A Factor VIII Minigene Comprising the Truncated Intron I of Factor IX Highly Improves the In Vitro Production of Factor VIII

2001 ◽  
Vol 86 (08) ◽  
pp. 596-603 ◽  
Author(s):  
Jean-Luc Plantier ◽  
Marie-Hélène Rodriguez ◽  
Nathalie Enjolras ◽  
Olivier Attali ◽  
Claude Négrier
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Haemophilia A ◽  
Intracellular Accumulation ◽  
In Vitro Production ◽  
Insertion Sites ◽  
Recombinant Fviii ◽  
Vitro Production

SummaryThe biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.

Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries

2016 ◽  
Vol 116 (07) ◽  
pp. 32-41 ◽  
Author(s):  
Anja Schmidt ◽  
Kerstin Brettschneider ◽  
Jörg Kahle ◽  
Aleksander Orlowski ◽  
Karin Becker-Peters ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tolerance Induction ◽  
Coagulation Factor ◽  
Cross Reactivity ◽  
Hybridoma Cells ◽  
Haemophilia A ◽  
Immune Tolerance Induction ◽  
Fviii Inhibitor ◽  
Antiidiotypic Antibody

SummaryFollowing replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated antiidiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitorspecific, high-affinity anti-idiotypes can be isolated from phagedisplayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

Bioengineering of coagulation factor VIII for improved secretion

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3412-3419 ◽  
Author(s):  
Hongzhi Z. Miao ◽  
Nongnuch Sirachainan ◽  
Lisa Palmer ◽  
Phillip Kucab ◽  
Michael A. Cunningham ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Mrna Levels ◽  
Golgi Transport ◽  
Recombinant Fviii ◽  
Therapy Strategies

Abstract Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

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.

scholarly journals Dental considerations in a patient with haemophilia

2016 ◽  
Vol 3 (1) ◽  
pp. 51-54 ◽  
Author(s):  
Chandra Khyati ◽  
Mavinakote Gowda Triveni ◽  
Rini Gopal ◽  
Ab. Tarunkumar ◽  
Suresh Hanagavadi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Dental Care ◽  
Coagulation Factor ◽  
Bleeding Risk ◽  
Factor Ix ◽  
Haemophilia A ◽  
Haemophilia B ◽  
Characteristic Features ◽  
Limited Health ◽  
Treatment Procedures

Abstract Haemophilia is a rare blood clotting disorder, characteristic features of which include extemporaneous and post-traumatic subcutaneous bleeding and mucosal haemorrhages. Genetic deficiency of coagulation factor VIII results in haemophilia A, while deficiency of factor IX leads to haemophilia B. The most common treatment for haemophilia A is administration of recombinant or plasma-derived factor VIII concentrate, to raise the levels of the deficient factor VIII. Tranexamic acid is also used as an anti-fibrinolytic agent that inhibits plasminogen activators present in oral secretion and stabilises the clot. Administration of factor IX is required in haemophilia B. Treatment leads to increased longevity and quality of life for patients. Dental conditions and treatments are more complicated and uncertain in patients with haemophilia due to bleeding risk, thus restorative dental care is of paramount importance for those with haemophilia. The fear of bleeding during treatment procedures is the primary cause of lack of proper dental care for people with haemophilia in countries with limited health care resources. This case report highlights the significance of clinical examination and investigation, and the importance of proper interaction between a haematologist and the periodontist for correct multidisciplinary and uneventful management of periodontal health of a patient with haemophilia.

scholarly journals A transgender person with haemophilia

2018 ◽  
Vol 5 (1) ◽  
pp. 147-151
Author(s):  
Caroline Valk
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Clotting Factor ◽  
Bilateral Mastectomy ◽  
Sex Reassignment Surgery ◽  
Haemophilia A ◽  
Cultural Changes ◽  
Transgender People ◽  
Specialist Doctor

