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.

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.

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.

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.

Thyroid hormone stimulation of plasma protein synthesis in cultured hepatocytes.

The direct effect of thyroid hormones on hepatocellular plasma protein synthesis has been studied in primary monolayer cultures derived from chick embryo liver. The chemically defined medium used for plating and maintaining the cultures contained no other hormones, protein, or serum supplement. Addition of physiological concentrations (10 nM) of triiodothyronine or thyroxine produced 3-fold or greater increases in the rates of synthesis of fibrinogen and three other major secreted proteins. By comparison albumin, transferrin, and total protein synthesis were not substantially increased. The enhanced synthesis of selected plasma proteins could be detected 6 h after initial addition of triiodothyronine. Exposure of the cells to the hormone for only 30 min was nearly as effective as continuous exposure in eliciting the ultimate response. Triiodothyronine exerted its half-maximal effect at a concentration of 1 nM. Diminished potency was associated with less iodination of the hormone; a marked reduction was noted with di-iodinated thyronine and no stimulatory activity at all with either mono- or non-iodinated thyronine.

Cloning of an EcoRI-generated fragment of the leucine operon of Salmonella typhimurium.

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.

Application of electroimmunoassay to the study of plasma protein synthesis in cultured hepatocytes.

Electroimmunoassay has been applied to the study of plasma protein synthesis and secretion in liver cell cultures. The assay is performed on unconcentrated samples of culture medium containing the secreted plasma proteins and yields results within 2 hours. The characteristics of plasma protein production by the cultured hepatocytes coupled with the sensitivity of this assay permit the study of plasma protein in synthesis and its regulation by hormones and other agents without the routine use of radioisotopes.

Fibrinogen synthesis in serum-free hepatocyte cultures: stimulation by glucocorticoids.

Fibrinogen synthesis was investigated in cultures of chicken embryo hepatocytes initiated and maintained in chemically defined, serum-free medium. 11-Hydroxy glucocorticoids caused a 3-fold stimulation of fibrinogen synthesis. Half-maximal stimulation was achieved with 1 nM corticosterone or hydrocortisone, as compared with only 0.1 nM dexamethasone. Increased fibrinogen production in the presence of these glucocorticoids was characterized by a 4-hr delay in onset, a sensitivity to actinomycin D, and a requirement for the continuous presence of the steroid. Crossed immunoelectrophoresis permitted analysis of the simultaneous effects of glucocorticoids on the synthesis of more than 20 plasma proteins secreted in culture. The absence of an effect on the synthesis of most of these proteins was in sharp contrast to the 3-fold increase in fibrinogen production. Sera from a variety of animals also stimulated an increase in fibrinogen synthesis that was similar in degree but less specific than that due to glucocorticoids and that partially masked the response of the cells to the steroid hormones. The presence of an anticoagulant in the medium was found to be necessary for detection of the fibrinogen secreted in culture. Although insulin was routinely included in the chemically defined medium, the cells synthesized fibrinogen and responded to glucocorticoids in the absence of hormonal supplementation of the medium. These findings are consistent with the thesis that variations in glucocorticoid levels contribute to the regulation of fibrinogen production in the intact animal.