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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Fu, Y. Articles by Grieninger, G. Search for Related Content PubMed PubMed Citation Articles by Fu, Y. Articles by Grieninger, G. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 92 No. 9 (November 1), 1998: pp. 3302-3308 Formation of the Human Fibrinogen Subclass Fib420: Disulfide Bonds and Glycosylation in Its Unique (E Chain) Domains By Yiping Fu, Jian-Zhong Zhang, Colvin M. Redman, and Gerd Grieninger From the Lindsley F. Kimball Research Institute of the New York Blood Center, New York, NY.     ABSTRACT Abstract Introduction Methods Results Discussion References COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel E chain and those of the more abundant subclass whose chains lack E's globular C-terminus. That region, referred to as the EC domain, is closely related to the ends of  and  chains of fibrinogen (C and C). Transfection of COS cells with E, , and  cDNAs alone results in secretion of the symmetrical molecule (E)2, also known as Fib420. Cotransfection with cDNA for the shorter chain yielded secretion of both ()2 and (E)2 but no mixed molecules of the structure E()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 E chain. No evidence from Cys  Ser replacements was found for interchain disulfide bridges involving the four cysteines of the EC domain. However, for fibrinogen secretion, the E, , and  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. © 1998 by The American Society of Hematology.     INTRODUCTION Abstract Introduction Methods Results Discussion References FIBRINOGEN, THE protein that forms the matrix of a blood clot, is a complex molecule composed of paired sets of three subunits (, , and ), each encoded by a separate gene. A few years ago we identified a subclass of native fibrinogen molecules based on  subunit differences.1 Molecules of this subclass are characterized by their greater mass (~420 kD) and lower plasma levels relative to the more abundant form (~340 kD), and we have coined the terms Fib420 and Fib340 to distinguish between them. Whereas the novel Fib420 molecules' three subunits all end in globular domains that resemble each other, those of the more commonly known subclass Fib340 have truncated  subunits that lack such a domain. Although Fib420 molecules represent only one of every 100 fibrinogen molecules in the blood of normal, healthy adults,2 counterparts have been found throughout the vertebrate kingdom.3,4 This implies an important function for the novel subclass that is distinct from that of the more abundant Fib340, but that is as yet undefined. Discovery of Fib420 in our laboratory evolved from the seminal finding that the complete sequence of the human gene encoding the  subunit contains an additional exon (exon VI) undetected by previous investigators.5,6 Alternative splicing to include exon VI gives rise to the isoform of the  chain (E) with a globular carboxy-terminal extension. It is the 236 residues encoded by exon VI (technically the EC domain, but often referred to as the VI-domain) that are as similar to the sequences of the C-terminal domains of the fibrinogen  and  chains (C and C) as the latter two are to each other (40% identity); the truncated C-terminus of the common  chain (C) has a very different character. Early investigations with transfected COS cells seemed to suggest the obligatory presence of common  chains for the assembly and secretion of recombinant E-containing fibrinogen.6 The subsequent unexpected finding that two E chains are incorporated per molecule of Fib4201 prompted the current investigation using batches of COS cells that more closely resemble hepatic cells in their overall efficiency of fibrinogen assembly. In this study, we show that such cells incorporate two E subunits per molecule of fibrinogen, whether the common  isoform is present or not. Further use of the system to explore the role of specific residues in Fib420 assembly and secretion was undertaken based on an extensive series of studies showing an absolute requirement for particular sets of interchain and intrachain disulfide bonds in assembly and secretion of the more abundant Fib340,7-10 which has 29 disulfide bonds in all and no free cysteine residues.11 In this study, we examine the roles of the four additional cysteines and two potential glycosylation sites contributed by each of the EC domains unique to Fib420.