The ultrastructure of fibrinogen-420 and the fibrin-420 clot.

Fibrinogen-420 is a minor subclass of human fibrinogen that is so named because of its higher molecular weight compared to fibrinogen-340, the predominant form of circulating fibrinogen. Each of the two Aalpha chains of fibrinogen-340 is replaced in fibrinogen-420 by an Aalpha isoform termed alphaE. Such chains contain a globular C-terminal extension, alphaEC, that is homologous with the C-terminal regions of Bbeta and gamma chains in the fibrin D domain. The alphaEC domain lacks a functional fibrin polymerization pocket like those found in the D domain, but it does contain a binding site for beta2 integrins. Electron microscopy of fibrinogen-340 molecules showed the major core fibrinogen domains, D-E-D, plus globular portions of the C-terminal alphaC domains. Fibrinogen-420 molecules had two additional globular domains that were attributable to alphaEC. Turbidity measurements of thrombin-cleaved fibrinogen-420 revealed a reduced rate of fibrin polymerization and a lower maximum turbidity. Thromboelastographic measurements also showed a reduced rate of fibrin-420 polymerization (amplitude development) compared with fibrin-340. Nevertheless, the final amplitude (MA) and the calculated elastic modulus (G) for fibrin-420 were greater than those for fibrin-340. These results suggested a greater degree of fibrin-420 branching and thinner matrix fibers, and such structures were found in SEM images. In addition, fibrin-420 fibers were irregular and often showed nodular structures protruding from the fiber surface. These nodularities represented alphaEC domains, and possibly alphaC domains as well. TEM images of negatively shadowed fibrin-420 networks showed irregular fiber borders, but the fibers possessed the same 22.5-nm periodicity that characterizes all fibrin fibers. From this result, we conclude that fibrin-420 fiber assembly occurs through the same D-E interactions that drive the assembly of all fibrin fibrils, and therefore that the staggered overlapping molecular packing arrangement is the same in both types of fibrin. The alphaEC domains are arrayed on fiber surfaces, and in this location, they would very likely slow lateral fibril association, causing thinner, more branched fibers to form. However, their location on the fiber surface would facilitate cellular interactions through the integrin receptor binding site.

The alternatively spliced alpha(E)C domain of human fibrinogen-420 is a novel ligand for leukocyte integrins alpha(M)beta(2) and alpha(X)beta(2).

The interaction of human plasma fibrinogen with leukocyte integrins alpha(M)beta(2) (CD11b/CD18, Mac-1) and alpha(X)beta(2) (CD11c/CD18, p150,95) is an important component of the inflammatory response. Previously, it was demonstrated that binding of fibrinogen to these integrins is mediated by gammaC, the globular C-terminal domain of the gamma chain. In this study, evidence was found of another fibrinogen domain that can serve as a ligand for the 2 leukocyte integrins: alpha(E)C, a homologous domain that extends the alpha chains in a recently discovered subclass of fibrinogen known as fibrinogen-420. Recombinant alpha(E)C supported strong adhesion and migration of cells expressing alpha(M)beta(2) and alpha(X)beta(2), including nonactivated and activated U937 and THP-1 monocytoid cells, and neutrophils. Cells transfected with complementary DNA for these integrins also bound alpha(E)C. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against alpha(M), alpha(X), and beta(2) subunits. Also, neutrophil inhibitory factor, a specific inhibitor of alpha(M)beta(2) and alpha(X)beta(2) function, efficiently blocked cell adhesion to alpha(E)C. In alpha(M)beta(2) and alpha(X)beta(2), the I domain is the binding site for alpha(E)C, since alpha(E)C bound to recombinant alpha(M) I and alpha(X)I domains in a dose-dependent and saturable manner. Synthetic peptides that duplicated sequences gamma190 to 202 and gamma377 to 395, previously considered putative binding sites in gammaC, effectively inhibited alpha(M)beta(2)- and alpha(X)beta(2)-mediated adhesion to alpha(E)C, suggesting that recognition of alpha(E)C by the I domain involves structural features in common with those of gammaC. These findings identify alpha(E)C as a second domain in fibrinogen-420 that binds alpha(M)beta(2) and alpha(X)beta(2) and can mediate leukocyte adhesion and migration.

Contribution of the alpha EC domain to the structure and function of fibrinogen-420.

