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PubMed1999

Collagen-bound von Willebrand factor has reduced affinity for factor VIII.

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

Animals↗
PubMed1984

Lack of correlation between factor VIII related antigen multimeric analysis pattern and parallel or non-parallel dose response curves in an ELISA factor VIII related antigen assay.

Factor VIII related antigen has been measured and epitope distribution has been explored by testing the degree of parallelism between standard and test plasma dose response curves using an enzyme immunoassay. Normal plasma, plasma fractions, and plasma from patients with haemophilia and von Willebrand's disease have been tested. All showed parallelism except for plasma from patients with the variant type IIA von Willebrand's disease, of which 10 had parallel and five had non-parallel dose response curves when compared with that of normal plasma. In one family plasma from seven members showed parallelism but from four others did not. An unrelated patient was tested on three occasions, and although the samples were parallel to each other, no sample was parallel to the standard. No correlation was found between parallelism as shown by the enzyme immunoassay and differences in factor VIII related antigen multimeric pattern, including triplet configuration, seen in the type IIA patients.

Adult↗
PubMed1989

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor.

Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VIII forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg. At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the 80-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor-VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin. In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45-50-70 kDa). The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.

Adsorption↗
PubMed1995

Measurement of anti-factor VIII IgG, IgG4 and IgM alloantibodies in previously untreated hemophilia A patients treated with recombinant factor VIII. Kogenate Japanese Clinical Study Group.

Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor VIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated with a recombinant factor VIII (rFVIII) preparation (Kogenate). Twelve pre- and post-treatment serum samples from 3 previously untreated patients who developed an inhibitor (alloantibody) were evaluated. Both rFVIII and plasma-derived factor VIII (pdFVIII) were used as antigen. Serum samples were diluted 37.5-fold. For the measurement of IgM antibody, protein A Sephadex suspension was added to the patients' serum samples and the supernatant was assayed. Anti-human IgG, IgG4, and IgM monoclonal antibodies labelled with peroxidase were added and absorbance at 450nm was determined. The reactivity of the IgG antibody with rFVIII and pdFVIII was extremely low. Samples containing the IgG4 inhibitor with a neutralizing activity of approximately 7.5 Bethesda units (BU)/ml resulted in absorbance values of 0.451-0.551, thus demonstrating a considerably high degree of sensitivity. The correlation between the neutralizing activity and reactions of the IgG4 antibody with rFVIII and pdFVIII antigens was high, with correlation coefficients of 0.912 and 0.966, respectively. Furthermore, the correlation coefficient between the measured absorbance values for the antibody reacted with pdFVIII and rFVIII was 0.961. No correlation was found between the reactivity of IgM to rFVIII and pdFVIII.

Adult↗
PubMed1997

Factor VIII Ise (R2159C) in a patient with mild hemophilia A, an abnormal factor VIII with retention of function but modification of C2 epitopes.

We found a patient with mild hemophilia A who had no detectable factor VIII antigen (FVIII:Ag), as shown by two-site ELISA using inhibitor alloantibodies (TK). We then analyzed A2, A2/B, and C2 antigen of the patient's DDAVP-induced FVIII using several anti-FVIII monoclonal antibodies. Factor VIII activity (FVIII:C) was increased from 12 to 42 U/dl by the administration of DDAVP. The DDAVP-induced increases in the A2 and A2/B antigens were 40 and 36 U/dl, respectively. However, the increase in the C2 antigen was only 7.5 U/dl. SSCP analysis and subsequent sequencing demonstrated an Arg to Cys transition at codon 2159. The anti-FVIII:C titer of monoclonal antibody, NMC-VIII/5 which recognized the C2 domain, against normal plasma was 450 Bethesda U/mg of IgG. However, the titer against DDAVP-treated patient's plasma was only 15 Bethesda U/mg. We also tested DDAVP-induced increase in the FVIII:Ag in another mild hemophilia A patient with the same mutation at Arg2159. Increase in his C2 antigen levels was only 19% of those in the A2 and A2/B antigen. We designate this abnormal FVIII as FVIII Ise. Our results show that a missense mutation at Arg2159 to Cys modifies the antigenicity of the C2 domain.