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References The original , , and  cDNAs of human fibrinogen were generous gifts from Dominic Chung (University of Washington, Seattle). Construction of vectors containing full-length cDNAs for the E, , , and  fibrinogen subunits has been described.6,12 If not otherwise indicated, pED4-Neo vectors (Genetics Institute, Cambridge, MA) were used.13 Cys  Ser mutations in the , , and  chains were described previously.7,9,10 The N-terminal and "C"-domain mutations of E were derivatized from the  mutants as done previously.6 The mutations Cys  Ser, Cys  Ala, and Asn  Gln in the E chain's VI-domain were generated by polymerase chain reaction (PCR)-directed mutagenesis according to standard procedures. As before, all constructs were verified by sequencing. Transient transfections of COS-1 cells were performed by the calcium phosphate method used earlier.12 To achieve E synthesis comparable to that of the other subunits, an excess of E-cDNA was generally added, as indicated. Qualitative evaluation of fibrinogen production was made by labeling the cells for 2 hours with [35S]methionine in the presence of 15 µg/mL heparin and 30 KIU aprotinin, then immunoprecipitating fibrinogen from cell lysates or culture medium with rabbit antibodies either to whole human fibrinogen (DAKO, Carpinteria, CA) or to the VI-domain of E.1,6,12 The intact fibrinogen species were separated by sodium dodecyl sulfate (SDS)/4%PAGE under nonreducing conditions; separation of the component subunits was achieved by SDS/7.5% PAGE under reducing conditions. Signals were detected by autoradiography. The results reported here were confirmed with several different passages of COS cells.     RESULTS Abstract Introduction Methods Results Discussion References COS cells transfected simultaneously with either the E// or the E/// sets of cDNAs synthesized all three or four fibrinogen subunits, respectively, as shown by SDS-PAGE analysis of cell lysates immunoprecipitated with antifibrinogen (Fig 1A). Predominantly E chains were immunoprecipitated by anti-VI, indicating that most of the intracellular E subunits are not assembled, ie, they exist as free polypeptides. Similar intracellular pools of free E chains have been found in the human hepatocarcinoma cell line HepG2 (Grieninger et al, in preparation). View larger version (83K): [in this window] [in a new window]   Fig 1. Synthesis and secretion of E- and -fibrinogen by transfected COS cells. Cells were transfected with stoichiometric proportions of pBC-vectors containing fibrinogen subunit cDNAs6,12 in combination as indicated below the lanes. When all four subunit cDNAs were cotransfected, E cDNA was included at a twofold excess. Fibrinogen was immunoprecipitated from cell lysates (A) and culture medium (B) with either antifibrinogen (f) or anti-VI (VI) as indicated above the lanes, and subunits were separated under reducing conditions. Migration of the  chain as a doublet in cell extracts (see also Figs 5A and 6A) may reflect the presence of the nonglycosylated precursor. Transfected COS cells secrete mostly fully assembled fibrinogen hexamers.12 To identify E-containing fibrinogen from among the other fibrinogen species, immunoprecipitation of culture medium with two antibodies was used. Anti-VI immunoprecipitated only those consisting of E, , and , whereas antifibrinogen collected every fibrinogen species, yielding a profile on reduced SDS-PAGE that included, when present, all four fibrinogen subunits E, , , and  (Fig 1B). In immunoprecipitates of the medium of cells transfected with E// cDNAs alone, the three fibrinogen subunits were detected at roughly similar molar ratios, irrespective of the antibody used (anti-VI or antifibrinogen). Because anti-VI is highly specific for the E chain (see Fig 1A, lanes 1 and 3; Fig 2, lane 3), immunoprecipitating  or  only when bound to E, it follows that the bulk of the E secreted by these cells is packaged in fully assembled fibrinogen molecules. Cells transfected additionally with the cDNA for common  (the E///-transfectants) secrete both -fibrinogen and E-fibrinogen as indicated by the presence of E, , and  chains in the anti-VI immunoprecipitates and all four chains in the antifibrinogen immunoprecipitates. Although E///-transfectants expressed significant amounts