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

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2799-2805 ◽  
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.

Rabbit Antibodies Against the Procoagulant Activity (VIII:C) of Human Factor VIII

1981 ◽  
Vol 46 (04) ◽  
pp. 699-705 ◽  
Author(s):  
T H Tran ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Gel Filtration ◽  
Procoagulant Activity ◽  
Solid Matrix ◽  
Factor Viii Activity ◽  
Physiological Conditions ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Factor Viii Antigen

SummaryThe procoagulant activity VIII:C was separated from factor VIII antigen (VIIIR:Ag) by gel filtration in the presence of 0.25 mol/l calcium chloride. Antibodies (anti-VIII:C) were obtained by immunization of rabbits with VIII:C. The last step of the purification procedure of antibodies consists of an adsorption on VIIIR:Ag-Sepharose 2 BCL as immunoadsorbent to remove contaminating traces of antibodies against VIIIR:Ag. The anti- VIII:C titer remains unchanged during this adsorption (29 Bethesda units per mg). In solution, anti-VIII:C neutralies factor VIII activity (in plasma, cryoprecipitate or in purified form) and the fragment VIII:C without reacting with VIIIR:Ag. Once immobilized on a solid matrix, i.e.2% agarose, it loses over 95% of its inhibitory capacity. The immobilized anti-VIIIR:Ag binds stoichiometrically the antigen and the activity of plasma factor VIII. These results together suggest that factor VIII is composed of 2 different entities, but undissociated under physiological conditions. Immunophysical analyses as a function of pH and temperature of anti-VIII:C and its complex with factor VIII show properties similar to those of homologous antibodies. The antigen determinants of VIII:C (VIII:CAg) are destroyed at low pHs or high temperatures, and VIII:C can no more form a complex with anti-VIII:C. Purified anti-VIII:C is also used in a two-stage assay to detect VIII:CAg or cross-reacting material in some severe haemophiliacs.

Immune Tolerance to Human Factor VIII Induced by Liver-Specific Expression of a Human Factor VIII Transgene in Hemophilic Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3387-3387 ◽  
Author(s):  
Pauline van Helden ◽  
Maria Sasgary ◽  
Sabine Unterthurner ◽  
Maria Schuster ◽  
Gerhard Antoine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Immune Responses ◽  
Factor Viii ◽  
Immune Tolerance ◽  
Preliminary Data ◽  
Human Factor ◽  
Hemophilia A ◽  
Specific Expression ◽  
Human Factor Viii

Abstract Therapy of hemophilia A has greatly benefited from the development of safe recombinant and plasmatic factor VIII (FVIII) concentrates. Current efforts to improve products focus on the extension of half-life by chemical and/or molecular modifications of FVIII. However, any modification of the FVIII protein poses the risk of creating neo-antigens that might cause FVIII inhibitors to be induced in patients. Therefore it is important to monitor the potential creation of neo-antigens during preclinical and clinical phases of drug development. Currently available animal models for hemophilia A develop high titers of anti-FVIII antibodies when treated with human FVIII. Using these models, it is difficult to differentiate between immune responses against native human FVIII and immune responses against human FVIII that carries neo-antigens. Considering these limitations, our aim is to develop a new model for hemophilia A that does not respond with antibodies to native human FVIII but develops antibodies against human FVIII that carries neo-antigens. We created a series of hemophilic mouse lines that carry a transgene for human FVIII that was placed under the control of an albumin promoter to direct liver-specific expression. Transgenic founder mice were generated by direct microinjection of the vector into the male pronucleus of fertilized oocytes obtained from mated female C57BL/6J mice after superovulation. Transgenic mice were crossed with hemophilic mice and bred to homozygousity for the expression of the human FVIII transgene. We analyzed the expression of human FVIII by real time PCR in lung, kidney, liver, heart, muscle, spleen, lymph nodes and reproductive organs. Gene expression analysis of bone marrow and thymus are currently ongoing. We selected three sublines (E, G and I) that show different levels of liver-specific expression of human FVIII for further analysis. We did not detect any FVIII antigen in the circulation in any of these three sublines when we used two different ELISA systems with detection limits around 1 ng/ml. We treated mice of sublines E, G and I intravenously with eight weekly doses of 200 ng of human FVIII (Advate) and analyzed the potential development of antibodies against native human FVIII. Our results indicate that transgenic mice of sublines E and I are immunologically tolerant to native human FVIII. They do not develop anti-FVIII antibodies (about 90% of all mice tested) or develop low titers (below 1:80 in 10% of mice tested) only. In contrast, mice of subline G develop high titers of anti-FVIII antibodies indicating that they are not immunologically tolerant to human FVIII. Preliminary data suggest that the degree of immunological tolerance against human FVIII correlates to a certain extent with the expression levels of the human FVIII transgen in liver and/or thymus. We are in the process of verifying these preliminary data. Furthermore, we have started to analyze FVIII-specific T-cell responses to define potential differences in the repertoire of FVIIIspecific T cells between the three sublines. We conclude that transgenic expression of human FVIII under the control of an albumin promoter is able to induce immune tolerance to human native FVIII in hemophilic mice. However, a certain threshold level of gene expression might be required for the induction of immune tolerance.