In addition to the conventional fibrinogen with its alpha, beta, and gamma subunit chains, there is a subclass of fibrinogen molecules, accounting for one percent of the total in human adults, in which both alpha chains have been replaced by extended alpha chains (alpha E) that sport a globular C-terminal domain (alpha EC) comparable to beta C and gamma C. Using nomenclature based on molecular weight, the subclass of alpha E-containing molecules has been named fibrinogen-420 to differentiate it from the better known fibrinogen, now referred to as fibrinogen-340. Review of the events leading to the discovery of fibrinogen-420 in the early 1990s and its subsequent characterization, culminating in the crystal structure of its unique alpha EC domains, highlights special aspects of its evolutionary history, outstanding features of its structure, and the perplexities of its biology. Various working hypotheses that have driven prior investigation are evaluated and practical insights are offered to spur further research into the role of fibrinogen-420.

The alpha(E)C domain of human fibrinogen-420 is a stable and early plasmin cleavage product.

Human fibrinogen-420, (alpha(E)betagamma)(2), was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (alphabetagamma)(2), were found to be similar, suggesting little impact of the unique alpha(E)C domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the alpha(E)C domain. One was a stable fragment (alpha(E)CX) comigrating with a 34-kd yeast recombinant alpha(E)C domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

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.

Crystal structure of a recombinant alphaEC domain from human fibrinogen-420.

The crystal structure of a recombinant alphaEC domain from human fibrinogen-420 has been determined at a resolution of 2.1 A. The protein, which corresponds to the carboxyl domain of the alphaE chain, was expressed in and purified from Pichia pastoris cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the beta- and gamma-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.

Fib420, the novel fibrinogen subclass: newborn levels are higher than adult.

Fib420 is a recently identified subclass of normal human fibrinogen in which two extended alpha chain isoforms (alphaE) replace the common alpha chains, yielding a molecule (ca. 420 kD) which is larger than the more abundant 340-kD form. Evidence for preservation of this subclass throughout vertebrate evolution suggests it performs some as yet unidentified vital function. A survey was undertaken to establish the range of plasma Fib420 levels in normal, healthy adults and in placental cord (fetal) blood. For measuring Fib420, a quantitative Western blot assay was developed using monoclonal antibody against the exon-VI encoded C-terminus of the molecule's unique alphaE chain. This alphaE chain signal was normalized to that of the beta chain, common to both fibrinogen forms. Analysis of plasma samples from the adult and newborn cohorts (n = 25 each; total fibrinogen ca. 2.6 mg/mL in both) revealed a statistically significant difference, with a mean level of 100 +/- 28 microg/mL in the neonate compared to 34 +/- 7 microg/mL in the adult. On average, 1 out of every 100 fibrinogen molecules in adult plasma belongs to the Fib420 subclass. Unlike in the newborn, adult Fib420 levels remained the same over a wide range of total plasma fibrinogen. The striking difference observed between these two cohorts suggests a changing developmental expression of the Fib420 subclass and a homeostatic control operating in later stages of life.

Fibrinogen alpha genes: conservation of bipartite transcripts and carboxy-terminal-extended alpha subunits in vertebrates.

All three well-studied subunits of the clotting protein fibrinogen (alpha, beta, gamma) share N-terminal structural homologies, but until recently only the beta and gamma chains were recognized as having similar globular C-termini. With the discovery of an extra exon in the human fibrinogen alpha gene (exon VI), a minor form of the alpha subunit (alpha E) with an extended beta- and gamma-like C-terminus has been identified (Fu et al., Biochemistry 31, 11968, 1992). In the present study, the polymerase chain reaction has been used to identify sequences that encode counterparts to alpha E in chicken, rabbit, rat, and baboon. The basic six-exon structure of the fibrinogen alpha genes is shown to be conserved among mammals and birds, as are the intron positions. Bipartite transcripts--still bearing an intron prior to the last exon--are found among the products of the various vertebrate fibrinogen alpha genes. The last exon represents the largest conserved segment of the gene and, in each species examined, encodes exactly 236 amino acids. The C-termini of these alpha E chains align without a single gap and are between 76 and 99% identical. Since the exon VI-encoded domain of alpha E is as well conserved as the corresponding regions of the beta and gamma chains, it follows that it is equally important and that alpha E-fibrinogen plays a vital, if as-yet unrecognized physiological role.

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.

Bipartite mRNA for chicken alpha-fibrinogen potentially encodes an amino acid sequence homologous to beta- and gamma-fibrinogens.