Abstract Introduction: Transgender people face many obstacles to accessing healthcare but cultural changes are likely to increase provision of sex reassignment surgery in countries with sufficient resources. Haemophilia services traditionally focus on providing factor replacement therapy for males and should therefore understand how the care they provide can be adapted to meet the needs of transgender people. Haemophilia is an X-linked congenital bleeding disorder, caused by deficiency of coagulation factor VIII (haemophilia A) or factor IX (haemophilia B). The condition is passed on through carrier females, the majority of whom have a factor level high enough to allow for normal blood clotting. However, around 10% of carrier females are symptomatic and at risk of abnormal bleeding. Case presentation: This case report describes a person with mild haemophilia A who, on first presentation to the haemophilia service, stated he was a transgender person in transition to becoming a male. Haemophilia was diagnosed when heavy bleeding occurred following bilateral mastectomy approximately 25 years previously. He now requested phalloplasty. Management and outcome: Phalloplasty was performed at a hospital geographically separate from the haemophilia centre, requiring careful coordination between the two services. A haemophilia specialist nurse provided education and training about haemophilia and its management to the surgical nurses. Twenty-four-hour support was available from the nurse and a specialist doctor. Preparation and administration of clotting factor was the responsibility of the haemophilia nurse until the surgical team was confident in its use. Clotting factor replacement was managed using standard procedures, successfully maintaining factor VIII above a target level of 100% with a twice daily dose. Surgery went well, but wound healing was delayed, in part, due to persistent bleeding. Discussion: Close collaboration between the haemophilia and surgical teams provided effective prophylaxis of bleeding during a complex procedure that presented new challenges. Both services now have better understanding of the needs of transgender people.

Further Studies on the “Dynia” Clotting Abnormality

1967 ◽  
Vol 17 (03/04) ◽  
pp. 365-380 ◽  
Author(s):  
L Pechet ◽  
F Cochios ◽  
D Deykin
Keyword(s):  
Factor Viii ◽  
Intermediate Product ◽  
Coagulation Factor ◽  
Normal Serum ◽  
Factor Ix ◽  
Factor X ◽  
Coagulation Abnormality ◽  
Coagulation Defect ◽  
Coumarin Drugs

Summary1. One patient with multiple bleeding episodes and 4 asymptomatic relatives in 3 generations reveal a coagulation abnormality that cannot be attributed to any known coagulation factor deficiency or to a circulating inhibitor. The abnormality is characterized by deficient generations of intrisic thromboplastin and intermediate product I, and can be corrected by normal plasma and serum.2. The ability of normal blood to correct the coagulation defect in vitro is heat labile; it is not removed from plasma by adsorption with BaSO4 or Al(OH)3. Neonatal blood and blood taken from patients either receiving coumarin drugs or with severe liver disease sustain the ability to correct the described defect. Ox, rabbit and dog blood are also corrective.3. The finding of normal factors XII, XI and IX activities, as well as normal serum thrombosis accelerator (STA) activity, places this abnormality beyond the stage of factor IX activation.4. The data suggest that the familial coagulation abnormality is located in the clotting sequence either in the activation of factor VIII, or in the activation of factor X by factor VIII. Either interpretation is in agreement with the finding of abnormal intermediate product I generation, which reflects primarily the intrinsic activation of factor X.5. The frequency of this defect is unknown. Conceivably some patients considered to have factor XI (PTA) deficiency because of a plasma and serum correctable defect in their thromboplastin generation, may actually have a coagulation defect similar to that described in the Dynia family.6. It is suggested that the name “Dynia defect” be temporarily assigned to this clotting abnormality.

scholarly journals Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2925-2935 ◽  
Author(s):  
DD Pittman ◽  
EM Alderman ◽  
KN Tomkinson ◽  
JH Wang ◽  
AR Giles ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Thrombin Activation ◽  
Recombinant Fviii

Abstract Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.

scholarly journals Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2925-2935 ◽  
Author(s):  
DD Pittman ◽  
EM Alderman ◽  
KN Tomkinson ◽  
JH Wang ◽  
AR Giles ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Thrombin Activation ◽  
Recombinant Fviii

Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.

Detection of the Carrier State in Hereditary Coagulation Disorders II

1968 ◽  
Vol 19 (03/04) ◽  
pp. 403-422 ◽  
Author(s):  
J. J Veltkamp ◽  
E. F Drion ◽  
E. A Loeliger
Keyword(s):  
Standard Deviation ◽  
Factor Viii ◽  
Normal Population ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Factor Xii ◽  
Haemophilia A ◽  
Coagulation Defects ◽  
The Individual

