Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies

Previously we created two strains of factor VIII-deficient mice by insertion of a neo gene into (1) the 3′ end of exon 16 and (2) exon 17 of the factor VIII gene. Affected mice of both strains have no plasma factor VIII activity, yet are healthy with no spontaneous bleeding. Factor VIII-deficient females bred with affected males survive pregnancy and delivery. We used reverse transcriptase-polymerase chain reaction of liver RNA to characterize factor VIII mRNA processing. Factor VIII mRNA of the exon 16 knockout strain contains neo sequences plus 17 bp of intron 16 due to use of a cryptic donor site in intron 16. All factor VIII mRNA of the exon 17 knockout strain lacks exon 17 and neo sequences. In skipping exon 17, the intron 16 donor site or a cryptic donor site 46 bp 3′ to the intron 16 donor site are used. Thus, factor VIII deficiency in exon 16 knockout mice is due to truncated protein, while in exon 17 knockout mice it is due to either truncated or partially deleted protein. After immunizing exon 16 knockout mice with human recombinant factor VIII, two monoclonal antibodies were obtained that recognize <7 100 pg of mouse factor VIII light chain. Assay of cryoprecipitate from the plasma of affected mice failed to show factor VIII light chain.

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Studies on the Characterization of Factor VIII and a Co-factor VIII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649167 ◽

1974 ◽

Vol 31 (02) ◽

pp. 328-338

Author(s):

M. M. P Paulssen ◽

H. L. M. A Vandenbussche-Scheffers ◽

P. B Spaan ◽

T de Jong ◽

M. C Planje

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

The Body ◽

Haemophilia A ◽

Von Willebrand's Disease ◽

Von Willebrand’S Disease ◽

Polysaccharide Content ◽

Plasma Factor ◽

Two Factors

SummaryFactor VIII occurs in the body in two different forms. In lymph factor VIII is bound to chylomicra. In plasma, factor VIII is bound to a protein.After delipidation of chylomicra we obtained a glycoprotein with a high polysaccharide content and a molecular weight of approx. 160,000.In plasma, factor VIII is attached to a protein which is present in normal concentrations in plasma of patients with haemophilia A and in serum (co-factor VIII).This factor is deficient in both the plasma and the serum of patients with von Willebrand’s disease.The binding between factor VIII and co-factor VIII is reversible.Some properties of these two factors are described.

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Ultrapure plasma factor VIII produced by anti-FVIIIc immunoaffinity chromatography and solvent/detergent viral inactivation. Characterization of the Method M process and Hemofil M antihemophilic factor (human)

Annals of Hematology ◽

10.1007/bf01703243 ◽

1991 ◽

Vol 63 (3) ◽

pp. 131-137 ◽

Cited By ~ 23

Author(s):

M. Griffith

Keyword(s):

Factor Viii ◽

Immunoaffinity Chromatography ◽

Viral Inactivation ◽

Plasma Factor ◽

Antihemophilic Factor

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Partial Characterization of the Factor VIII-Stimulating Material of Rabbit Liver Perfusates: Relationships to Rabbit Plasma Factor VIII

British Journal of Haematology ◽

10.1111/j.1365-2141.1975.tb00879.x ◽

1975 ◽

Vol 31 (4) ◽

pp. 437-447 ◽

Cited By ~ 3

Author(s):

R. E. Benson ◽

W. Jean Dodds

Keyword(s):

Factor Viii ◽

Rabbit Liver ◽

Partial Characterization ◽

Rabbit Plasma ◽

Plasma Factor

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Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-013-0338-4 ◽

2013 ◽

Vol 171 (1) ◽

pp. 10-19 ◽

Cited By ~ 2

Author(s):

Sudheer Reddy A.R. ◽

Padikara Kutty Satheeshkumar ◽

Mookambeswaran A. Vijayalakshmi

Keyword(s):

Pichia Pastoris ◽

Factor Viii ◽

Light Chain ◽

Coagulation Factor ◽

Biologically Active ◽

Coagulation Factor Viii ◽

In Vitro Characterization

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Characterization of Antibodies Induced by Human Factor VIII in a Murine Knockout Model of Hemophilia A

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614124 ◽

2000 ◽

Vol 84 (11) ◽

pp. 826-832 ◽

Cited By ~ 53

Author(s):

Birgit Reipert ◽

Rafi Ahmad ◽

Peter Turecek ◽

Hans Schwarz

Keyword(s):

Factor Viii ◽

Knockout Mice ◽

Hemophilia A ◽

Subcutaneous Administration ◽

Type I ◽

Thrombin Cleavage ◽

Human Factor Viii ◽

Inhibitory Antibodies ◽

Igg1 Subclass

SummaryTo investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy, knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

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Immunologic Characterization of 12 Factor VIII Inhibitors

Blood ◽

10.1182/blood.v34.1.85.85 ◽

1969 ◽

Vol 34 (1) ◽

pp. 85-90 ◽

Cited By ~ 25

Author(s):

DONALD I. FEINSTEIN ◽

SAMUEL I. RAPAPORT ◽

MAY N. Y. CHONG

Keyword(s):

Factor Viii ◽

Light Chain ◽

Specific Antisera

Abstract Twelve Factor VIII inhibitors were characterized as immunoglobulins by highly specific antisera. All 12 inhibitors were characterized as belonging to the γG class of immunoglobulins. Ten inhibitors contained only K type light chain determinants and two contained only γ chain determinants. Therefore, these inhibitors were homogeneous by these criteria. Homogeneity cannot be established, however, until H-chain subtyping can be carried out.

