scholarly journals Structure of Blood Coagulation Factor VIII in Complex With an Anti-C2 Domain Non-Classical, Pathogenic Antibody Inhibitor

2021 ◽  
Vol 12 ◽  
Author(s):  
Estelle K. Ronayne ◽  
Shaun C. Peters ◽  
Joseph S. Gish ◽  
Celena Wilson ◽  
H. Trent Spencer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Domain Architecture ◽  
Coagulation Factor ◽  
Factor Ix ◽  
C2 Domain ◽  
C2 Domains ◽  
Domain Interactions ◽  
Blood Coagulation Factor Viii ◽  
Multiple Domain

Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.

Structural Basis for Hemophilia A Caused by Mutations in the C Domains of Blood Coagulation Factor VIII

2000 ◽  
Vol 83 (01) ◽  
pp. 78-85 ◽  
Author(s):  
Andrew Gale ◽  
Jean-Luc Pellequer ◽  
Elizabeth Getzoff ◽  
John Griffin
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Three Dimensional ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Structural Basis ◽  
Head Orientation ◽  
Missense Mutations ◽  
C2 Domains ◽  
Blood Coagulation Factor Viii

SummaryThree dimensional homology models for the C1 and C2 domains of factor VIII (FVIII) were generated. Each C domain formed a β-sandwich, and C1 was covalently connected to C2 in a head-to-head orientation. Of the >250 missense mutations that cause FVIII deficiency and hemophilia A, 34 are in the C domains. We used the FVIII C1-C2 model to infer the structural basis for the pathologic effects of these mutations. The mutated residues were divided into four categories: 15 conserved buried residues that affect normal packing of the hydrophobic side chains, 2 non-conserved buried residues that affect structure, 11 conserved exposed residues and 6 non-conserved exposed residues. The effects of all 34 missense mutations can be rationalized by predictable disruptions of FVIII structure while at most four mutations (S2069F, T2154I, R2209Q/G/L and E2181D) may affect residues directly involved in intermolecular interactions of FVIII/VIIIa with other coagulation factors or vWF.

scholarly journals The 3.2 Å structure of a bioengineered variant of blood coagulation factor VIII indicates two conformations of the C2 domain

2019 ◽  
Vol 18 (1) ◽  
pp. 57-69 ◽  
Author(s):  
Ian W. Smith ◽  
Anne E. d'Aquino ◽  
Christopher W. Coyle ◽  
Andrew Fedanov ◽  
Ernest T. Parker ◽  
...  
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
C2 Domain ◽  
Blood Coagulation Factor Viii

scholarly journals The tertiary structure and domain organization of coagulation factor VIII

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1240-1247 ◽  
Author(s):  
Betty W. Shen ◽  
Paul Clint Spiegel ◽  
Chong-Hwan Chang ◽  
Jae-Wook Huh ◽  
Jung-Sik Lee ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Tertiary Structure ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Factor Viii Gene ◽  
C2 Domains ◽  
Activated Platelets ◽  
Membrane Bound ◽  
Domain Stabilization

AbstractFactor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 domains. The structure permits unambiguous modeling of the relative orientations of the 5 domains of r-fVIII. Comparison of the structures of fVIII, fV, and ceruloplasmin indicates that the location of bound metal ions and of glycosylation, both of which are critical for domain stabilization and association, overlap at some positions but have diverged at others.

scholarly journals The Sequence GluLys of Human Blood Coagulation Factor VIII Comprises a Binding Site for Activated Factor IX

1996 ◽  
Vol 271 (4) ◽  
pp. 1935-1940 ◽  
Author(s):  
Peter J. Lenting ◽  
Jan-Willem H. P. van de Loo ◽  
Marie-José S. H. Donath ◽  
Jan A. van Mourik ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Blood Coagulation ◽  
Human Blood ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Factor Viii ◽  
Blood Coagulation Factor Viii ◽  
Human Blood Coagulation

scholarly journals МЕТОДОЛОГІЧНІ ПІДХОДИ ОДЕРЖАННЯ ФАКТОРА VIII ЗСІДАННЯ КРОВІ

2020 ◽  
Vol 79 (1-2) ◽  
pp. 102-108
Author(s):  
N. O. Shurko ◽  
V. L. Novak
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Modern Technology ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Protein Therapy ◽  
Biotechnological Approach