Overlapping cDNAs derived from the chicken alpha-fibrinogen mRNA have been sequenced, beginning from within the coding region for the signal peptide of this subunit and terminating within the poly(A) extension. The predicted size of chicken alpha-fibrinogen is 54,187 daltons, which is the smallest of any alpha chain reported; the oligopeptide repeats that characterize the central regions of the other alpha subunits were conspicuously absent. A further unexpected finding was the presence on the mRNA of a separate, long open reading frame (752 nucleotides), beginning 312 nucleotides downstream from the alpha-fibrinogen coding sequence and containing intron-like features near its 5' end. The protein sequence predicted from this second open reading frame lacks an initiating methionine but is homologous to the C-terminal regions of all known beta- and gamma-fibrinogens as well as the C termini of two nonfibrinogen proteins: cytotactin (tenascin), an extracellular matrix protein, and pT49, a putative protein specific to cytotoxic T cells. The intron-like features of the second open reading frame immediately precede the region of common homology, and the beginnings of the corresponding homologous segments in the beta- and gamma-fibrinogen sequences are marked by aligned intron positions. Based on these findings, it is proposed that fibrinogen gene evolution included a fusion of two distinct ancestral genes.

Glycogen metabolism in cultured chick hepatocytes: a morphological study.

Ultrastructural and autoradiographic observations of cultured chick hepatocytes under the following conditions are described: Induction of glycogen synthesis with glucose alone and glucose plus insulin, and glucagon-induced glycogen breakdown. Profiles of hepatocytes cultured in medium containing 10 mM glucose showed typical cellular organelles and occasionally a few glycogen granules. After incubation of hepatocytes with 3H-glucose, silver grains were found over these sparse glycogen granules, indicating a low level of glycogen synthesis by a few cells. After addition of 75 mM glucose for 1 hr about 3% of the profiles of cells showed glycogen, and by 24 hr half of the hepatocytes had glycogen. Addition of insulin plus glucose induced glycogen synthesis in 82% of the cells after 6 hr, and by 24 hr almost every cellular profile showed glycogen particles. Morphologically, glycogen accumulation was similar whether the cells were stimulated by high glucose or by glucose plus insulin: glycogen granules appeared in restricted regions of the cytoplasm, which were rich in smooth endoplasmic reticulum (SER), and peroxisomes were found close to the newly deposited glycogen particles. At maximum glycogen accumulation the association of SER and peroxisomes with glycogen was less obvious. Glycogenolysis induced by incubation of glycogen-rich hepatocytes with glucagon resulted in proliferation of SER in the glycogen regions of the cells. These observations are compatible with the concept of regions in the hepatocyte cytoplasm specialized for glycogen metabolism. Possible roles for SER and peroxisomes found near glycogen particles and other organelles in hepatic glycogen metabolism are discussed.

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.

Hemopexin is a developmentally regulated, acute-phase plasma protein in the chicken.

Identity has been established between chicken hemopexin and alpha 1-globulin "M," a plasma known for the hormone responsiveness of its synthesis in monolayer cultures of embryonic chicken hepatocytes (Grieninger, G., Plant, P. W., Liang, T. J., Kalb, R. G., Amrani, D., Mosesson, M. W., Hertzberg, K. M., and Pindyk, J. (1983) Ann. N. Y. Acad. Sci. 408, 469-489). Identification was based on immunological cross-reactivity, electrophoretic behavior on sodium dodecyl sulfate-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes. Electroimmunoassays were used to investigate plasma protein levels, particularly those of hemopexin, in the acute-phase response and embryonic development. Acute-phase plasma protein production, elicited by injection of chickens with turpentine, bore many similarities to the pattern of hepatocellular plasma protein synthesis produced in response to the addition of specific hormones in culture. The response of the stressed chickens included elevated levels of hemopexin and fibrinogen (5- and 2-fold, respectively) accompanied by a 50% drop in albumin. Hemopexin levels of developing chick embryos were measured for several days before and after hatching. Onset of hemopexin production occurred around the time of hatching, and was followed by a steep increase (more than 1000-fold over 4 days). Similarly, it was not until the 12th h of culture that hepatocytes isolated from both early and late stage chicken embryos began to produce hemopexin, although, from their initiation in culture, they secreted a number of other plasma proteins in quantity. After 12 h, hepatocellular output of hemopexin rapidly accelerated. This precocious induction ex vivo required no hormonal or macromolecular medium supplements. These observations indicate that the embryonic chicken hepatocyte culture system will provide a useful model for studying the regulation of hemopexin biosynthesis in hepatic development and the acute-phase response.