of the common  chain, none was found in their anti-VI-immunoprecipitated culture medium, suggesting that no mixed molecules of the structure E()2 were exported. View larger version (45K): [in this window] [in a new window]   Fig 2. Secretion of Fib420 by transfected COS cells in the presence and absence of the common chain. Cells were transfected with pED4-Neo-vectors containing fibrinogen subunit cDNAs in combination as indicated below each lane; E cDNA was included at a fivefold excess. Fibrinogen was immunoprecipitated from culture medium with either antifibrinogen (f) or anti-VI (VI) as indicated and run under nonreducing conditions. Traces of incompletely assembled or single subunits could be seen in the lower part of the gel only after overexposure of the film, confirming that most of the chains detected in the medium upon reduction of the immunoprecipitates in Fig 1B were indeed components of hexameric fibrinogen molecules. The issue of mixed versus symmetrical molecules was explored further by separating the secreted fibrinogen species under nonreducing conditions. As seen in Fig 2, E//-transfectants secreted a high molecular weight species (Fib420) that is significantly larger than that of the //-transfectants (Fib340). Most striking, however, is that only one E-containing species is secreted when all four chains are cotransfected (E///-transfectants); this product comigrates with Fib420. No mixed molecules, containing E as well as  and therefore migrating at a position intermediate between Fib420 and Fib340, were detected. These findings with heterologous host cells support our earlier hypothesis that formation of symmetrical fibrinogen molecules, (E)2, is energetically favored over that of the mixed molecules E()2,1 driven perhaps by alternative disulfide bond configurations either between the two E chains' VI-domains to form a fourth nodule and/or connecting the VI-domains to N-terminal cysteines that could belong, in principle, to any subunit chain, even E itself. The latter would serve to tether the VI-domains to the central nodule, consistent with the tetranodular images observed in many published electron micrographs of fibrinogen.14-16 To test this alternative disulfide bond hypothesis, we looked for qualitative changes in the fibrinogen species secreted by COS cells that had been transfected with subunit cDNAs coding for either wild-type chains or chains in which specific cysteines were converted to serines (or alanines) by site-directed mutagenesis. All four cysteines in the VI-domain of human E were substituted, individually and in combination; changes in the N-terminus and in the "C" region of the E chain were also introduced (illustrated in Fig 3). In addition, N-terminal substitution mutants at sites in the  and  chains (65S and 8S/9S) were evaluated for their potential contribution to Fib420 formation. View larger version (5K): [in this window] [in a new window]   View larger version (5K): [in this window] [in a new window]   Fig 3. Schematic representation of targeted amino acid positions in the E subunit for site-directed mutagenesis. Residues are numbered as done previously.6 Arrows or simple vertical lines indicate positions of those cysteines converted, either singly or in combination, to serine or alanine by site-directed mutagenesis. (A) The three regions of the E chain: the N-terminus (NT) containing a large -helical segment, the so-called "C" region, and the C-terminal globular VI-domain (EC domain); (B) the VI-domain, potential glycosylation sites in the wild-type sequence at Asn667 and Asn812 are marked, respectively, with closed and open diamonds. The glycosylation site at Asn791 introduced by the Cys793  Ser change is marked with a striped diamond. Putative loops connecting cysteines E613/644 as well as E780/793 are drawn by analogy with the intrachain loops formed by homologous cysteines in the C and C domains. To establish whether any of the above cysteine mutations per se affected Fib420 secretion, they were initially examined in E//-transfectants, ie, in the absence of the major  chain (Fig 4). All mutant fibrinogen subunits were expressed, as shown by analysis of cell lysates (Fig 4A). Despite some variation in the amount of fibrinogen secreted by the different mutants, it is clear that E613S and E780S/793S substitutions abolished secretion of Fib420 (Fig 4B). Although the double