Adolescent↗
PubMed2004

Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays.

Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing one-stage assays with two-stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a B-domain deleted recombinant FVIII (BDDrFVIII, ReFacto). In order to alleviate this assay variance, a product-specific reference standard [the ReFacto Laboratory Standard (RLS)], was established for laboratory use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto-containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the one-stage clotting assay for the determination of FVIII:C in ReFacto-containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one-stage clotting and chromogenic substrate assays. Thirty-one clinical laboratories worldwide participated in the study of severe-hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL(-1). FVIII:C levels were determined against both the RLS and the local in-house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one-stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one-stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one-stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one-stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto-containing SHP samples by clinical laboratories using a product-specific standard in one-stage clotting assays. In addition, the product-specific reference standard significantly reduced the discrepancy between the one-stage clotting and the chromogenic substrate assay for ReFacto.

Blood Coagulation↗
PubMed1995

Inhibitors to factor VIII in a family with mild hemophilia: molecular characterization and response to factor VIII and desmopressin.

Inhibitor development in patients with mild hemophilia is a rare event. We report the occurrence of a persistent, high-responding inhibitor in two affected members of a mild hemophilia A family and discuss the therapeutic approaches employed in these patients in terms of their efficacy and effect on antibody titer. Desmopressin was an effective option for bleeding management, because endogenous factor VIII released by DDAVP was less immunogenic than exogenous factor VIII replacement, which invariably triggered anamnestic responses. Genetic analysis performed to investigate whether or not a peculiar molecular lesion accounted for this particular phenotype identified a G-A transversion at nucleotide 6507 in exon 23. This missense mutation has been already described in mild hemophilia A, but not in patients with inhibitors.

Adult↗
PubMed1984

Acquired factor VIII inhibitor in a non-haemophilic patient: successful treatment with plasma exchange associated with factor VIII concentrate and immunosuppressors.

We report the successful treatment of a 55-year-old man with an acquired factor VIII inhibitor, the appearance of which followed surgery for a carcinoma of the biliary tract. The patient, who had recurrent life-threatening bleeding episodes, was treated with intensive plasmapheresis, factor VIII concentrates, azathioprine and methylprednisolone. Disappearance of the inhibitor was observed after the second series of plasma exchanges. Controls 2 and 3 months after treatment confirmed the complete absence of the inhibitor and normal levels of factor VIII. The crucial role of plasma exchange is considered and discussed.

Autoantibodies↗
PubMed1987

On the unreliability of one-stage factor VIII:C clotting assays after infusion of factor VIII concentrates.

A multicentre study was undertaken in order to determine the reliability of the methods of assay of F VIII:C and the position of the peak value obtained after infusion of F VIII:C concentrates. Blood samples were drawn before and 10, 30 and 60 min after injection of F VIII concentrates in six haemophiliacs in one of the centres. Coded, frozen plasma samples were dispatched to the laboratories of the other four centres. F VIII:C was determined by different one-stage methods using the same international standard but with different activators. The results of the different laboratories differed widely and no agreement was reached on the existence of a lag period. To reach a valid conclusion not only the same sample has to be analysed, as in this study, but also the same laboratory technique has to be used by all participating investigators. To reach agreement on in vivo recovery and on elimination curves for different F VIII concentrates multicentre studies must be based on reliable methods of assay.

Adult↗
PubMed1976

Factor VIII and human platelet aggregation. I. Evience that the platelet aggregating activity of bovine factor VIII is a property of its 'carrier protein' subunit.

