Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 2

The deduced primary amino acid sequence of human factor VIII determined from the cloned cDNA demonstrated that factor VIII is encoded by a precursor protein of 2,351 amino acid residues from which a 19-amino-acid signal peptide is cleaved. Plasma factor VIII is a heterodimer processed from a larger precursor polypeptide. It consists of a COOH-terminal-derived light chain of 80,000 MW in a metal ion-dependent association with an NH2-terminal-derived heavy chain fragment of 90,000–200,000 MW. MLID85061549 MLID85061550  16,18 In the plasma this complex is stabilized by association through hydrophilic and hydrophobic interactions with a 50-fold excess of vWF. The amino acid sequence revealed an organization of three structural domains that occur in the order A1:A2:B:A3:C1:C2, as shown in Figure 105-1.  MLID85061549 MLID85061550 16,18 The A1 (amino acid residues 1–329) and A2 (380–711) domains of factor VIII are located in the heavy chain and the A3 (1,649–2,019) domain is located in the light chain. The A domains have 30% homology to each other, to the triplicated A domains of ceruloplasmin and factor V. MLID85061549 MLID87260886 MLID86259737  16,19,20 The residues implicated for copper binding in ceruloplasmin are conserved in the first and third A domains of factor VIII, suggesting that the A domains of factor VIII may be involved in metal ion binding. However, these residues are not conserved in the A domains of factor V, although copper ions were detected in purified preparations of factor V MLID85030396  21 and factor VIII. 22 The C1 (residues 2,020–2,172) and C2 (residues 2,173–2,332) domains are located in the terminus of the factor VIII light chain and exhibit homology to milk fat globule protein and to A, C, and D chains of discoidin 1, which are all capable of binding glycoconjugates and negatively charged phospholipids. MLID82170475 MLID91046008  23,24 The B domain is encoded by a single large exon of 3,100 nucleotides, has no known homology to other proteins, and contains 18 of the 25 potential asparagine (N)-linked glycosylation sites within factor VIII. The cloning of the factor V cDNA and gene revealed a high degree of amino acid conservation between the A and C domains with no detectable homology within the B domains, MLID87260886  19 although both B domains are encoded by large single exons. The domain organization and homologies between factors V and VIII suggest that these genes evolved from a primordial ferroxidase gene by triplication of the A domain, insertion of the B domain, and addition of the two C domains. After duplication of the primordial cofactor gene, the factor V and factor VIII genes likely evolved by extensive divergence of amino acid residues within the B domain, while amino acid residues within the A and C domains were conserved.

In addition to the A, B, and C domains, there are three acidic amino acid-rich regions in the factor VIII protein molecule at the junction of the A1/A2 (residues 331–372), A2/B (residues 700–740), and B/A3 (residues 1,649–1,689) domains that are juxtaposed to sites of thrombin cleavage (Fig. 105-1). All these acidic regions also contain the post-translationally modified amino acid tyrosine sulfate at residues 364, 718, 719, 721, 1,664, and 1,680. MLID92207952  25 The murine factor VIII cDNA is highly homologous to the A and C domains of human factor VIII, whereas the acidic regions and B domain show partial homology. MLID93300511  26 However, all thrombin cleavage sites and sulfated tyrosine residues are conserved. The murine factor VIII B domain also contains 19 potential N-linked glycosylation sites, although in different positions than in the human sequence, suggesting that glycosylation in the B domain is important for factor VIII expression or function, or for both.

Biosynthesis and Metabolism of Factor VIII

The natural cell type(s) that produces factor VIII has not been definitively identified. However, evidence obtained from liver transplantation in factor VIII-deficient dogs MLID69087279 MLID71186448  27,28 and several hemophiliac patients MLID85163654 MLID87214539  29,30 strongly implies that the liver is a major site of factor VIII synthesis. In addition, immunochemical localization by light microscopic MLID84178963  31 or electron microscopic MLID86040432  32 examination detected the factor VIII antigen in hepatocytes. However, RNA hybridization analysis has detected factor VIII mRNA in hepatocytes and in many other cells and tissues. MLID86040431  33 To date there are no known established cell lines that express factor VIII. Thus, it has not been possible to study the biosynthesis of factor VIII in its natural host cell. However, the expression of factor VIII in mammalian cells transfected with the factor VIII gene allowed analysis of the biosynthesis and processing of this glycoprotein. MLID88198183  34