scholarly journals A Molecular Model for the Triplicated A Domains of Human Factor VIII Based on the Crystal Structure of Human Ceruloplasmin

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2413-2421 ◽  
Author(s):  
S. Pemberton ◽  
P. Lindley ◽  
V. Zaitsev ◽  
G. Card ◽  
E.G.D. Tuddenham ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hemophilia A ◽  
Molecular Model ◽  
Significant Advance ◽  
Molecular Defect ◽  
Missense Mutations ◽  
Sequence Identity ◽  
Mutation Database ◽  
Human Factor Viii ◽  
A Domains

Abstract The hemophilia A mutation database lists more than 160 missense mutations: each represents a molecular defect in the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from mild to severe. Without a three-dimensional FVIII structure it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has been solved, and the A domains of FVIII share approximately 34% sequence identity with hCp. This advance has enabled the building of a molecular model of the A domains of FVIII based on the sequence identity between the two proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as the binding-sites for factor IXa and activated protein C; it has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the prediction of hypothetical links to dysfunction in all but a few cases. A computer-generated molecular model such as that reported here cannot substitute for a crystal structure. However, until such a structure for FVIII becomes available, the model represents a significant advance in modeling FVIII; it should prove a useful tool for exploiting the increasing amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation of the structure-function relationships via mutagenesis and expression in vitro.

The Immunological Properties of Factor VIII

1966 ◽  
Vol 16 (03/04) ◽  
pp. 559-573 ◽  
Author(s):  
L Uszyński
Keyword(s):  
Factor Viii ◽  
Inhibitory Activity ◽  
Hemophilia A ◽  
Severe Form ◽  
Molecular Defect ◽  
Bovine Plasma ◽  
Human Factor Viii ◽  
Antigenic Properties ◽  
Coagulation Tests ◽  
Normal Human

SummaryRabbits immunized against human AHG fibrinogen-free preparations, were shown to produce anti-AHG antibodies. The inhibitory activity of these antibodies was tested by thromboplastin generation test, thrombelastography, and the specific anti-AHG antibodies neutralization test. The latter test permitted quantitative determination of antigenic form of factor VIII. The inhibitory activity of anti-FI-O-Ta serum resulted exclusively from the anti-AHG antibodies which in coagulation tests behaved like circulating anticoagulants directed against factor VIII.The anti-AHG antibodies were neutralizable by normal human serum or plasma even contained only trace of AHG activity after storage. There was no antigenic form of factor VIII in the severely affected patients with hemophilia A, von Willebrand’s disease nor in the normal plasma adsorbed on bentonite. The presented results suggest a molecular defect of factor VIII in patients with hemophilia A. The severe form of this disease depends, probably, on a major impairment of AHG biosynthesis, leading to changes in the antigenic properties of the molecule. The AHG from rabbit, porcine and bovine plasma respectively did not neutralize the anti-AHG antibodies formed in rabbits immunized against human factor VIII preparations.

scholarly journals Moderation of hemophilia A phenotype by the factor V R506Q mutation

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1183-1187 ◽  
Author(s):  
WC Nichols ◽  
K Amano ◽  
PM Cacheris ◽  
MS Figueiredo ◽  
K Michaelides ◽  
...  
Keyword(s):  
Protein C ◽  
Hemophilia A ◽  
Clinical Phenotype ◽  
Point Mutations ◽  
Molecular Genetic Analysis ◽  
Von Willebrand Disease ◽  
Clinical Severity ◽  
Molecular Defect ◽  
Factor V ◽  
Missense Mutations

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.

scholarly journals Molecular Characterization of Hemophilia a in Southeast Bulgaria

2008 ◽  
Vol 11 (1) ◽  
pp. 55-60 ◽  
Author(s):  
E Sukarova Stefanovska ◽  
P Tchakarova ◽  
G Petkov ◽  
G Efremov
Keyword(s):  
Molecular Characterization ◽  
Hemophilia A ◽  
Single Strand Conformation Polymorphism ◽  
Molecular Defect ◽  
Missense Mutations ◽  
Nucleotide Substitutions ◽  
Intron 1 ◽  
Gradient Gel Electrophoresis ◽  
Exon 11