mutation E613S/644S, which knocks out the putative first loop in the VI-domain, has no discernable effect on Fib420 secretion, the single E613S mutant and its mutated disulfide bond partner (E644S; Table 1) are inhibitory. In either of these single mutations, a reactive thiol group introduced by an unpaired cysteine appears to interfere nonspecifically with the assembly and/or secretion process. Of note, the E780S/793S mutation creates a neo-N-glycosylation site (NNS) at Asn791. That carbohydrate is attached to this site can be seen from the upward mobility shift of E780S/793S in Fig 4A, lane 5. To test whether the extra carbohydrate chain is responsible for blocking secretion, we changed Cys793 to alanine instead of serine in mutant E780S/793A. This particular mutation also blocked secretion (Table 1), indicating that the missing (second) intrachain loop and not the carbohydrate attachment is responsible for lack of Fib420 export in E780S/793S mutants. View larger version (101K): [in this window] [in a new window]   View larger version (77K): [in this window] [in a new window]   Fig 4. Cys  Ser changes in the E, , and  subunits: Negative and neutral effects on the secretion of Fib420. Cys  Ser changes were made in the fibrinogen subunits at the positions indicated above the lanes. COS cells were transfected with pED4-vectors containing E, , and  (either wild-type or mutant) cDNAs as shown; E constructs were included at a fivefold excess. Fibrinogen was immunoprecipitated, from cell lysates (A) and culture medium (B), with antifibrinogen and run under reducing conditions. The size of protein markers (lane 1) is displayed in kD in the left margin.   View this table: [in this window] [in a new window]   Table 1. Effect of E Subunit Mutations on Secretion of Fib420 in COS Cells Transfected With E, , and  cDNAs In addition to the VI-domain cysteines, we evaluated substitutions of cysteines in other parts of the Fib420 molecule that might be available for alternative disulfide bonding with the VI-domain, reasoning that those cysteines were not found to be critical for export of Fib340. The first pair of these was further upstream in the E chain, Cys442 and Cys472, which correspond to positions in the C region of the common  chain known to form an intrachain loop10; mutant E442S/472S had no effect on Fib420 secretion in E//-transfectants (Fig 4). Other candidate cysteines that we evaluated, located in the N-termini of the constituent fibrinogen chains (E Cys28 and Cys36,  Cys65, and  Cys8 and Cys9), are involved in forming the symmetrical disulfide bridges that hold the trimers together; only one out of the four symmetrical bonds is absolutely required for Fib340 hexamer assembly and secretion.7,8,10 None of these mutations, E28S/36S, 65S, or 8S/9S, when cotransfected with complementary wild-type chains, interfered with proper secretion of Fib420 molecules in the E//-transfectants (Fig 4). Having established that Fib420 production was possible despite substitution of particular residues, the same mutations were examined in E///-transfectants. This test of the alternative disulfide bond configuration hypothesis could be expected to yield secretion of mixed molecules (containing both E and ) whenever cysteines involved in bonds favoring homodimer formation were replaced. For each set of mutations, it was first shown that simultaneous transfection with all four cDNAs leads to expression of all four subunits (Fig 5A) and that for the subunits comprising Fib420, ie, the E, , and  chains, the levels of expression were comparable to those of the E//-transfectants (Fig 4A). The medium of the transfected cells was then immunoprecipitated with anti-VI for detection of secreted E-containing fibrinogen (Fig 5B). Although secretion with mutated subunits was less efficient, in no case were mixed molecules detectable as species migrating faster than that of the E//-transfectant. It follows that selective incorporation of two E subunits in the presence of the abundant  chain is not dependent on disulfide bridges involving cysteines in the VI-domain of E or on available cysteine partners further upstream in the E chain itself or in the other two subunits present in the central core of the fibrinogen molecule. These results with recombinant molecules are consistent with trypsin digests of -fibrinogen,4 the lamprey equivalent of Fib420, which