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Loss of Tolerance to Exogenous and Endogenous Factor VIII in a Mild Hemophilia A Patient With an Arg593 to Cys Mutation

Blood ◽

10.1182/blood.v90.5.1902 ◽

1997 ◽

Vol 90 (5) ◽

pp. 1902-1910 ◽

Cited By ~ 59

Author(s):

Arthur R. Thompson ◽

Michael E.P. Murphy ◽

MiaoLiang Liu ◽

Evgueni L. Saenko ◽

John F. Healey ◽

...

Keyword(s):

Factor Viii ◽

Light Chain ◽

Hemophilia A ◽

High Titer ◽

Factor Viii Inhibitor ◽

Dna Encoding ◽

Spontaneous Bleeding ◽

Factor Viii Concentrates ◽

Loss Of Tolerance ◽

A2 Domain

AbstractA 42-year-old patient with mild hemophilia A developed spontaneous muscle hematomas 1 month after intense therapy with factor VIII concentrates. Factor VIII clotting activity was less than 1% and his factor VIII inhibitor was 10 Bethesda units (BU)/mL. The titer peaked at 128 BU despite daily infusions of factor VIII; 1 year later, the titer was 13 BU with no spontaneous bleeding for 4 months. The plasma inhibitor was 95% neutralized by factor VIII A2 domain but less than 15% neutralized by light-chain or C2 domain. His inhibitor did not cross-react with porcine factor VIII and was at least 10-fold less reactive to a series of hybrid factor VIII proteins in which human residues 484-508 are replaced by the homologous porcine sequence (Healey et al, J Biol Chem 270:14505, 1995). The inhibitor patient's DNA encoding his A2 domain and flanking sequences showed a C-T transition predicting Arg593 to Cys. Thirteen patients from 5 unrelated families with Cys593 have not developed inhibitors. Factor VIII clotting activity from one of them was inhibited similarly to diluted normal plasma by inhibitor patient plasma. In an homologous structure, ceruloplasmin (Zaitseva et al, J Biol Inorgan Chem 1:15,1996), the residue equivalent to Arg593, is in a loop distinct from residues 484-508. On solution phase immunoprecipitation with labeled factor VIII fragments, A2, light chain, and C2 domains bound. In contrast to typical immune responses to factor VIII in patients with severe hemophilia A, this patient's inhibitor was almost entirely reactive with common epitopes within the A2 domain whereas by more sensitive immunoprecipitation testing antibodies to light chain epitopes were also present. Accordingly, immune responsiveness to exogenous factor VIII (antigen burden) appears to be more critical than his endogenous, hemophilic factor VIII to his developing high-titer anti–factor VIII antibodies and loss of tolerance to both native and hemophilic factor VIII proteins.

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Haemophilic Pelvic Pseudotumour: A New Surgical Option

Healthcare ◽

10.3390/healthcare9101269 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1269

Author(s):

Gianluigi Pasta ◽

Roberta Ruggieri ◽

Salvatore Annunziata ◽

Alessandro Gallese ◽

Vincenzo Pio Gagliardi ◽

...

Keyword(s):

Case Report ◽

Factor Viii ◽

Rare Complication ◽

Clinical Manifestations ◽

Clotting Factor ◽

Recurrent Bleeding ◽

Spontaneous Bleeding ◽

Polyglycolic Acid Mesh ◽

Rod System ◽

Plasma Factor

Background: Haemophilia is an inherited coagulopathy caused by the absence or dysfunction of clotting factor VIII or IX. Clinical manifestations are generally secondary to recurrent bleeding episodes mainly in the musculoskeletal system. Bleeding symptoms appear early in life and, when the disease is severe (when plasma factor VIII or IX activity is <1% of normal), joint and muscle bleeding may occur spontaneously. A pseudotumour is a recurrent, chronic, encapsulated, slowly expanding, muscle hematoma. Haemophilic pseudotumour is a rare complication of haemophilia which occurs, as a condition either from repeated spontaneous bleeding or coming from a traumatic origin, in 1–2% of haemophilic patients. Case report: A 32-year-old man with severe haemophilia A referred to our Clinic with a massive right iliac wing pseudotumour complicated by Staphylococcus aureus superinfection and skin fistulisation. In this report we describe the medical management and surgical treatment by the adoption of a novel surgical technique which involves the use of a pedicle-screw and rod system (PSRS), a polyglycolic acid MESH and bone cement in order to build up an artificial ilium-like bony mass. This case report highlights the importance of interdisciplinary approach and the efficacy of eradicating surgery as treatment, especially in the case of large and long-lasting lesions.

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