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  Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practicehttp://gateway.ut.ovid.com/gw1/ovidweb.cgi1 de 8224/06/2006 01:29 p.m. Editors: Colman, Robert W.; Clowes, Alexander W.; Goldhaber, Samuel Z.; Marder,Victor J.; George, James N.Title: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 5thEdition Copyright ©2006 Lippincott Williams & Wilkins > Table of Contents > Part I - Basic Principles of Hemostasis and Thrombosis > Section A - Coagulationand its Regulation > Chapter 3 - The Blood Coagulation Factors: their Complementary Dnas, Genes, andExpression Chapter 3 The Blood Coagulation Factors: theirComplementary Dnas, Genes, and Expression Daniel L. GreenbergEarl W. Davie The complementary DNAs (cDNAs) and genes corresponding to all the proteins known toparticipate in the blood coagulation cascade have been cloned (1). Most of these coagulationproteins are synthesized in the liver and secreted into the plasma. Accordingly, the cloningof these proteins has been made possible by the isolation of liver messenger ribonucleic acid(mRNA), the preparation of cDNAs corresponding to the liver mRNA, and the insertion of these cDNAs into plasmids or phage for their amplification, identification, and sequenceanalysis. These experiments have provided a fruitful approach to the study of the structure,function, and biosynthesis of the coagulation proteins and the organization of their genesfrom both healthy individuals and patients with clotting disorders. More recently, substantialprogress has been made in the understanding of the liver-specific expression of thesegenes. The pharmaceutical production of factors VIII, IX, and VIIa have greatly enhancedtreatment options for patients with hemophilia and other clotting disorders. Additionally,animal models for gene therapy have been encouraging for both hemophilia A and B. ThecDNAs and genes for these various proteins are described in this chapter, with primaryemphasis on those of human srcin. FIBRINOGEN Fibrinogen ( M  r  340,000) participates in the final stages of the blood coagulation process(1). It is a complex glycoprotein consisting of two sets of three different polypeptide chains,including two α chains ( M  r  67,600), two β chains ( M  r  52,300), and two γ chains ( M  r  48,900)(2). Fibrinogen also contains four carbohydrate chains, including one on each of the β and γchains (3,4). The α chain is free of carbohydrates (5). The complete amino acid sequence forhuman fibrinogen has been determined by amino acid sequence analysis and cDNA andgenomic cloning (6,7,8,9,10,11,12,13,14,15,16,17,18).A single copy of the gene for each of the three polypeptide chains of fibrinogen is presenton the long arm of human chromosome 4 at q23-q32 (15). The three genes are groupedtogether in approximately 50 kb of DNA and range in size from 5.4 to 8.4 kb (see Fig. 3-1)(16). The three genes occur in the order of γ, α, and β, with approximately 14 kb of DNAseparating the genes for the γ and α chains and approximately 13 kb of DNA separating thegenes for the α and β chains. The gene for the β chain is oriented in the oppositetranscriptional direction from the genes for the γ and α chains (15).  Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practicehttp://gateway.ut.ovid.com/gw1/ovidweb.cgi2 de 8224/06/2006 01:29 p.m. P.22The genes for the three chains of human fibrinogen have been sequenced in their entirety(16,18). The gene coding for γ the chain is 8.5 kb long and contains nine introns (A throughI) and 10 exons (see Fig. 3-2) (16,17,18). The introns vary considerably in size, rangingfrom 96 to 1,638 nucleotides (see Table 3-1). The poly(A) addition site as determined bythe cDNA sequence for the chain is 203 nucleotides downstream from the stop codon of TAAand follows the typical processing or polyadenylation sequence of AATAAA by 19 nucleotides(19).The gene coding for the α chain of human fibrinogen is 5.4 kb long and contains four introns(A through D) and five exons (Fig. 3-2) (16). The introns range in size from 459 to 1,132nucleotides (Table 3-1). The fifth exon is unusually large: It codes for more than 75% of theα chain (amino acids 153 through 625). This exon also codes for eight tandem repeats of 13amino acids (residues 270 through 374) that are unique to the α chain (see Table 3-2).These repeats also typically contain the sequence of Gly-Ser-Ser, which is coded by thenucleotide