Bovine factor VIII is a potent inducer of aggregation of human platelets. Upon gel filtration of five-thousand-fold purified material in 0.5 M CaCl2, bovine factor VIII is separated into high and low molecular weight components; the former contains both a 'carrier protein' and platelet aggregating activity, the latter the procoagulant activity (low molecular weight factor VII, LMW-FVIII). Upon removal of Ca2+ ions, LMW-F VIII recombines with the 'carrier protein'. LMW-F VIII modifies aggregation by 'carrier protein', but not aggregation by undissociated bovine factor VII, adenosine-5'-diphosphate or adrenaline. This finding indicates that the platelet aggregating activity in indeed a property of the 'carrier protein'.

Animals↗
PubMed2006

Successful use of recombinant factor VIII devoid of von Willebrand factor during multiple teeth extractions in a patient with type 3 von Willebrand disease.

We report a 55-year-old patient with type 3 von Willebrand disease who underwent multiple tooth extractions with successful hemostatic management using recombinant factor VIII. The patient was previously misdiagnosed and treated incorrectly then at 53 years old, he was diagnosed with type 3 von Willebrand disease. As he had avoided dental treatments for two decades due to severe bleeding after dental extraction, multiple severe caries and marginal periodontitis were revealed. The patient refused the use of blood products in hemostatic management because he was afraid of blood-borne diseases and development of anti-von Willebrand factor alloantibodies. After close consultation, we therefore decided to use recombinant factor VIII. Four teeth extraction procedures were executed twice. Before extraction, bolus recombinant factor VIII (50 IU/kg) was administered intravenously followed by continuous infusion (5-10 IU/kg per h) for approximately 48 h. The factor VIII:C level increased from about 1 to 20-32% 30 min after bolus infusion. During continuous infusion (10 IU/kg/h), factor VIII:C was maintained at more than 10%. Little bleeding occurred during and after the multiple teeth extractions and during suture removal. On frequent examinations during a 1-year follow-up, neither von Willebrand factor nor factor VIII inhibitors were detected.

Blood Loss, Surgical↗
PubMed2002

Long-term persistence of anti-factor VIII antibody-secreting cells in hemophilic mice after treatment with human factor VIII.

The analysis of anti-factor VIII (FVIII) antibody-secreting cells (ASC) at different anatomic sites provides valuable information about the nature of the anti-FVIII immune response in hemophilic mice after treatment with human FVIII. An Elispot system is described that is suitable for analyzing frequencies and IgG subclasses of anti-FVIII ASC at the single-cell level. Hemophilic mice were treated with four doses of FVIII. Anti-FVIII antibodies in blood as well as anti-FVIII ASC in spleen and bone marrow were analyzed after each dose of FVIII and subsequently up to 22 weeks after termination of the FVIII treatment. Anti-FVIII ASC first appeared in the spleen where they were detectable after two intravenous doses of FVIII. Their appearance correlated with that of anti-FVIII antibodies in blood plasma. Anti-FVIII ASC in bone marrow were detectable after three doses of FVIII and were probably cells that initially formed in the spleen and subsequently migrated to the bone marrow. Whereas the frequency of anti-FVIII ASC in the spleen increased up to the fourth dose of FVIII and declined thereafter, in the bone marrow it remained constant for up to at least 22 weeks after the termination of the FVIII treatment. Titers of anti-FVIII antibodies in blood plasma increased up to the fourth dose of FVIII, then remained high constantly for 14 weeks and decreased but the antibodies were still detectable for up to at least 22 weeks after the fourth dose of FVIII. The IgG-subclass distribution of anti-FVIII ASC was similar in spleen and bone marrow and matched the subclasses of anti-FVIII antibodies in blood plasma indicating that both organs contribute to circulating antibodies in the blood.

Animals↗
PubMed1979

Development of factor VIII antibody in haemophilic monozygotic twins. European Study Group of Factor VIII Antibody.

The immunologic consequences of replacement therapy have been studied in 2 pairs of young haemophilic monozygotic twins. In both pairs one cotwin only developed a factor VIII specific antibody. This non-concordance illustrates the influence upon a specific immune response of non-genetic factors since the blood products, administered to each brother-pair, were not at all time identical nor were the regimens of transfusion therapy.

Antibody Formation↗