appears to be a similarly symmetrical molecule but which has E-like chains derived from a separate gene17 instead of being alternatively spliced  gene products as in higher vertebrates.3,6 View larger version (95K): [in this window] [in a new window]   View larger version (95K): [in this window] [in a new window]   Fig 5. Effect of E, , and  subunit mutations on Fib420 secretion in presence of the wild-type subunit: No secretion of mixed molecules. Cys  Ser changes were made in the fibrinogen subunits at the positions indicated above the lanes. COS cells were transfected with either wild-type or mutant E, , and  cDNAs together with wild-type cDNA in the combinations shown; E constructs were included at a fivefold excess. Fibrinogen was immunoprecipitated from cell lysates (A) with antifibrinogen and run under reducing conditions; from the culture medium (B), fibrinogen was immunoprecipitated with anti-VI and run under nonreducing conditions. As described previously,1 due to the differential proteolytic susceptibility of and E subunits in cell lysates, a proteolytic fragment derived only from the chain, not from E, appears as a band migrating below the  chain doublet in panel A (compare also Fig 1A). In contrast to the predominant human  chain, which has no carbohydrate, human E is N-glycosylated.1 We knocked out each of the two potential glycosylation sites, E667 and E812, by changing asparagines to glutamines. These mutated E chains showed a mobility shift comparable in magnitude to that fortuitously generated in mutant E780S/793S with its second active glycosylation site. Examination of lysates of E//-transfectants in Fig 6 revealed that the mutant E812Q comigrates with wild-type E, whereas E667Q migrates faster, suggesting that normally carbohydrate is attached to Asn667 and not to Asn812. E780S/793S, as noted above, is larger than wild-type E because of an additional carbohydrate moiety at Asn791. Thus, the stepwise increase in molecular weight of E667Q, E812Q, and E780S/793S reflects the attachment of zero, one, and two carbohydrate chains, respectively. View larger version (88K): [in this window] [in a new window]   Fig 6. Mutation of potential N-glycosylation sites in the E VI-domain: Determination of carbohydrate attachment site. The VI-domain's two potential glycosylation sites, at Asn667 and Asn812, were changed to Gln; a new site at Asn791 had been introduced by the Cys793  Ser change. COS cells were transfected with either wild-type or these mutant E cDNAs together with the  and  subunit cDNAs. Fibrinogen was immunoprecipitated from cell lysates and culture medium with antifibrinogen and run under reducing conditions. Upon overexposure of the film, Fib420 subunits are clearly detectable in the culture medium of the Q667 mutant. In mutant E667Q, secretion of Fib420 is reduced to less than 20% of wild-type levels (Fig 6; upon overexposure of the film the subunits are clearly detectable), suggesting possible involvement of carbohydrate attachment at this site in secretion. However, from an earlier finding that Fib420 secretion was not appreciably inhibited when tunicamycin blocked N-glycosylation in HepG2 cells,1 it is apparent that reduced secretion of E667Q mutant Fib420 results not from the absence of sugar but rather from a conformational change introduced by replacing Asn with Gln.     DISCUSSION Abstract Introduction Methods Results Discussion References This study shows, in transfected COS cells, that formation of secreted E-containing fibrinogen hexamers is possible with the E, , and  subunit building blocks alone. Importantly, the predominant secreted species of E-containing fibrinogen is the homodimeric (E)2, even in the presence of the common  chain (ie, in E///-transfectants); no mixed molecules of the composition E()2 are observed (Figs 1 and 2). In other words, the heterologous COS cell system mimics the hepatic Fib420 assembly process as evaluated in the cell line HepG2, where E-homodimeric fibrinogen molecules are formed preferentially in the presence of an abundant supply of  chains, the E: chain ratio being roughly 1:20.1 This investigation of human E-containing fibrinogen contradicts prior notions regarding the obligatory presence of common  chains for assembly and secretion.6 Inability of cells used in previous COS experiments to match the performance of hepatoma cells (HepG2), which assemble E