sequence of GGG-AGC-TCT.The DNA sequence coding the gene for the β chain of fibrinogen is 8 kb long and containsseven introns and eight exons (Fig. 3-2) (16). The size and location of the introns in thegene are shown in Table 3-1. Three polyadenylation sites are present in the regions, whichcan lead to slightly different lengths in the mRNAs. The gene for the β chain also containstwo Alu repetition sequences, including one in intron E and one in the region of the gene.The location of the introns in the three chains of fibrinogen relative to the coiled-coilregions present in the intact molecule are illustrated in Figure 3-2.Fibrinogen is synthesized primarily in the hepatic parenchymal cells, where the individualpolypeptides are processed and assembled (20,21). Its synthesis is markedly increasedduring an acute-phase state induced by tissue damage, inflammation, or stress. Synthesis of the three chains also appears to be under coordinate control in that defibrination byinjection of Malayan pit viper venom results in a coordinate increase in liver mRNA for eachof the three chains (22). Studies in rats suggest that the expression of the three chains of fibrinogen is under coordinate regulation at the transcriptional level (23).Recent epidemiologic data has indicated that high levels of circulating fibrinogen areassociated with an increased risk of myocardial infarction and stroke (24,25). These studieshave prompted an interest in the regulation the expression fibrinogen by hepatocytes.Fibrinogen biosynthesis is unusual in that it is an acute-phase reactant (APR) although it isconstitutively expressed at a high level. Fibrinogen belongs to the class II group of APRsbecause it is induced by the inflammatory cytokine IL-6. Inflammatory cells containreceptors for fibrinogen and fibrinogen degradation products, and will produce IL-6 inresponse to these products (25,26). Glucocorticoids stimulate the synthesis of class II APRsthrough increasing the production of IL-6 by inflammatory cells. The IL-6 promoter containsglucocorticoid response elements that result in increased transcription of the hormone in thepresence of glucocorticoids. Theincreased production of IL-6 by inflammatory cells then stimulates the hepatocytes toincrease the synthesis of class II APRs. IL-6 enhances the gene expression of many of thetype II APRs through the IL-6 activated transcription factor STAT 3 (signal transducer andactivator of transcription 3) (27). In rats, activated STAT 3 binds to IL-6 response elementsand is required for the upregulation of fibrinogen genes during an acute-phase response(28). However, in humans it is unclear which nuclear transcription factor(s) regulate theIL-6 response elements in the α and β chains. In the human γ chain, IL-6 activated STAT 3bound weakly to the promoter element, and this binding decreased the functional activity of the promoter as measured by reporter gene (29). Accordingly, the mechanism of IL-6–dependent regulation of the fibrinogen gene requires further investigation.  Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practicehttp://gateway.ut.ovid.com/gw1/ovidweb.cgi3 de 8224/06/2006 01:29 p.m. FIGURE 3-1.  Abbreviated scheme illustrating the biosynthesis of human fibrinogen inliver. The arrangement of the and genes on human chromosome 4 is indicated by the solid wavy lines . The arrows  above each gene indicate the direction of transcription. Theindividual messenger RNAs (mRNAs) are processed and transported from the nucleus tothe cytoplasm, where they form the membrane-bound polysomes for the biosynthesis of each polypeptide chain. Additional processing of each polypeptide chain and assembly of the three chains into a mature molecule generate a fibrinogen that is secreted into theplasma. The intrachain and interchain disulfide bonds shown in the mature molecule arediscussed in the text.An interesting relation is developing between fibrinogen gene expression and lipidmetabolism. Oxysterols, which inhibit cholesterol synthesis by a feedback mechanism,diminish fibrinogen expression in both primary and transformed hepatic cells (30).Additionally, activation of the hepatic nuclear hormone receptor peroxisomeproliferation-activated receptor-α (PPAR-α) with fenofibrate, a lipid-lowering agent, reducesthe IL-6–mediated induction of most, if not all, of the type II APRs (31). This suppression of IL-6–regulated APR genes produced by the liver is due to downregulation