sepharose and Hemophilia-A

Hemophilia A is routinely treated by administration of exogenous coagulation factor VIII (FVIII). As safety and efficacy of FVIII products have improved over the years, development of FVIII-neutralizing antibodies (FVIII inhibitors) has emerged as the most serious complication. The new human cell line-derived recombinant human FVIII (human-cl rhFVIII) is the first recombinant FVIII product produced in a human cell line without additive animal proteins, with a goal of minimizing the risk of inhibitor development.. Biochemical analyzes of purity, molecular and functional attributes of the novel human-cl rhFVIII were undertaken for product characterization.. Human-cl rhFVIII was shown to be highly pure, with host-cell protein and DNA traces comparable to, or lower than, currently marketed recombinant FVIII (rFVIII) products. Human-cl rhFVIII was shown to have high specific FVIII activity and characteristics similar to full-length rFVIII products. Furthermore, no significant discrepancy between one-stage and chromogenic assay results were observed for human-cl rhFVIII, indicating potency ratios of these assays comparable to the full-length rFVIII products. In functional tests, human-cl rhFVIII exhibited physiological thrombin generation and a normal rate of inactivation by activated protein C. Importantly, human-cl rhFVIII displayed higher binding capacity with von Willebrand factor than comparator products, thus minimizing circulating unbound FVIII and further reducing the potential risk of inhibitor development.

Topics: Blotting, Western; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Factor VIII; Hemophilia A; Humans; Protein C; Recombinant Proteins; Sepharose; Surface Plasmon Resonance; Thrombin; von Willebrand Factor

Extracorporeal immunoadsorption of factor VIII (FVIII) antibodies using Sepharose matrix columns coupled with staphylococcal Protein-A was reported two decades ago. The efficiency of this technique for removing FVIII antibodies of the IgG subtypes was clearly demonstrated. The recent widespread use of a variety of apheresis techniques for the management of a multitude of haematological and oncological conditions has made this technology more accessible and affordable. For the treatment of patients with FVIII inhibitors, the use of porcine FVIII makes it possible to control haemostasis with a therapeutic product for which in vitro testing can help predict the in vivo efficacy. By lowering the level of FVIII inhibitors, immunoadsorption can make the use of pFVIII concentrate possible in situations otherwise untreatable with FVIII preparations. Moreover, lowering the level of FVIII inhibitors by immunoadsorption allows adequate haemostasis to be achieved with much lower doses of FVIII leading to significant saving. Our preliminary data suggest that immunoadsorption combined with the use of pFVIII should be considered early in the treatment plan for controlling haemostasis in patients with FVIII inhibitors.

Topics: Adult; Animals; Autoantibodies; Factor VIII; Female; Hemophilia A; Humans; Immunosorbent Techniques; Immunosuppressive Agents; Isoantibodies; Male; Middle Aged; Pregnancy; Pregnancy Complications, Hematologic; Sepharose; Swine

Diagnosis of von Willebrand disease Type 2N (vWD 2N), which mimics hemophilia A and its carrier state, is important for accurate genetic counseling and appropriate therapy. To make testing for the disorder more clinically applicable, we developed a simplified method for measurement of factor VIII (FVIII) binding to von Willebrand factor (vWF) using commercially available reagents and standard clinical assays. FVIII binding to vWF was measured by capture of patient vWF by polyclonal antibodies on cyanogen bromide-activated Sepharose beads, reaction with recombinant FVIII, and assay of unbound FVIII by clotting methods. Unbound vWF was measured in patient plasma after capture by the Laurell method. The ratio of bound FVIII/bound vWF was normal in hemophilia A, vWD Type 1, and vWD Type 3 patients, and abnormal in 5 subjects from two families, all of whom had vWD 2N mutations. Patient 1, with FVIII 8 U/dl, vWF: Ag 61 U/dl, vWF:RC 74 U/dl, and FVIII binding nil, was homozygous for the Arg91 Gln mutation. She was followed during pregnancy and delivered an unaffected heterozygous son. Patient 2 had FVIII 8 U/dl, vWF:Ag 73 U/dl, and vWF:RC 71 U/dl, and very low FVIII binding. She was heterozygous for Arg91Gln, as were her mother and sister; no second vWD 2N mutation was found. Her brother, with FVIII 14 U/dl, vWF:Ag 113 U/dl, and vWF:RC 72 U/dl, has no evidence of vWD 2N. With an X-linked inheritance pattern of bleeding tendency, this family is the first reported with combined hemophilia A and vWD 2N.

