Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.

Church, William R.; Jernigan, Robert L.; Toole, John J.; Hewick, Rodney M.; Knopf, John L.; Knutson, Gaylord J.; Nesheim, Michael E.; Mann, Kenneth G.; Fass, David N.

doi:10.1073/pnas.81.22.6934

Cited by 162 publications

(91 citation statements)

References 14 publications

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“…Factor VIII and factor V share homologous structures and functions (30). Although factor V shows no acidic region separating the A1 and A2 domains, a functionally important acidic-rich residue region was recently localized to Nterminal region of the A2 domain of factor Va (residues 323-331) (31).…”

Section: Discussionmentioning

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: Discussionmentioning

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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“…The FVIII protein consists of 2,351 amino acids, which are subdivided into a leader peptide, three A domains, a B domain, and two C domains. The A domains, which all share approximately 30% sequence identity among themselves, are homologous to the triplicate A domains present in factor V (FV) [2] and in ceruloplasmin (hCp) [3]. Murine hephaestin (He) [4] and haphaestin (Ha) [5] share a similar percentage of identity with human FVIII.…”

Section: Introductionmentioning

confidence: 99%

Small FVIII gene rearrangements in 18 hemophilia A patients: Five novel mutations

et al. 2005

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Hemophilia A (HA) is a disorder caused by mutations of the FVIII gene, which is located on the tip of the long arm of the X chromosome. In a cohort of 18 unrelated Italian patients affected with HA of varying severity, we performed mutational screening of the gene by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing of abnormal peaks. We identified five novel mutations and 9 previously reported DNA alterations. Two of the 9 previously reported alterations were each common to 3 unrelated patients. Six different mutations were characterized as missense alterations, while 8 were non-missense mutations. Among the new gene alterations, one created a stop codon, one consisted of an out-of frame deletion, and one was a splice-site mutation. The last two were missense alterations. In an attempt to better understand the causative effect of the mutations and the clinical variability of the patients, we investigated the consequences of each missense mutation and visualized the effect of the amino acid change on structural FVIII models. Am.

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“…fVIII contains a domain structure designated A1-A2-B-ap-A3-C1-C2 that is defined based on internal sequence homology (1,2). The fVIII A domains share homology with the copper-binding protein ceruloplasmin (3,4), which has an A1-A2-A3 domain structure in which the three A domains are arranged along a pseudo-3-fold axis of symmetry (5). Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junction into A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits.…”

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confidence: 99%

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Doering

Healey

Parker

et al. 2004

Journal of Biological Chemistry

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Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency. Factor VIII (fVIII)1 is a plasma protein that functions in proteolytically activated form as a cofactor within the intrinsic pathway of blood coagulation to increase the rate of proteolytic activation of factor X by activated factor IX. fVIII contains a domain structure designated A1-A2-B-ap-A3-C1-C2 that is defined based on internal sequence homology (1, 2). The fVIII A domains share homology with the copper-binding protein ceruloplasmin (3, 4), which has an A1-A2-A3 domain structure in which the three A domains are arranged along a pseudo-3-fold axis of symmetry (5). Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junction into A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits. fVIII circulates in the plasma as an inactive heavy chain/light chain heterodimeric procofactor that is non-covalently bound to von Willebrand factor. Proteolytic activation of fVIII by thrombin results from cleavages at Arg-372 between the A1 and A2 domains, Arg-740 between the A2 and B domains, and Arg-1689 between the ap and A3 domains. During this process, the covalent linkage between the A1 and A2 domains is lost, and the B domain and 41-residue ap are released, producing a heterotrimeric,

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Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.

Cited by 162 publications

References 14 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

Small FVIII gene rearrangements in 18 hemophilia A patients: Five novel mutations

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

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