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.

Overlapping cDNAs derived from the chicken a-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 a-fibrinogen is 54,187 daltons, which is the smallest of any a chain reported; the oligopeptide repeats that characterize the central regions of the other a 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 a-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 (3-and y-fibrinogens as well as the C termini of two nonflibrinogen proteins: cytotactin (tenascin), an extraceilular 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 .8-and y-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.Fibrinogen, an acute-phase plasma protein, acts in the final step of blood coagulation by forming a fibrin clot after its N-terminal fibrinopeptides are removed by thrombin cleavage (1). It is a multichain molecule composed of two sets of a, /3, and y subunits in all vertebrate species studied, from human to the most primitive existing vertebrate, lamprey, representing lineages that diverged 400-500 million years ago (2, 3). In spite of their functional distinctions, the three fibrinogen subunits share many structural features, suggesting a common evolutionary history. In particular, within the N-terminal region of each polypeptide, there is a fixed-length a-helical segment, with distinctive Cys-Xaa-Xaa-Xaa-Cys motifs at each end. These join with parallel regions on the other subunit polypeptides to form a disulfide-ring interlocked supercoil (4).The genetic information for the a, ,B, and y subunits resides on three separate genes that are normally transcribed in a stoichiometric and primarily liver-specific manner (5, 6). The human genes are clustered on 50 kilobases (kb) of chromosome 4 (7) and contain two common intron positions in their N-terminal regions (8)(9)(10). These facts reinforce the hypothesis that the three subunit genes arose from the same ancestral gene. Based on the closer similarity of the 83-and

The crystal structure of a recombinant ␣ E C domain from human fibrinogen-420 has been determined at a resolution of 2.1 Å. The protein, which corresponds to the carboxyl domain of the ␣ E 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 ␤-and ␥-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.

In fibrinogen, aE chains form a subpopulation of asubunits that are distinguished by a carboxyl extension homologous to the C termini of the other two constituent chains: 13 and y. The molecular mass of aE is >50% greater than that of the common a subunit, due in part to an extra 236 amino acids. These residues are encoded by exon VI, a recently discovered extension of the fibrinogen a 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 aE migration on SDS/PAGE at "110 kDa rather than at its calculated mass of 92,843 Da. An antibody specific for the exon VI-encoded domain of aE (anti-VI) and capable of recognizing ag-containing fibrinogen in both native and denatured form was generated using a recombinant protein as immunogen. Its use in Western blot analysis offractions ofnormal human blood (plasma and preparations of fibrinogen) revealed a single, sharp, aE-containing band migating behind the position of the broad, predominant fibrinogen band, (aPY)2. Designation of the upper band as Fib42o, an =420-kDa homodimer of the formula (aEPY)2, is based on the overwhelming proportion of aE subunits (>80% of the total a chains) found in anti-VIimmunoprecipitable material from hepatoma cell medium. Several lines of evidence suggest that the aE subunit, alone or incorporated into fibrinogen, is more stable than the common a chain, a feature of potential clinical importance.The major clotting protein, fibrinogen, is composed of paired sets of three subunits (a, (3, and 'y) that are encoded on separate genes (1, 2). The ( and y subunits display strong homology between their C-terminal domains, which is not shared by the a subunit in its most abundant form. However, recent discovery of an extra exon (exon VI) in the fibrinogen a gene has led to identification of an extended a subunit (aE), a naturally occurring species with a strikingly (-and ylike globular C terminus (3). The first evidence for the exon, which encodes the 236 amino acids ofthe carboxyl extension, was an a-chain transcript in normal liver that is "bipartite"-i.e., its a coding region is followed by an untranslated region and a long, second open reading frame (4). Additional splicing of the bipartite transcript-removal of the untranslated region (intron E, separating exons V and VI)-leads to expression of the extra sequence as the C-terminal domain of aE. In contrast, the predominant a chain, which lacks this homology to the E and y chains, derives its C terminus from translation of 14 codons into intron E.Using human hepatoma (HepG2) cells as well as transfected COS cells, it has been shown that aE is assembled into fibrinogen molecules (3). Based on studies using a specific antibody to the exon VI-encoded carboxyl extension, we now report the presence of the aE chain in a fibrinogen variant of human blood. We show that most of the variant molecules have aE in place of both a chains to produce a significa...