The article deals with basic methods used by modern technology to obtain coagulation factor VIII (FVIII). The blood plasma fractionation remains the only biotechnological approach to make life-saving protein therapy to treat human diseases. The biological medicines from human plasma play a vital role in the treatment of patients with different diseases. These products include a range of coagulation factors (FVIII, FIX, the prothrombin complex, Von Willebrand factor, fibrinogen etc.), immunoglobulins, protease inhibitors, anticoagulants and albumin. Four plasma proteins are commercially important for production: albumin, IgG, factor VIII, and factor IX. VIII is a coagulation factor in the blood, which is missing or defective in patients with Hemophilia A. Replacement therapy with FVIII concentrates constitutes the basis for hemophilia care. Cryoprecipitate was described in the mid 60's of the XX century as a first concentrate of antihemophilic FVIII.The main indications for the clinical use of cryoprecipitate were hypofibrinogenemia or disfibrinogenemia. Previously, cryoprecipitate was used for treatment of hemophilia A and von Willebrand’s disease. Traditional FVIII production methods included deposition steps, which were aimed at elimination of protein impurities such as fibrinogen, fibronectin and immunoglobulins. These technologies could use the combination of methods at low temperatures or the addition of protein precipitating substances (PEG, polyvinylpyrrolidone, dextran, ficol, percol etc.). Using chromatographic methods in FVIII production technology allowed receiving high purity and specific activity concentrate of FVIII. Ion exchange chromatography techniques are often used in order to isolate coagulation FVIII. These techniques include methods of affinity chromatography as well as the use of monoclonal antibodies to bind of FVIII. Nowadays, production of plasma concentrate of FVIII is used in combination with different chromatographic techniques.

Structure of Blood Coagulation Factor VIII in Complex with an Anti-C1 Domain Pathogenic Antibody Inhibitor

Blood ◽  
2021 ◽  
Author(s):  
Joseph S Gish ◽  
Lexi Jarvis ◽  
Kenneth C Childers ◽  
Shaun C Peters ◽  
Connor S Garrels ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Domain Antibody ◽  
C1 Domain ◽  
Group A ◽  
Surface Loops ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Blood Coagulation Factor Viii

Antibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a Group A anti-C1 domain inhibitor, termed 2A9, in complex with a B domain-deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at three different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156 and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor (vWF). Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti-C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.

scholarly journals A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2335-2340
Author(s):  
Toufik Abache ◽  
Alexandre Fontayne ◽  
Dominique Grenier ◽  
Emilie Jacque ◽  
Alain Longue ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Viiia

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 μg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

MOLECULAR ANALYSIS OF COAGULATION FACTOR VIII AND IX GENES BY DNA PROBES

1987 ◽  
Author(s):  
C de la Salle ◽  
M J Baas ◽  
L Grunebaum ◽  
R Gialeraki ◽  
T Mandalaki ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Cdna Probe ◽  
Coagulation Factor ◽  
Molecular Study ◽  
Large Deletion ◽  
Factor Ix ◽  
Hemophilia B ◽  
Factor Viii Gene ◽  
Genomic Probe

About 250 individuals belonging to 44 families with hemophilia A or B were studied in our laboratory. The detection of carriers was first established by pedigree analysis of each family . and coagulation and immunological assays of factor VIII or IX. The availability of specific probes for the molecular study of these two genes makes possible a diagnosis with certainty in the case of informative families. 25 families of hemophilia A were studied. For each person, blood was collected into EDTA and leucocyte DNA was extracted, digested by restriction endonucleases, electrophoresed in 0.9 % agarose gels and transferred to nitrocellulose filters by Southern blotting. Two probes were used for the analysis of factor VIII gene. The St 14 probe (J.L. Mandel) located on the q28 region of the X chromosome and closely linked to the gene, determines a restriction fragment length polymorphism (RFLP) when the DNA is digested by the enzyme TaqI. The p114-12 genomic probe (Genentech) corresponding to the exons 17 and 18 of the factor VIII gene, reveals a RFLP in the DNA digested by the enzyme BclI. 19 families -of hemophilia B were studied. A total factor IX cDNA probe was used for the screening of potential deletions in the case of hemophiliacs with circulating antibodies. A genomic probe containing the exons II, III and IV of factor IX was used to detect the TaqI RFLP. For the study of factor VIII gene, the extragenic probe St 14 gives a very high percentage of informativity (about 90 %) but recombination can occur between the probe and the gene. The p 114-12 probe, which is used to confirm the results given by the St 14 probe, gives about 20 % informativity. In our study, we were able to diagnose carrier state with certainty in 92 % of the families. For hemophilia B, the genomic probe gives about 40 % informativity. A large deletion of the region of the factor IX gene has been found in one family and remains to be mapped. In conclusion, carrier detection and prenatal diagnosis can be established with certainty by molecular studies in most cases of hemophilia A using the St 14 probe, with a 5 % risk of recombination when the BclI RFLP cannot confirm. This diagnosis is possible in about 40 % of the cases of hemophilia B.

scholarly journals Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice

Blood ◽  
2012 ◽  
Vol 119 (22) ◽  
pp. 5294-5300 ◽  
Author(s):  
Aleksandra Wroblewska ◽  
Simon D. van Haren ◽  
Eszter Herczenik ◽  
Paul Kaijen ◽  
Aleksandra Ruminska ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Major Complication ◽  
Human Monocyte ◽  
Cell Responses ◽  
Chromogenic Assay ◽  
C1 Domain ◽  
Blood Coagulation Factor Viii

Abstract Development of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 domain of FVIII. We created a C1 domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4+ T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.

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