta 2 gene in fibroblasts and monocytes. "Uninduced" human FS-4 fibroblasts as well as those induced with interleukin-1 alpha, tumor necrosis factor, or bacterial lipopolysaccharide secrete at least five forms of IFN-beta 2 of apparent molecular mass in the range from 23 to 30 kDa which can be resolved by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The three higher molecular mass forms are not observed when FS-4 cells are induced in the presence of tunicamycin, suggesting that these forms are N-glycosylated (gp28, gp29, and gp30). Although secretion of the two lower molecular mass forms is resistant to tunicamycin, they are labeled by [3H]glucosamine (gp23-gp25). The inclusion of cycloheximide during the [35S]methionine labeling of induced FS-4 cells results in the preferential synthesis and secretion of the 29-kDa triplet. Human monocytes induced with bacterial lipopolysaccharide also secrete several distinct forms of IFN-beta 2 in the size range from 23 to 30 kDa which co-migrate in polyacrylamide gels with those obtained from FS-4 cells. Our observations help relate previous descriptions of multiple forms of hepatocyte-stimulating factor to specific proteins derived from the IFN-beta 2 gene.

Regulation of apo-A-I processing in cultured hepatocytes.

Apo-A-I, the major protein component of high density lipoproteins, appears intracellularly as an intermediate precursor (pro-apo-A-I) with a hexapeptide extension (RHFWQQ) at its amino terminus. Proteolytic processing of pro-apo-A-I to apo-A-I has been shown to occur extracellularly in cell and organ cultures from rat and human tissues. Recently, however, intracellular conversion has been detected in chickens. To determine what distinguishes and regulates these two processing methods, the proteolytic processing and secretion of apo-A-I was studied by metabolic labeling in chick hepatocytes and in Hep-G2 cells (derived from a human hepatocellular carcinoma). The proportions of intracellular and secreted pro-apo-A-I and apo-A-I were measured by sequencing NH2-terminal portions of the proteins and determining the location of radio-labeled amino acids. Chick hepatocytes cultured in the absence of hormones or fetal bovine serum secreted primarily processed apo-A-I (83%). In the presence of serum these cells secreted only pro-apo-A-I, whereas incubation with a combination of hormones (insulin, triiodothyronine, dexamethasone) resulted in secretion of a nearly equal mixture of the pro- and processed forms of the protein. In contrast, Hep-G2 cells, maintained in the absence of serum, secreted only pro-apo-A-I; when grown in the presence of serum these cells secreted a mixture of pro- and processed apo-A-I. Under conditions in which chick hepatocytes and Hep-G2 cells secreted both forms of the protein, a mixture of pro- and processed apo-A-I was also found intracellularly; when only the pro-form was secreted, the cells likewise contained only pro-apo-A-I. Under all the above conditions, the secreted apo-A-I exhibited similar isoform patterns in two-dimensional gel electrophoresis. These data show that both chick hepatocytes and human hepatoma cells are capable of intracellularly processing pro-apo-A-I to apo-A-I, and that the extent of intracellular processing is controlled by the cell's hormonal environment.

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions.

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.

Selective intracellular degradation of fibrinogen and its reversal in cultured hepatocytes.

Primary monolayer cultures of chick embryo hepatocytes were employed in pulse-chase experiments to examine plasma protein synthesis and secretion. The fates of [35S]methionine-labeled fibrinogen and transferrin were monitored in cell extracts and in spent culture media. It was found that hepatocytes, which were maintained in the absence of added hormones or serum, released into the medium virtually all of the label of transferrin but only 30% of the label in fibrinogen. The remainder of the labeled fibrinogen was retained by the cells, gradually disappearing in a manner suggestive of its intracellular degradation. To stimulate fibrinogen production on as many levels as possible, fetal bovine serum was added to the medium of the cultured cells. Serum elicited an increase in the level of fibrinogen mRNA which was accompanied by a 7-fold increase in the rate of fibrinogen synthesis as well as the complete release of fibrinogen label, resulting in an overall 20-fold enhancement in the hepatocellular output of this protein. Thus, both the amount of fibrinogen synthesized as well as the amount ultimately secreted are subject to modulation by the hepatocellular environment.

Glycosylated A alpha chains in chicken fibrinogen.

Fibrinogen immunoprecipitated from cultured chick embryo hepatocytes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with fibrinogen from chicken plasma. The character and relatedness of the constituent polypeptide bands were established on the basis of enzymatic treatment, peptide analysis, metabolic labeling with [14C]glucosamine, and inhibition of glycosylation with tunicamycin. Hepatocyte-derived fibrinogen resolved into five polypeptides that, in order of decreasing apparent molecular weight, were identified as glycosylated A alpha, non-glycosylated A alpha, B beta, gamma', and gamma. Fibrinogen immunoprecipitated directly from chicken plasma yielded an identical profile except for an additional smaller A alpha chain. This small A alpha chain appears to be the product of partial proteolysis in the circulation and was the only A alpha band found in purified plasma fibrinogen (fraction I-2). The observation of glycosylated A alpha chains is novel. The gamma/gamma' chain heterogeneity appears to be due to an amino acid extension similar to that observed in mammalian fibrinogens. Fibrinogen from cells exposed to fetal bovine serum, a potent stimulator of fibrinogen production, was enriched in glycosylated A alpha chains, which constituted approximately one-third of the A alpha chain population. Serum did not affect the gamma/gamma' chain distribution.

Hormonal regulation of fibrinogen synthesis in cultured hepatocytes.

Most of what was originally known of the effects of hormones on fibrinogen synthesis was based, as noted above, on experiments involving surgical removal of endocrine glands. Some caution should be exercised when using such in vivo experiments to derive the hormonal requirements of fibrinogen synthesis, however, since multiple hormonal alterations often occur in these animals. The development of a variety of ex vivo systems has allowed investigators to more carefully control the hepatocellular environment. The work of several laboratories, including our own, has now made it clear that hormones and other agents directly stimulate hepatocellular synthesis of fibrinogen. From the studies summarized here, using chick embryo hepatocytes as a model, several generalizations emerge: Fibrinogen synthesis may be considered to be a "constitutive" liver function, since hepatocytes cultured without serum, hormones or other macromolecular supplements synthesize this protein at a basal rate for several days. Addition of certain hormones (e.g. T3, dexamethasone, insulin), individually and in physiological concentrations, elicits an increase in fibrinogen production, varying with each agent in onset, dose, minimum exposure required and accompanying effects on the synthesis of other plasma proteins. Glucocorticoids and thyroid hormones are similar in the selectivity of their stimulation (neither affects albumin or transferrin synthesis) but differ in that thyroid hormones need to be present for just a short "triggering" period. The stimulation of fibrinogen synthesis by insulin occurs only following prolonged exposure to concentrations 10-times higher than the very low doses to which albumin synthesis responds rapidly.