sepharose and von-Willebrand-Diseases

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

We developed a simple and fast method for studying the heparin binding of von Willebrand factor (vWF) in the plasma milieu. Using plasma from patients with von Willebrand disease (vWD) subtype II, we found that the heparin binding was impaired when compared with a normal plasma control. Further experiments performed with purified vWF of various multimeric composition, obtained either by gradual reduction or gel filtration, confirmed that heparin binding is dependent on the multimerization of vWF and that high molecular weight (HMW) multimers of vWF are required for normal heparin binding. After reduction of plasma vWF by 1.5 mM DTT, the vWF monomer still binds to heparin but to a lower extent. Under these conditions, no significant differences were obtained between control and patients showing that the heparin binding domain located on the vWF subunit is not altered in the subtypes IIA, IIB and IIC studied.

Topics: Antibodies, Monoclonal; Binding Sites; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Agar Gel; Heparin; Humans; Plasma; Protein Binding; Protein Conformation; Sensitivity and Specificity; Sepharose; von Willebrand Diseases; von Willebrand Factor