Molecular Characterization of Hemophilia a in Southeast BulgariaThe results of molecular characterization of Hemophilia A in 50 patients from Southeast Bulgaria are presented. Southern blot analysis for the detection of inversions in intron 22, and polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) or de-naturing gradient gel electrophoresis (DGGE) for screening of the coding sequences of the Factor VIII (FVIII) gene were used. A molecular defect was found in 35 (70%), the most frequent being an inversion in intron 22, found in 19 (38%) patients; an intron 1 inversion was not detected. In one severely affected patient, an Alu insert was found, which disrupted exon 14 at codon 1224. Nucleotide substitutions were found in 15 (30%) patients, the most frequent being an Arg531→Cys missense mutation in exon 11. The same nonsense mutation (codon -5,CGA>TGA) was found in two patients with a severe phenotype. Seven missense mutations (Asn90→Thr, Arg 372→His, Glu456→Val, Tyr473→His, Arg1689→ Cys, Arg2159→Cys and Arg2163→His) were detected in isolated families. Two of these (Asn90→Thr and Glu456→ Val) are being reported for the first time.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

Reassessment of theTP53 mutation database in human disease by data mining with a library ofTP53 missense mutations

2004 ◽  
Vol 25 (1) ◽  
pp. 6-17 ◽  
Author(s):  
Thierry Soussi ◽  
Shunsuke Kato ◽  
Pierre P. Levy ◽  
Chikashi Ishioka
Keyword(s):  
Data Mining ◽  
Human Disease ◽  
Missense Mutations ◽  
Mutation Database

scholarly journals Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3468-3478 ◽  
Author(s):  
Adoración Venceslá ◽  
María Ángeles Corral-Rodríguez ◽  
Manel Baena ◽  
Mónica Cornet ◽  
Montserrat Domènech ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Low Density Lipoprotein ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Missense Mutations ◽  
Factor X ◽  
Novel Mutations ◽  
Large Deletions ◽  
Function Relationship ◽  
The Impact

Abstract Hemophilia A (HA) is an X-linked bleeding disorder caused by a wide variety of mutations in the factor 8 (F8) gene, leading to absent or deficient factor VIII (FVIII). We analyzed the F8 gene of 267 unrelated Spanish patients with HA. After excluding patients with the common intron-1 and intron-22 inversions and large deletions, we detected 137 individuals with small mutations, 31 of which had not been reported previously. Eleven of these were nonsense, frameshift, and splicing mutations, whereas 20 were missense changes. We assessed the impact of the 20 substitutions based on currently available information about FV and FVIII structure and function relationship, including previously reported results of replacements at these and topologically equivalent positions. Although most changes are likely to cause gross structural perturbations and concomitant cofactor instability, p.Ala375Ser is predicted to affect cofactor activation. Finally, 3 further mutations (p.Pro64Arg, p.Gly494Val, and p.Asp2267Gly) appear to affect cofactor interactions with its carrier protein, von Willebrand factor, with the scavenger receptor low-density lipoprotein receptor–related protein (LRP), and/or with the substrate of the FVIIIapi•FIXa (Xase) complex, factor X. Characterization of these novel mutations is important for adequate genetic counseling in HA families, but also contributes to a better understanding of FVIII structure-function relationship.

Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A

2001 ◽  
Vol 85 (01) ◽  
pp. 125-133 ◽  
Author(s):  
Huiyun Wu ◽  
Mark Reding ◽  
Jiahua Qian ◽  
David Okita ◽  
Ernie Parker ◽  
...  
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Th2 Cells ◽  
Tween 20 ◽  
Sorbitan Monolaurate ◽  
Human Factor Viii ◽  
Ifn Γ ◽  
Th1 And Th2

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

Identification of a Novel Missense Mutation in Exon 4 of the Human Factor VIII Gene Associated with Sever Hemophilia a Patient

2007 ◽  
Vol 10 (23) ◽  
pp. 4299-4302 ◽  
Author(s):  
Habib Onsori ◽  
Mohammad Ali Hossein . ◽  
Sheideh Montaser-Kou . ◽  
Mohammad Asgharzadeh . ◽  
Abbas Ali Hosseinpou .
Keyword(s):  
Factor Viii ◽  
Missense Mutation ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Exon 4

scholarly journals Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4671-4677 ◽  
Author(s):  
S Connelly ◽  
JM Gardner ◽  
RM Lyons ◽  
A McClelland ◽  
M Kaleko
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
High Dose ◽  
Specific Enzyme ◽  
Adult Mice ◽  
Human Factor Viii

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

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