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

Blood ◽  
1973 ◽  
Vol 42 (3) ◽  
pp. 437-444 ◽  
Author(s):  
J.-P. Allain ◽  
D. Frommel
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Direct Consequence ◽  
Antigenic Stimulation ◽  
Factor Viii Activity ◽  
Human Factor Viii ◽  
Antigen Antibody ◽  
Stimulation Of

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.

scholarly journals Dose Requirement for Substitution Therapy in Hemophilia A.

1977 ◽  
Author(s):  
J.P. Allain
Keyword(s):  
Factor Viii ◽  
Clinical Result ◽  
Hemophilia A ◽  
Factor Viii Activity ◽  
Dose Requirement ◽  
Plasma Factor ◽  
Post Infusion ◽  
The Relationship ◽  
Different Doses

In order to determine the relationship between different doses of factor VIII and their clinical effect,70 children with severe hemophilia A treated with single doses of cryoprecipitate were studied. Plasma factor VIII levels were tested by a one stage assay on samples taken before, and 30 and 60 min. post-infusion. Clinical failure was defined as an absence of clinical improvement or the occurrence of bleeding in another site within 24 hours post-treatment. The relationship between plasma factor VIII levels or doses injected in u/kg and clinical result followed an exponential curve. Calculated from 153 factor VIII recoveries, plasma factor VIII levels of 0.35 and 0.52 u/ml corresponded to 95 and 99% satisfactory treatment, respectively. The same clinical result was obtained with 20 and 31 u of factor VIII/kg when studied in 1043 infusions of lyophilized cryoprecipitate. The in vivo recovery being 0.015 u factor VIII activity/ml for 1 u/kg injected, these dosages provided plasma factor VIII levels of 0.30 and 0.47 u/ml respectively. Since home treatment is largely based on single infusions of factor VIII, it is suggested that most current bleedings can be adequately treated with a dose of factor VIII providing a plasma factor VIII activity of 0.30-0.35 u/ml.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

The Fractionation Properties of Human Factor VIII (Antihaemophilic Factor)

1960 ◽  
Vol 04 (02) ◽  
pp. 253-260 ◽  
Author(s):  
Franco Gobbi
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Maximum Yield ◽  
Factor Vii ◽  
Precipitation Method ◽  
Salting Out ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Two Factors ◽  
Thromboplastic Activity

SummaryThe fractionation properties of human Factor VIII (antihaemophilic factor, AHF, antihaemophilic globulin) have been studied using a plasma of congenital afibrinogenaemia as a starting material.From a fibrinogen-free plasma, Factor VIII does not precipitate with ethanol at a final concentration of 8%; on the contrary the maximum yield is reached at an ethanol concentration of 25%.With a precipitation method carried out by a one to ten dilution of plasma with distilled water and acidification by N/10 hydrochloric acid to a pFI 5.2, Factor VIII does not precipitate with the euglobulin fraction; when normal plasma is used, such a precipitation is almost complete.With the salting-out fractionation method by ammonium sulphate, Factor VIII precipitates at a concentration between 25 and 33% of saturation either from fibrinogen-free and from normal human plasma.A non-specific thromboplastic activity appears in the fractions prepared by every method. This activity, which is probably due to the activation of seric accelerators, is easily removed by Al(OH)s adsorption. Thus, in order to insure the specificity of Factor VIII assays, the preliminary adsorption of the fractions is indispensable before testing their antihaemophilic activity.Fibrinogen and Factor VIII have different and definite precipitation patterns. When these two factors are associated the fractionation properties of AHF appear quite modified, showing a close similarity to those of fibrinogen. This fact can explain the technical difficulties encountered in the attempt to purify the antihaemophilic factor, and the lack of reproducible procedures for removing fibrinogen without affecting Factor VII.