of important IL-6signal transduction proteins and the ubiquitous transcription factorsCCAAT-enhancer/binding proteins (see subsequent text) (32).The 5′-flanking regions of all three of the chains making up fibrinogen have beeninvestigated. In keeping with the observation that fibrinogen is upregulated by IL-6, allthree of the transcriptional regulatory regions of these genes contain a functional IL-6response element (see Fig. 3-3) (33). There is a unique difference between the IL-6responsive element in the gene for the γ chain and those in the genes for the and α and βchains. In the latter two genes, the IL-6 responsive element is associated with an adjacentfunctional CCAAT-enhancer binding protein (C/EBP) site, which is essential fortransactivation by IL-6 (35,36,37,38). However, in the gene for the γ chain, the IL-6response element is not associated with an active C/EBP site (34).The genes for the α and β chains are apparently expressed in a liver-specific manner by ahepatocyte nuclear factor-1 (HNF-1)–dependent mechanism. In contrast, liver-specific  Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practicehttp://gateway.ut.ovid.com/gw1/ovidweb.cgi4 de 8224/06/2006 01:29 p.m. P.23expression of the gene for the chain is apparently mediated by an upstream stimulatoryfactor (USF). USF and HNF-1 are expressed in many different tissues, as well as by the liver,indicating that tissue-specific cofactors or accessory proteins are also involved. DNA bindingand functional reporter gene assays have demonstrated that both HNF-1 and USF must alsoassociate with other tissue-specific transacting proteins to achieve liver-specific expression(see subsequent text). All three fibrinogen genes contain TATA-like sequences thatpresumably facilitate the choice of transcription initiation sites by RNA polymerase II.The 5′-flanking region of the gene coding for the human chain has been isolated, sequenced,and characterized (36). The principal site of transcription initiation is located 55 bpupstream from the initiator methionine codon, or 13,399 bp downstream from thepolyadenylation site of the gene coding for the γ chain. The promoter and IL-6 responseelement lie within the region from to the transcription start site, located at Although sixsequences with homology to the consensus IL-6 response element are present, a singlesequence of CTGGGA localized from to was shown to be a functional element in IL-6induction (36,37). A C/EBP site is located at to Mutation of this C/EBP site decreases IL-6response by threefold, indicating that C/EBP is among the regulatory proteins associatedwith the IL-6 response. Recent studies suggest that the IL-6 response element of the αchain also binds to the novel transcription factor A α-core protein, which is homologous tothe mitochondrial single-stranded DNA binding protein P16 (39,40). An HNF-1 binding site,present from to in combination with other upstream elements, is essential for liver-specificexpression (36). An additional positive element (-133 to -1393 bp) and a negative element(-749 to -133 bp) have also been characterized by reporter gene assays. A series of patientswith hypofibrinogenemia were recently found to be heterozygous for the single base pairsubstitution C to T at in the region of the chain (41). Functional in vitro  experimentsanalyzing this mutation showed a corresponding reduction in hepatocyte reporter geneexpression.The 5′-flanking region containing the regulatory sequences for the liver-specific expressionof the β chain has also been partially characterized (34,35). HNF-1 binds at -91 to -79 bprelative to the transcription start site. A C/EBP site located at -132 to -124 bp, and thefunctional IL-6 response element is located at to The liver-specific expression of the β chaingene appears to be mediated in part by the presence of an accessory protein designated asdimerization cofactor of HNF-1 (DCoH) (42). The IL-6 response element is located at toadjacent to the C/EBP site, and contains the core sequence CTGGAAA. Full IL-6 responseactivity is dependent on C/EBP binding and other accessory or cofactors, including DCoH.Two common single-base substitution polymorphisms, one at -455 bp and the other at -854bp, independently increase transcription of the β chain, and this is correlated with theobserved increase in plasma fibrinogen in middle-aged men who have the polymorphism(43).

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