Expression of active human factor VIII from recombinant DNA clones

Wood, William I.; Capon, Daniel J.; Simonsen, Christian C.; Eaton, Dan; Gitschier, Jane; Keyt, Bruce A.; Seeburg, Peter H.; Smith, Douglas H.; Hollingshead, P G; Wion, Karen L.; Delwart, Eric; Tuddenham, Edward G. D.; Vehar, Gordon A.; Lawn, Richard M.

doi:10.1038/312330a0

Cited by 675 publications

(365 citation statements)

References 45 publications

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“…Factor VIII is synthesized as a multi-domain single chain molecule (A1-A2-B-A3-C1-C2) consisting of 2332 amino acid residues with a molecular mass of ϳ300 kDa (2,3). Factor VIII is processed to a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B-domain linked to a light chain consisting of the A3, C1, and C2 domains (2)(3)(4).…”

Section: From the Departments Of ‡Biochemistry And Biophysics And ¶Mementioning

confidence: 99%

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Nogami

Lapan

Zhou

et al. 2004

Journal of Biological Chemistry

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We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC 50 ‫؍‬ 11 M) and partially blocked cleavage at the A2-B junction (IC 50 ‫؍‬ 100 M). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of ϳ50% and an activation rate constant of ϳ30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.Factor VIII, a plasma protein deficient of defective in individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa (1). Factor VIII is synthesized as a multi-domain single chain molecule (A1-A2-B-A3-C1-C2) consisting of 2332 amino acid residues with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B-domain linked to a light chain consisting of the A3, C1, and C2 domains (2-4).Factor VIII is converted into an active form, factor VIIIa, following limited proteolysis catalyzed by either thrombin or factor Xa (5). Cleavages at Arg 372 and Arg 740 of the heavy chain produce the 50-kDa A1 and 40-kDa A2 subunits. Cleavage of the 80-kDa light chain at Arg 1689 produces a 72-kDa A3-C1-C2 subunit. Additionally, cleavage by factor Xa at Arg 1721 produces a 67-kDa A3-C1-C2 subunit. Proteolysis at Arg 372 and Arg 1689 is essential for generating factor VIIIa cofactor activity (see for review see Ref. 6). Cleavage at the former site ...

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Section: From the Departments Of ‡Biochemistry And Biophysics And ¶Mementioning

confidence: 99%

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Nogami

Lapan

Zhou

et al. 2004

Journal of Biological Chemistry

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“…Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6,71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3, 4,17,191, have been described.…”

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confidence: 99%

The effect of von Willebrand factor on activation of factor VIII by factor Xa

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12,20/21,23,27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3 -51.Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3,4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-VIII -von-Willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII.In a previous paper ...

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“…This is probably due to the functional and physiological differences of various regions of the fallopian tube. No MspI sites exist within this region of the F8 gene Toole et al, 1984;Wood et al, 1984); therefore, MspI/HpaII Southern blot methods were not informative for examining methylation patterns at this locus (data not shown).…”

Section: Cell Type-specific Rfmps In Human and Mouse Genesmentioning

confidence: 99%

“…Southern blots were also made from 4.5% non-denaturing polyacrylamide gels in TBE buffer (0.089 M Tris-borate, 0.089 M boric acid, 2 mM EDTA, pH 8.0) by electrotransfer to nylon membrane as previously described for DGG blots (Laprise et al, 1998). Blots were hybridized as described previously (Laprise et al, 1998;Reindollar et al, 2000) with several genomic and cDNA clones: plasmid p625.8, containing sequences 30 kb downstream from the human F8 gene Wood et al, 1984); mouse F8 cDNA (Elder et al, 1993); human ADRB2 cDNA (Kobilka et al, 1987); human GCCR genomic fragments (Reindollar et al, 2000); human MYB genomic and cDNA fragments (Franchini et al, 1983); human COL2AI genomic fragments (Cheah et al, 1985); human TP53 cDNA (Nogueira, 1997); human AMH cDNA (Cate et al, 1986); and mouse Th genomic fragments (Iwata et al, 1992).…”

Section: Preparation and Hybridization Of Dgg And Southern Blotsmentioning

confidence: 99%

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Laprise

Gray

2007

Gene

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Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, laborintensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.

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Expression of active human factor VIII from recombinant DNA clones

Cited by 675 publications

References 45 publications

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

The effect of von Willebrand factor on activation of factor VIII by factor Xa

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

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