Topics: Adult; Antibodies; Blood Coagulation Tests; Child; Exons; Factor VIII; Female; Hemophilia A; Heterozygote; Homozygote; Humans; Male; Mutation; Pedigree; Pregnancy; Protein Binding; Sepharose; von Willebrand Diseases; von Willebrand Factor

A new approach for removing the anti-factor VIII antibodies in hemophilic patients by immunoadsorption is proposed. The method is based on the fact that the anti-factor VIII antibodies were predominantly of the IgG4 subclass; anti-human IgG4 antibodies were covalently linked to agarose and large amounts of anti-factor VIII antibodies can be eliminated. A study of 21 blood samples from hemophilic patients with anti-factor VIII antibodies allows us to confirm the large predominance of IgG4 in the anti-factor VIII population. In some samples, the presence of IgG3 related anti-VIII:C was checked by adsorption on an anti-IgG3 column. In a majority of cases, after IgG4 (or IgG4 + IgG3) immunoadsorption, the substitution therapy becomes possible or easier.

Topics: Antibody Specificity; Autoantibodies; Factor VIII; Hemophilia A; Humans; Immunoglobulin G; Immunosorbent Techniques; Protein Binding; Sepharose

The agarose gel method for the detection and quantitation of Factor VIII inhibitors was investigated and compared with the Bethesda method. The agarose gel method was standardized for this study by modifications of the original method of Bird. (Bird P: Coagulation in an agarose gel and its application to the detection and measurement of factor VIII antibodies. Br J Haematol 1975; 29:329-340) Precision studies on values obtained by the agarose gel method indicated it is a reproducible assay. Random error was evident in both methods, but variance appeared to be greater in the Bethesda method for most of the samples evaluated. Comparison of the two methods with split patient samples indicated that proportional error is present in the agarose gel method, and that standardization of the method requires additional study. The agarose gel assay detected low levels of inhibitor (down to 3.4 Bethesda units) and all positive samples were identified. A screening procedure modification of the agarose gel method provided the sensitivity to detect Factor VIII inhibitor levels of less than 1.0 Bethesda units. The results indicated that the agarose gel method is a feasible alternative to the Bethesda method for both quantitative and qualitative determination of Factor VIII inhibitors. Furthermore, since reproducibility is better, it may be useful in detecting the "true" fluctuations of inhibitor titers in a given patient.

Topics: Blood Coagulation; Factor VIII; Gels; Hemophilia A; Humans; Methods; Partial Thromboplastin Time; Prothrombin Time; Sepharose; Time Factors

Plasmas from 14 patients with factor VIII inhibitors, 10 haemophiliacs and four non-haemophiliacs, were assayed by both the agarose gel and Bethesda methods. Good correlation was observed in 34 samples from 13 patients, but there was poor correlation in three samples from a single haemophilic patient. The sensitivity of the method was increased by diluting normal platelet rich plasma (PRP) with congenital factor VIII deficient plasma. With this modification, as little as 0.4 of a Bethesda unit (Bu) could be measured accurately. The agarose method is easier to perform and requires much less technician time than the Bethesda assay (Ba). Inhibitory activity can be measured even in the presence of large amounts of transfused factor VIII or factor IX concentrates. To study the effect of factor IX concentrates on the inhibitor, the method was modified by incorporating into the gels plasmas specifically deficient in either factor VII, IX or X. Our data suggest that factor VII is probably responsible for the 'bypassing' activity of factor IX concentrates.