The Immunological Properties of Factor VIII

1966 ◽  
Vol 16 (03/04) ◽  
pp. 559-573 ◽  
Author(s):  
L Uszyński
Keyword(s):  
Factor Viii ◽  
Inhibitory Activity ◽  
Hemophilia A ◽  
Severe Form ◽  
Molecular Defect ◽  
Bovine Plasma ◽  
Human Factor Viii ◽  
Antigenic Properties ◽  
Coagulation Tests ◽  
Normal Human

SummaryRabbits immunized against human AHG fibrinogen-free preparations, were shown to produce anti-AHG antibodies. The inhibitory activity of these antibodies was tested by thromboplastin generation test, thrombelastography, and the specific anti-AHG antibodies neutralization test. The latter test permitted quantitative determination of antigenic form of factor VIII. The inhibitory activity of anti-FI-O-Ta serum resulted exclusively from the anti-AHG antibodies which in coagulation tests behaved like circulating anticoagulants directed against factor VIII.The anti-AHG antibodies were neutralizable by normal human serum or plasma even contained only trace of AHG activity after storage. There was no antigenic form of factor VIII in the severely affected patients with hemophilia A, von Willebrand’s disease nor in the normal plasma adsorbed on bentonite. The presented results suggest a molecular defect of factor VIII in patients with hemophilia A. The severe form of this disease depends, probably, on a major impairment of AHG biosynthesis, leading to changes in the antigenic properties of the molecule. The AHG from rabbit, porcine and bovine plasma respectively did not neutralize the anti-AHG antibodies formed in rabbits immunized against human factor VIII preparations.

scholarly journals Further Experience on the Effect of High Pre-Donation Factor VIII Levels in Blood Donors on the Factor VIII Content of Small-Pool Fractions

1977 ◽  
Author(s):  
H. Beeser ◽  
H. Eqli
Keyword(s):  
Factor Viii ◽  
High Plasma ◽  
Activity Levels ◽  
Factor Viii Activity ◽  
Factor Viii Level ◽  
Plasma Factor ◽  
Viii Level ◽  
Factor Viii Concentrates ◽  
Small Pool ◽  
Concentrate Preparation

Because of the well known wide normal range of the factor VIII activity between 60 to 170% I man, selecting of donors with high activity levels would be of advantaae for the preparation of factor VIII concentrates. This is especially true for preparing small-pool fractions, as for technical reasons the final product cannot be controlled for its factor VIII content. In preliminary investigations, we reported on elsewhere, high factor VIII activity in donors estimated before a donation had been rarely reproducible before a second donation after 8-12 weeks. So as a preliminary result of finding a donor’s factor VIII level varying from donation to donation selecting of plasmas with high factor VIII content for concentrate preparation could only be establishedby re-estimating the activity before each donation. Proceeding in this way would be much too troublesome. To get more reliable information whether a healthy subject’s high factor VIII plasma level is distinctly varying or rather constant we assayed the plasma of 200 donors with factor VIII activity > 120% two times more before donation. The results confirmed our preliminary findings, especially the fact that a high plasma factor VIII activity in experienced donors was rarely reproducible when re-estimated before a second and third donation. As a consequence selecting of donors with high factor VIII procoaqulant activity for preparing small-pool factor VIII concentrates is impracticable.

scholarly journals Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 823-831 ◽  
Author(s):  
VT Turitto ◽  
HJ Weiss ◽  
TS Zimmerman ◽  
II Sussman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Human Factor ◽  
Rabbit Aorta ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.

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