A Soluble Recombinant Factor VIII Fragment Containing the A2 Domain Binds to Some Human Anti-Factor VIII Antibodies that Are not Detected by Immunoblotting

1992 ◽  
Vol 67 (06) ◽  
pp. 665-671 ◽  
Author(s):  
Dorothea Scandella ◽  
Lisa Timmons ◽  
Mora Mattingly ◽  
Norma Trabold ◽  
Leon W Hoyer
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Human Factor ◽  
Binding Capacity ◽  
Amino Acid Residues ◽  
Protein Fragment ◽  
Immunoprecipitation Assay ◽  
Human Factor Viii ◽  
A2 Domain

SummaryHuman factor VIII (f VIII) inhibitors are pathologic antibodies that inactivate fVIII. A cDNA clone was modified to encode f VIII amino acid residues 373-740 for expression in a baculovirus vector in insect cells. The encoded protein fragment H2 was produced as a soluble, secreted protein, and it was used to test inhibitor plasmas for the presence of antibodies that were not detected by immunoblotting. Seven of 13 inhibitors that bound, only to the fVIII light chain by immunoblotting also bound to fragment H2 in an immunoprecipitation assay. Thus multi-chain inhibitor reactivity of inhibitors is more frequent than previously reported. One of these inhibitors was shown to share the epitope for other inhibitors that bind to H2 within amino acid residues 373-541 in immunoblotting assays. The sensitive immunoprecipitation assay described allows determination of relative H2 binding capacity of the total IgG and epitope localization of inhibitors that cannot be similarly characterized by immunoblotting.

Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1618-1626 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
S de Graaf ◽  
CA Fulcher
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Factor Viii Inhibitor ◽  
Type I ◽  
Protein Fragments ◽  
Human Factor Viii ◽  
Circulating Antibodies ◽  
A1 Domain ◽  
A2 Domain

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

scholarly journals The cDNA and derived amino acid sequence of porcine factor VIII

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4209-4214 ◽  
Author(s):  
JF Healey ◽  
IM Lubin ◽  
P Lollar
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Hemophilia A ◽  
Sequence Determination ◽  
C2 Domains ◽  
Protein Alignments ◽  
A2 Domain

The cDNA corresponding to 137 bp of the 5′ untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337–372 and 1649–1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

1996 ◽  
Vol 75 (01) ◽  
pp. 036-044 ◽  
Author(s):  
Thomas L Ortel ◽  
Karen D Moore ◽  
Mirella Ezban ◽  
William H Kane
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Human Factor ◽  
Secreted Protein ◽  
Factor V ◽  
Minimal Effect ◽  
Repetitive Domain ◽  
Human Factor Viii ◽  
A1 Domain ◽  
A2 Domain

SummaryFactor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

1987 ◽  
Author(s):  
Richard J Jenny ◽  
Debra D Pittman ◽  
John J Toole ◽  
Ronald W Kriz ◽  
Randal J Kaufman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Human Factor ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Factor V ◽  
Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 540-545 ◽  
Author(s):  
Edward N. van den Brink ◽  
Ellen A. M. Turenhout ◽  
Christine M. C. Bank ◽  
Karin Fijnvandraat ◽  
Marjolein Peters ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Factor Viii ◽  
Gene Segment ◽  
Amino Acid Residues ◽  
Germline Gene ◽  
Single Chain ◽  
Factor Viii Activity ◽  
A2 Domain ◽  
Acidic Region

One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The VHdomain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg484–Ile508 , a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The VH domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp712–Ala736 in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp712–Ala736for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor.

scholarly journals Residues 484-508 Contain a Major Determinant of the Inhibitory Epitope in the A2 Domain of Human Factor VIII

1995 ◽  
Vol 270 (24) ◽  
pp. 14505-14509 ◽  
Author(s):  
John F. Healey ◽  
Ira M. Lubin ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Leon W. Hoyer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Major Determinant ◽  
Human Factor Viii ◽  
A2 Domain

Functional mapping of the A2 domain from human factor VIII

2012 ◽  
Vol 107 (02) ◽  
pp. 315-327 ◽  
Author(s):  
Didier Saboulard ◽  
Jean-Luc Pellequer ◽  
Jean-Luc Plantier ◽  
Claude Négrier ◽  
Marc Delcourt
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Human Factor ◽  
Coagulation Factor ◽  
Factor X ◽  
Structural Element ◽  
Human Factor Viii ◽  
Coagulant Activity ◽  
Factor X Activation ◽  
A2 Domain

SummaryCoagulation factor VIII (FVIII) is a multidomain glycoprotein in which the FVIII A2 domain is a key structural element. We aimed at identifying residues within FVIII A2 domain that are crucial for the maintenance of the cofactor function. A high number (n=206) of mutants were generated by substituting original residues with alanine. The mutants were expressed in COS-1 cells and their antigen levels and procoagulant activities were measured. The residues were classified in three categories: those with a non-detrimental alteration of their activities (activity >50 % of control FVIII; n=98), those with a moderate alteration (15 %<activity<50%; n=45) and those that were severely affected (activity<15%; n=63). The mutants sensitive to mutation were retrieved in the HAMSTeRS database with a higher percentage than those that were not affected (58.8% vs. 9.2%). The results revealed the existence of clusters of residues that are sensitive (Arg418-Phe436, Thr459-Ile475, Ser535-Gly549, Asn618-Ala635) or not (Leu398-Arg418, Pro485-Asp500, Gly506-Gly520, Pro596-Asp605) to mutations. The stretches of residues sensitive to mutations were buried within the molecule suggesting that these amino acids participate in the maintenance of the A2 domain structure. In contrast, residues resistant to mutations formed external loops without well- defined structures suggesting that these loops were not crucial for the process of factor X activation. This study provided a detailed map of the FVIII A2 domain between residues 371 and 649, identifying residues crucial for maintaining FVIII function and residues that can be mutated without jeopardising the coagulant activity.

scholarly journals The diversity of the immune response to the A2 domain of human factor VIII

Blood ◽  
2013 ◽  
Vol 121 (14) ◽  
pp. 2785-2795 ◽  
Author(s):  
Rebecca C. Markovitz ◽  
John F. Healey ◽  
Ernest T. Parker ◽  
Shannon L. Meeks ◽  
Pete Lollar
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Entire Surface ◽  
Human Factor Viii ◽  
Key Points ◽  
A2 Domain

Key Points The Abs to the human fVIII A2 domain in a murine hemophilia A model inhibit fVIIIa and activation of fVIII Epitopes targeted by hemophilia A mouse Abs cover nearly the entire surface of the human fVIII A2 domain

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