Topics: Factor IX; Factor VIII; Hemophilia A; Humans; Immunodiffusion; Reference Values; Sepharose

Antibodies against factor-VIII coagulant activity can appear in haemophilic patients and, although infrequently, can affect individuals not suffering from Haemophilia A. The Oxford and Bethesda methods are presently the most commonly used techniques for measuring these antibodies. Both methods are time-consuming and not suitable for the screening of large risk groups. An appropriate method for screening these coagulation inhibitors is that described by P. Bird in 1975. It is based on inhibition of the coagulation produced when plasma samples containing inhibitors diffuse in agarose gels mixed with normal platelet rich plasma (PRP). However, this technique is highly dependent on the variability derived from the use of PRP (amount of coagulant factor-VIII, number of platelets, etc.). In an attempt to avoid these disadvantages, Bird's method has been modified by using standardized commercial reagents (lyophilized plasma with 100% factor-VIII coagulant activity, purified fibrinogen, and platelet Factor 3) instead of PRP. The sensitivity reaches 0.8 Bethesda units and the correlation with the Bethesda method is r = 0.964, p less than 0.001. This newly developed method is as simple as Bird's, and appears to be at least, as accurate and reproducible as the Bethesda method.

Topics: Blood Coagulation Tests; Factor VIII; Gels; Hemophilia A; Humans; Isoantibodies; Reference Standards; Sepharose

Topics: Adolescent; Adult; Aged; Antibodies; Child; Chromatography, Affinity; Complement System Proteins; Factor IX; Factor VIII; Hemophilia A; Humans; Immunoglobulin G; Male; Plasma Exchange; Plasmapheresis; Sepharose

Plasma samples from eighteen patients (9 hemophilic, 9 nonhemophilic) with antibodies to Factor VIII were purified by protein A gel chromatography. This procedure separates the IgG3 from the IgG1, IgG2, and IgG4 subclasses. The partially purified anti--Factor VIII fractions were typed for their heavy and light chain composition by immunoneutralization assay. Residual anti--Factor VIII activity was revealed by inhibition of fibrin formation in agarose gel plates. The combination of these two techniques permitted the study of a relatively large number of partially purified samples, at several dilutions of each, against each anti-heavy or anti-light chain antiserum obtained from various sources. A high degree of subclass and light chain restriction was found in the antibodies from the hemophilic patients, eight were predominantly of IgG4 kappa type, the ninth was IgG4 but of mixed light chains. In contrast, a heterogeneous distribution was found in the ten nonhemophilic patients: two IgG4 kappa, one IgG4 lambda, one IgG4 with both kappa and lambda, two a mixture of IgG1 and IgG4 with only kappa light chains, and three a mixture of IgG1 and IgG4 as well as both light chains.

Topics: Antibodies, Anti-Idiotypic; Chromatography, Affinity; Factor VIII; Hemophilia A; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Neutralization Tests; Sepharose; Staphylococcal Protein A

Topics: Antibodies; Factor VIII; Gels; Hemophilia A; Humans; Male; Polysaccharides; Sepharose

Topics: Alkylation; Amino Acids; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Factor VIII; Hemophilia A; Humans; Immunoelectrophoresis; Isoelectric Focusing; Male; Oxidation-Reduction; Peptides; Plasmapheresis; Sepharose; Sodium Dodecyl Sulfate

Evidence has been presented that fibrin formation may be detected directly in agarose gels containing citrated plasma when they are treated with thrombin or calcium chloride. A new assay is described for haemophilic factor-VIII antibody based on inhibition of fibrin formation within an agarose gel containing normal plasma.

Topics: Antibodies; Antibody Formation; Blood Coagulation Tests; Calcium Chloride; Factor VIII; Fibrin; Gels; Hemophilia A; Humans; Polysaccharides; Sepharose; Time Factors

A method for detecting factor-VIII clotting activity in agarose is described. It is based on factor VIII promoting coagulation in a mixture of haemophilic plasma in agarose which is detected by a change in opacity. When this test was used to detect factor-VIII clotting activity in a one-dimensional Laurell electroimmunoassay for factor VIII-related antigen all the factor-VIII activity was found in the same position as the factor VIII-related antigen immunoprecipitate. Factor-VIII clotting activity did not appear to be simply trapped in this immunoprecipitate and therefore it has been concluded that the molecule containing factor-VIII clotting activity carries factor VIII-related antigen determinants.

Topics: Animals; Antigens; Binding Sites, Antibody; Blood Coagulation; Factor VIII; Hemophilia A; Humans; Immunoelectrophoresis; Rabbits; Sepharose