and  chains into fibrinogen with equal efficiency, may account for the anomalous earlier findings. Exploiting the current COS cells' fidelity with regard to Fib420 synthesis and secretion, studies were extended to analyze the molecule's glycosylation sites as well as the requirement for specific cysteines in connection with its secretion. Of the two potential glycosylation sites in the Fib420's unique EC domain, the tripeptide at Asn667 is conserved in all vertebrates, including lamprey, whereas the one at Asn812 is conserved only in mammals.3,17 By substituting Gln for Asn we have now shown that it is the highly conserved Asn667 site that has carbohydrate attached in the E chain of human Fib420 (Fig 6). Thus, Fib420 has a total of six carbohydrate moieties among its six component chains. All of the sites have the consensus tripeptide sequence NXT, with X being a positively charged residue: arginine in and , lysine in . Whereas the  chain bears carbohydrate in a more upstream region of the chain, both E and  chains use sites in the C-terminal region. Although the EC and C attachment sites do not align with each other, they are both located between the homologous first and second disulfide loops. With its four extra cysteines per EC domain, Fib420 has the potential to form 33 disulfide bonds. The positions of the four cysteines in EC are invariant among E homologs throughout vertebrate evolution and align precisely with cysteines in the C and C domains,3,6,17 each of which has two intrachain disulfide bridges. The results from substituting serine or alanine residues for the VI-domain cysteines (Figs 4 and 5, Table 1) are consistent with the existence of similar bonds between the cysteine pairs at E613/E644 and at E780/E793. The finding that the individual E613S and E644S mutations block secretion but not the double mutation E613S/644S strongly suggests that there is an intrachain loop formed by Cys613 and Cys644. Fib420 secretion is prevented by double mutation of the cysteines presumably forming the domain's second disulfide loop (E780/793), indicating a vital role for them, and not those of the potential first loop, in Fib420 export from the cell. A number of protein sequences have been identified that bear C-terminal domains homologous to EC, C, and C. In this family of fibrinogen-related domains (FREDs),18 which contains at least 14 unique members at latest count, the cysteines of the first and second disulfide loops are preserved. With only one exception,19 the 12 residues of the second loop are highly conserved; those of the first loop, varying in number from 24 to 30, constitute the sequence with the lowest conservation in the entire family of FREDs. In the context of our findings regarding Fib420 secretion, the second loop's higher degree of conservation may reflect a more critical role in secretion of the parent molecule. It should be mentioned that the C-terminal region of the  chain actually has three intrachain disulfide bonds. In the large loop formed by cyteines 201/286, which is essential for secretion,10 only  Cys286 is part of the C domain proper.6 Although the loop is conserved among all vertebrate fibrinogen  chains, there is no cysteine corresponding to  Cys286 in any other member of the FRED family, implying a highly specific function. Structurally, the E chain differs significantly from the  and  chains by virtue of the large "C" region (identical to the truncated C-terminus of the common  chain) that tethers the subunit's globular C-terminus (EC) to its -helical N-terminus. Overall, the "C" tether, encoded by exon V of the  gene, is poorly conserved among the fibrinogens of higher vertebrates.20 However, the disulfide loop it contains, homologous to the human cysteines at positions 442/472, is highly conserved, although it does not play a role in either Fib340 or Fib420 secretion10 (Figs 4 and 5). An apparent conformational change in Fib420 occurs in the E442S/472S mutant as a result of eliminating a disulfide-bridged loop, and it is reflected, even in this high-molecular-weight range, in distinctly slower migration on SDS-PAGE relative to all the other species that were generated (Fig 5). This is consistent with the slower migration noted previously of the 442S/472S mutant relative to wild-type  chains under nonreducing SDS-PAGE conditions10 and gives credence to the hypothesis that the loops in the "C"-region of E make the Fib420 molecule more compact. It remains to be seen whether the 442S/472S Fib420 mutant displays neoepitopes and/or is more susceptible to proteolytic attack. Based on sequence comparison,6 the globular fold of the EC domain is expected to be very similar to the folds of the C and C domains, which were recently determined at atomic resolution and which were found to be virtually superimposable.21-23 However, the dependence of secretion on the integrity of the two homologous intradomain disulfide bridges is distinctly different for each subunit. In the C region, only the first loop is required; in the EC region, it is the second loop; and in the C region both loops play a critical part. These observations support different structural roles for the subunits in the process of fibrinogen assembly, a notion put forward more than a decade ago.24,25 The mechanism that favors formation of the symmetrical Fib420 molecule over that of a mixed E/-containing molecule in the presence of excess  chains is not known at present. We previously speculated that alternative disulfide bridges, either between the VI-domains and/or the VI-domain and the center of the molecule, might provide the impetus for homodimer formation. Without directly determining disulfide bridges between particular cysteines, the experiments using E///-transfectants (Fig 5) do not support that hypothesis; specific mutations in E's VI-domain and the N-termini of all the E, , and  chains, designed to remove cysteines potentially available for alternative disulfide bridges, failed to reduce production of (E)2 in favor of mixed E()2 molecules. Thus, a different mechanism based on noncovalent interactions must be advanced to explain symmetrical incorporation of E chains into Fib420 against all stoichiometric odds. Given the high negative charge borne by each EC domain,6 it is conceivable that chaperone proteins associated with nascent fibrinogen26,27 may play a critical role in balancing the spatial charge distribution.     NOTE ADDED IN PROOF As this report was being processed for publication, the assignments of bound cysteine pairs and carbohydrate attachment site in the EC domain were confirmed by x-ray crystallographic analysis of a recombinant version of the domain.28     FOOTNOTES    Submitted April 28, 1998; accepted July 6, 1998.    Supported in part by the National Institutes of Health through grants to G.G. (HL 51050) and C.M.R. (HL 37457) and by the American Heart Association through a grant to G.G.    Address reprint requests to Gerd Grieninger, PhD, New York Blood Center, 310 E 67th St, New York, NY 10021; e-mail: ggrien{at}server.nybc.org.    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.     ACKNOWLEDGMENT We thank our colleague K.M. Hertzberg for many valuable contributions to the manuscript.     REFERENCES Abstract Introduction Methods Results Discussion References 1. Fu Y, Grieninger G: Fib420: A normal human variant of fibrinogen with two extended  chains. Proc Natl Acad Sci USA 91:2625, 1994[Abstract/Free Full Text] 2. Grieninger G, Lu X, Cao Y, Fu Y, Kudryk BJ, Galanakis DK, Hertzberg KM: Fib420, the novel fibrinogen subclass: Newborn levels are higher than adult. Blood 90:2609, 1997[Abstract/Free Full Text] 3. Fu Y, Cao Y, Hertzberg KM, Grieninger G: Fibrinogen  genes: Conservation of bipartite transcripts and carboxy-terminal-extended  subunits in vertebrates. Genomics 30:71, 1995[Medline] [Order article via Infotrieve] 4. Shipwash E, Pan Y, Doolittle RF: The minor form chain from lamprey fibrinogen is rapidly crosslinked during clotting. Proc Natl Acad Sci USA 92:968, 1995[Abstract/Free Full Text] 5. Weissbach L, Grieninger G: Bipartite mRNA for chicken -fibrinogen potentially encodes an amino acid sequence homologous to - and -fibrinogens. Proc Natl Acad Sci USA 87:5198, 1990[Abstract/Free Full Text] 6. Fu Y, Weissbach L, Plant PW, Oddoux C, Cao Y, Liang TJ, Roy SN, Redman CM, Grieninger G: Carboxy-terminal-extended variant of the human fibrinogen  subunit: A novel exon conferring marked homology to  and  subunits. Biochemistry 31:11968, 1992[Medline] [Order article via Infotrieve] 7. Zhang JZ, Kudryk B, Redman CM: Symmetrical disulfide bonds are not necessary for assembly and secretion of human fibrinogen. J Biol Chem 268:11278, 1993[Abstract/Free Full Text] 8. Huang S, Cao Z, Davie EW: The role of amino-terminal