sodium-dodecyl-sulfate and Hemophilia-A

A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

Topics: Aminocaproates; Anticoagulants; Blood Coagulation Tests; Calcium; Carbohydrates; Cellulose; Chromatography, Gel; Citrates; Electrophoresis, Polyacrylamide Gel; Factor VIII; Fibrinolysin; Glycine; Hemophilia A; Immunoelectrophoresis; Plasminogen; Sodium Dodecyl Sulfate; Thrombin; Trypsin; von Willebrand Diseases

Topics: Animals; Anticoagulants; Binding Sites; Blood Coagulation; Blood Platelets; Carbohydrates; Chromatography; Disulfides; Electrophoresis, Polyacrylamide Gel; Factor VIII; Glycoproteins; Goats; Hemophilia A; Humans; Immune Sera; Macromolecular Substances; Male; Molecular Weight; Platelet Aggregation; Precipitin Tests; Rabbits; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases

A family is described in which a syndrome resembling moderately severe classic hemophilia was apparently inherited as an X chromosome-linked trait. In two affected individuals, the titer of functional antihemophilic factor varied dramatically from time to time, while the conversion of prothrombin to thrombin was impaired in no apparent relationship to AHF functional activity. A transfusion of 200 ml of fresh-frozen plasma did not correct the serum prothrombin times in either patient. In vitro, the additions of 10% of normal plasma or serum or washed plain or frozen platelets also did not normalize the serum prothrombin times. No inhibitor could be demonstrated in the blood of either patient. In one patient, RH, dissipation of infused cryoprecipitated AHF was abnormally slow, and, after an intensive course of transfusion of cryoprecipitate and whole blood, the titer of functional AHF remained at normal levels for at least 1 wk. The plasma of RH inhibited a human antibody against AHF in proportion to its titer of functional AHF (i.e., the defect was CRM-) despite the presence of relatively greater amounts of antigenic material recognized by heterologous antiserum. No qualitative abnormality of the AHF-like material in RH's plasma was identified. Inheritance of the abnormality appears superficially to be X chromosome-linked; on this assumption, three of four obligate carriers of the disorder were recognized by the presence of excess amounts of AHF-like antigens relative to AHF functional activity. This coagulation disorder has been designated Heckathorn's disease and may presage the discovery of other examples of hemophilia-related syndromes.

Topics: Adolescent; Adult; Antibodies; Antigens; Arthroplasty; Binding, Competitive; Blood Coagulation Tests; Blood Transfusion; Child; Child, Preschool; Chromatography, Gel; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Factor VIII; Female; Fibrinogen; Hemophilia A; Humans; Immunoelectrophoresis; Male; Middle Aged; Pedigree; Platelet Aggregation; Prothrombin Time; Sodium Dodecyl Sulfate

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

Normal human antihemophilic factor (AHF, factor VIII) and the protein antigenically related to it in hemophilic plasma both appeared in the void volume of columns of agarose (Sepharose 4B) during purification of these agents. On ultracentrifugation upon sucrose gradients, both agents had sedimentation characteristics similar to those of an S30 marker. After reduction, the polypeptide chains of purified normal AHF and of the nonfunctional agent from hemophilic patients had an apparent molecular weight of 200,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These observations suggest that AHF, purified as described, exists as a large molecule with subunits of molecular weight of approximately 200,000. Antisera to normal AHF and the nonfunctional agent from hemophilic plasma appeared to be directed against antigens of similar electrophoretic mobility and precipitating characteristics, present in normal and hemophilic plasma but deficient in severe von Willebrand's disease plasma. Both antisera neutralized the AHF clot-promoting activity present in normal plasma, and this property was removed by absorption of the antisera with concentrates of normal or hemophilic plasma but to a greatly reduced extent by concentrates of von Willebrand's disease plasma. These findings suggest that the antigen detected in normal plasma by the antisera appears on a molecule participating in the AHF clot-promoting reaction.

Topics: Animals; Antigens; Blood Coagulation; Centrifugation, Density Gradient; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Factor VIII; Hemophilia A; Humans; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Molecular Weight; Rabbits; Sodium Dodecyl Sulfate; Ultracentrifugation; von Willebrand Diseases

Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by beta-mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 h. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.

Topics: Amino Acids; Blood Coagulation Tests; Chromatography, DEAE-Cellulose; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Factor VIII; Hemophilia A; Humans; Immune Sera; Immunodiffusion; Isoelectric Focusing; Mercaptoethanol; Molecular Weight; Sodium Dodecyl Sulfate; Thrombin; Thromboplastin; Urea

Topics: Amino Acids; Animals; Binding Sites, Antibody; Blood Coagulation Tests; Carbohydrates; Cattle; Chromatography, Gel; Dogs; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Factor VII; Factor VIII; Freezing; Hemophilia A; Humans; Immunoelectrophoresis; Molecular Weight; Protein Denaturation; Rabbits; Sodium Dodecyl Sulfate; Thrombin; Ultracentrifugation