Summary and ConclusionsSpecific determination of the coagulation factors makes it possible to demonstrate that heterozygotes of hereditary coagulation defects on the average display about 50% of the normal activity of the relevant coagulation factor. For the autosomally inherited defects, the published values of the coefficient of variation of the carrier population and the normal population show little disparity. It appears that discrimination of carriers of the factor II, V, VII, X, or XI deficiencies can be carried out with reasonable success. An exception, however, is the discrimination of carriers of haemophilia A and B. The large overlapping of the normal and the carrier distributions has been attributed to technical inadequacies in the assessment of factor VIII and IX activity and also to the possibly disturbing effect of the random inactivation of one of the two X-chromosomes (Lyon hypothesis). The statistical approaches described in the literature for the calculation of chance of carriership in individual cases are not accepted.Throughout the present investigation a one-stage method for the measurement of coagulation factor activity was used; this method was based on established principles, and adapted for use in Schnitger’s coagulometer. The experimental error (which with the two-stage method amounted to 25%) is 5.7% for factor VIII, 8.1 % for factor IX, and 7.3% for factor XII, expressed as the coefficient of variation of the result. This drastic improvement of the accuracy of the method was accomplished chiefly by automatic registration. The procedure is also more rapid and less tiring to perform than the two-stage method.The average factor VIII activity found in 30 proven carriers of haemophilia A was antilog 1.71 % (51 %) of normal. The standard deviation for this population was 0.21, as calculated from logarithms of the factor VIII activities, and the values were symmetrically distributed. For the normal population, which consisted of 60 persons, the standard deviation was 0.12. The biological variation of the factor VIII activity within the individual was 0.084 for carriers and 0.061 for normals; these values do not differ significantly.For the carriers of haemophilia B the average factor IX activity was antilog 1.7% (50%) and the standard deviation 0.13. The average activity in the normal population of 65 persons was antilog 2.007% (100.4%) and the standard deviation 0.105. There was a significant increase in factor IX activity with increasing age, at least for females. The variation within the individual was 0.066 for carriers and 0.0577 for normals.The wide variation within the populations of carriers of haemophilia A and B as compared to the normal population as well as to the populations of heterozygotes of autosomally inherited coagulation defects could be interpreted as support for the hypothesis of Lyon. This phenomenon could be caused, however, by different normal iso-alleles and the influence of other genetic or environmental factors.On the basis of our observations, it is possible to make the discrimination of carriers and normals on a 95% confidence level in the population of potential carriers of haemophilia A in 34% and of haemophilia B in 60% of the cases. The chance of carriership can be calculated for individual cases, with Bayes’ theorem, from the known chance of carriership on genetic grounds and the observed factor VIII or IX activity.The bimodal distribution of the results obtained in 50 obligatory heterozygotes for Hageman trait reflect the occurrence of two normal iso-alleles for the Hageman locus, one with 23% and the other 60% factor XII activity. The whole group still shows a mean activity of about 50% as compared to the normal population.Laboratory methods for measuring blood coagulation factors, adequate for the detection of heterozygotes of most autosomally inherited coagulation defects, have been shown to make only a modest contribution to the improvement of genetic counselling in haemophilia A and B. More fundamental approaches to the problem of discrimination of haemophilia carriers are required, because further standardization of the laboratory procedures cannot substantially improve the results obtained in this study. Theoretically, this problem will be easily solved when new common X-linked markers, closely linked to the haemophilia loci, are discovered.

Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains

2009 ◽  
Vol 101 (05) ◽  
pp. 834-839 ◽  
Author(s):  
Soheila Gharagozlou ◽  
Ramazan Sharifian ◽  
Jalal Khoshnoodi ◽  
Katayoon Karimi ◽  
Monica Milani ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Synthetic Peptides ◽  
Coagulation Factor ◽  
Phosphatidyl Serine ◽  
Factor Ix ◽  
Peptide Sequence ◽  
Haemophilia A ◽  
Antigen Specificity ◽  
Acquired Haemophilia

SummaryDevelopment of antibodies (Ab) that either block the function of coagulation factor VIII (FVIII) (inhibitors) or clear it from circulation, seriously complicate the treatment of haemophilia A patients with FVIII products. Autoantibodies which develop in subjects without congenital FVIII defects, cause acquired haemophilia, a rare but life-threatening coagulopathy. Identification of the FVIII epitopes to which inhibitor Abs bind will help understanding the mechanisms of inhibitor activity, and perhaps development of new therapies. Here, we examined the FVIII peptide sequence regions recognised by anti-FVIII Ab in the plasma of six congenital and one acquired haemophilia patients with high inhibitor titers (24.4–2000 BU/ml). We used indirect ELISA and overlapping synthetic peptides, 20 residues long, spanning the sequence of the A and C FVIII domains. None of the plasma samples reacted with A1, A3 or C1 domain peptides. Six plasmas reacted with A2 and/or C2 peptides. Peptides spanning residues A2–521–690 and C2–2251–2332 were recognised most frequently and strongly. They include residues that contribute to the binding sites for activated factor IX and phosphatidyl serine/ von Willebrand factor. These results suggest that anti-FVIII Abs share a pattern of antigen specificity in our panel of patients, and that exposed regions of the FVIII molecule that form functionally important binding sites elicit an intense Ab response.

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