Antibodies to Factor VIII. I. Variations in Stability of Antigen-Antibody Complexes in Hemophilia A

Abstract Human factor VIII-anti-factor VIII complexes were formed in vitro in slight antigen excess, using plasma of hemophiliacs who were found to have antibodies neutralizing AHF activity. These complexes, stable at +37°C and pH 7.4, were submitted to classical procedures known to favor dissociation of antibody from antigen. The methods used to obtain dissociation, incubations at +56°C and at pH 4.2, inactivated both unbound factor VIII and that released as a consequence of dissociation. The extent of dissociation was measured by the recovery of anti-factor VIII activity. Increasing resistance of complexes towards dissociation was observed in the plasma of the patients whose titer of inhibitor was increasing after recent transfusions. These observations suggested the emergence, as a direct consequence of renewed antigenic stimulation, of a population of different antibodies characterized by higher combining strength.

Download Full-text

Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614449 ◽

1999 ◽

Vol 81 (02) ◽

pp. 234-239 ◽

Cited By ~ 19

Author(s):

Sheila Connelly ◽

Julie Andrews ◽

Angela Gallo-Penn ◽

Luigina Tagliavacca ◽

Randal Kaufman ◽

...

Keyword(s):

Factor Viii ◽

Adenoviral Vector ◽

Human Factor ◽

Hemophilia A ◽

Adenoviral Vectors ◽

Full Length ◽

Human Factor Viii ◽

Immunohistochemical Analyses

SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

Download Full-text

Impaired Prothrombin Consumption in Bernard-Soulier Syndrome Is Corrected In Vitro by Human Factor VIII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655972 ◽

1997 ◽

Vol 77 (02) ◽

pp. 383-386 ◽

Cited By ~ 8

Author(s):

S Bellucci ◽

J P Girma ◽

M Lozano ◽

D Meyer ◽

J P Caen

Keyword(s):

Factor Viii ◽

Human Factor ◽

Platelet Membrane ◽

Giant Platelets ◽

Prolonged Bleeding Time ◽

Human Factor Viii ◽

Von Willebrand ◽

Willebrand Factor ◽

Bernard Soulier Syndrome

SummaryThe Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein lb (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII.'C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612915 ◽

2001 ◽

Vol 85 (01) ◽

pp. 125-133 ◽

Cited By ~ 55

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

Download Full-text

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

Pakistan Journal of Biological Sciences ◽

10.3923/pjbs.2007.4299.4302 ◽

2007 ◽

Vol 10 (23) ◽

pp. 4299-4302 ◽

Cited By ~ 3

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

Download Full-text

Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽

10.1182/blood.v87.11.4671.bloodjournal87114671 ◽

1996 ◽

Vol 87 (11) ◽

pp. 4671-4677 ◽

Cited By ~ 69

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.

Download Full-text

The Activation of Factor VIII by Thrombin

Blood ◽

10.1182/blood.v26.4.500.500 ◽

1965 ◽

Vol 26 (4) ◽

pp. 500-509 ◽

Cited By ~ 17

Author(s):

A. H. ÖZGE-ANWAR ◽

G. E. CONNELL ◽

J. F. MUSTARD

Keyword(s):

Factor Viii ◽

Human Factor ◽

Experimental Approach ◽

Factor Viii Activity ◽

Clotting Factors ◽

Activation Effect ◽

Activation Reaction ◽

Human Factor Viii

Abstract The activation of human factor VIII by thrombin has been demonstrated by a new experimental approach. This method permitted investigation of the interaction of thrombin and factor VIII in the absence of most other clotting factors. The activation effect of thrombin is susceptible to inhibition by diisopropylfluorophosphate. Trypsin cannot replace thrombin in the activation reaction, and it destroys factor VIII activity rapidly.

Download Full-text

Correction of the coagulation defect in hemophilia A mice through factor VIII expression in skin

Blood ◽

10.1182/blood.v95.9.2799.009k23_2799_2805 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2799-2805 ◽

Cited By ~ 42

Author(s):

Steven S. Fakharzadeh ◽

Yue Zhang ◽

Rita Sarkar ◽

Haig H. Kazazian

Keyword(s):

Transgenic Mice ◽

Factor Viii ◽

Hemophilia A ◽

Systemic Disease ◽

Factor Viii Activity ◽

Human Factor Viii ◽

Plasma Factor ◽

Transgenic Lines ◽

Rag 1 ◽

Involucrin Promoter

To test the hypothesis that factor VIII expressed in the epidermis can correct hemophilia A, we generated transgenic mice in a factor VIII–deficient background that express human factor VIII under control of the involucrin promoter. Mice from 5 transgenic lines had both phenotypic correction and plasma factor VIII activity. In addition to the skin, however, some factor VIII expression was detected in other tissues that have stratified squamous epithelia. To determine whether an exclusively cutaneous source of factor VIII could correct factor VIII deficiency, we grafted skin explants from transgenic mice onto mice that are double knockouts for the factor VIII and RAG-1 genes. Two graft recipients had plasma factor VIII activity of 4% to 20% of normal and improved whole blood clotting compared with factor VIII–deficient mice. Thus, expression of factor VIII from the epidermis can correct hemophilia A mice, thereby supporting the feasibility of cutaneous gene therapy for systemic disease.

Download Full-text

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽

10.1182/blood-2010-05-282707 ◽

2011 ◽

Vol 117 (3) ◽

pp. 798-807 ◽

Cited By ~ 109

Author(s):

Natalie J. Ward ◽

Suzanne M. K. Buckley ◽

Simon N. Waddington ◽

Thierry VandenDriessche ◽

Marinee K. L. Chuah ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Viral Vectors ◽

Hemophilia A ◽

Lentiviral Vector ◽

Wild Type ◽

Fviii Activity ◽

Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Download Full-text

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

Blood ◽

10.1182/blood.v74.5.1618.bloodjournal7451618 ◽

1989 ◽

Vol 74 (5) ◽

pp. 1618-1626 ◽

Cited By ~ 1

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.

Download Full-text