disulfide bonds in the structure and assembly of human fibrinogen. Biochem Biophys Res Commun 190:488, 1993[Medline] [Order article via Infotrieve] 9. Zhang JZ, Redman CM: Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen. J Biol Chem 269:652, 1994[Abstract/Free Full Text] 10. Zhang JZ, Redman C: Fibrinogen assembly and secretion. Role of intrachain disulfide loops. J Biol Chem 271:30083, 1996[Abstract/Free Full Text] 11. Henschen A: Number and reactivity of disulfide bonds in fibrinogen and fibrin. Arch Kemi 22:355, 1964 12. Roy SN, Procyk R, Kudryk BJ, Redman CM: Assembly and secretion of recombinant human fibrinogen. J Biol Chem 266:4758, 1991[Abstract/Free Full Text] 13. Kaufman RJ, Davies MV, Wasley LC, Michnick D: Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res 19:4485, 1991[Abstract/Free Full Text] 14. Erickson HP, Fowler WE: Electron microscopy of fibrinogen, its plasmic fragments and small polymers. Ann NY Acad Sci 408:146, 1983[Medline] [Order article via Infotrieve] 15. Gollwitzer R, Bode W, Schramm HJ, Typke D, Guckenberger R: Laser diffraction of oriented fibrinogen molecules. Ann NY Acad Sci 408:214, 1983[Medline] [Order article via Infotrieve] 16. Veklich YI, Gorkun OV, Medved LV, Nieuwenhuizen W, Weisel JW: Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated alpha C fragments on polymerization. J Biol Chem 268:13577, 1993[Abstract/Free Full Text] 17. Pan Y, Doolittle RF: cDNA sequence of a second fibrinogen  chain in lamprey: An archetypal version alignable with full-length  and  chains. Proc Natl Acad Sci USA 89:2066, 1992[Abstract/Free Full Text] 18. Doolittle RF: A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives. Protein Sci 1:1563, 1992[Medline] [Order article via Infotrieve] 19. Xu X, Doolittle RF: Presence of a vertebrate fibrinogen-like sequence in an echinoderm. Proc Natl Acad Sci USA 87:2097, 1990[Abstract/Free Full Text] 20. Murakawa M, Okamura T, Kamura T, Shibuya T, Harada M, Niho Y: Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen A-chains. Characterization of the partial nucleotide and deduced amino acid sequences in five mammalian species; Rhesus monkey, pig, dog, mouse and Syrian hamster. Thromb Haemost 69:351, 1993[Medline] [Order article via Infotrieve] 21. Yee VC, Pratt KP, Cote HC, Trong IL, Chung DW, Davie EW, Stenkamp RE, Teller DC: Crystal structure of a 30 kDa C-terminal fragment from the  chain of human fibrinogen. Structure 5:125, 1997[Medline] [Order article via Infotrieve] 22. Pratt KP, Cote HCF, Chung DW, Stenkamp RE, Davie EW: The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro. Proc Natl Acad Sci USA 94:7176, 1997[Abstract/Free Full Text] 23. Spraggon G, Everse SJ, Doolittle RF: Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Nature 389:455, 1997[Medline] [Order article via Infotrieve] 24. Yu S, Sher B, Kudryk B, Redman CM: Intracellular assembly of human fibrinogen. J Biol Chem 258:13407, 1983[Abstract/Free Full Text] 25. Yu S, Sher B, Kudryk B, Redman CM: Fibrinogen precursors. Order of assembly of fibrinogen chains. J Biol Chem 259:10574, 1984[Abstract/Free Full Text] 26. Roy S, Yu S, Banerjee D, Overton O, Mukhopadhyay G, Oddoux C, Grieninger G, Redman C: Assembly and secretion of fibrinogen. Degradation of individual chains. J Biol Chem 267:23151, 1992[Abstract/Free Full Text] 27. Roy S, Sun A, Redman C: In vitro assembly of the component chains of fibrinogen requires endoplasmic reticulum factors. J Biol Chem 271:24544, 1996[Abstract/Free Full Text] 28. Spraggon G, Applegate D, Everse SJ, Zhang J-Z, Veerapandian L, Redman C, Doolittle RF, Grieninger G: Crystal structure of a recombinant EC domain from human fibrinogen-420. Proc Natl Acad Sci USA 95:9099, 1998[Abstract/Free Full Text] © 1998 by the American Society of Hematology.   0006-4971/98/92-0039$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: D. Applegate, L. S. Steben, K. M. Hertzberg, and G. Grieninger The alpha EC domain of human fibrinogen-420 is a stable and early plasmin cleavage product Blood, April 1, 2000; 95(7): 2297 - 2303. [Abstract] [Full Text] [PDF] D. Applegate, L. Haraga, K. M. Hertzberg, L. S. Steben, J.-Z. Zhang, C. M. Redman, and G. Grieninger The alpha EC Domains of Human Fibrinogen420 Contain Calcium Binding Sites But Lack Polymerization Pockets Blood, November 15, 1998; 92(10): 3669 - 3674. 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