sodium-dodecyl-sulfate and von-Willebrand-Diseases

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; Cats; Cattle; Dog Diseases; Dogs; Electrophoresis, Agar Gel; Goats; Horses; Immunoenzyme Techniques; Macromolecular Substances; Reference Values; Sodium Dodecyl Sulfate; Swine; von Willebrand Diseases; von Willebrand Factor

We have identified a patient with von Willebrand's disease (vWD) resembling type IIB vWD, with increased ristocetin induced platelet aggregation (RIPA), the absence of the large multimers of von Willebrand factor (vWF) in plasma, and the presence of the large multimers in platelets in whom a family study indicated a probable double heterozygous inheritance pattern. The propositus was a 12-year-old boy with frequent epistaxis and bruising. Abnormal hemostatic findings included a prolonged bleeding time (BT), decreased levels of factor VIII coagulant activity (VIIIC), von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (RCof), and an increased RIPA. In the presence of ristocetin, binding of the patient's plasma vWF to normal platelets was increased but binding of normal vWF to his platelets was normal. SDS-agarose gel (1.5%) electrophoresis revealed that plasma vWF lacked the large multimers, and 3.0% gel electrophoresis revealed that the multimers had a 5-band pattern similar to normal. The above findings were consistent with type IIB vWD, but 1-deamino[8-D-arginine]-vasopressin (DDAVP) infusion resulted in a shortened BT and the transient appearance of large multimers without a decrease in the platelet count. Family studies revealed that his mother has mild bleeding symptoms, decreased VIIIC, vWF:Ag, and RCof levels and normal to slightly reduced RIPA with a multimer pattern consistent with type I vWD. In contrast, the father, sister, and paternal grandfather were asymptomatic, with a slightly decreased VIIIC level but a normal BT and vWF:Ag and RCof levels. Their RIPA and vWF binding to normal platelets were increased, but unlike the propositus their plasma contained large multimers. We concluded that the propositus is a type IIB-like variant differing from previously reported IIB variants in two ways: 1) his response to DDAVP and 2) a possible double heterozygous mode of inheritance rather than the usual dominant route.

Topics: Blood Platelets; Child; Deamino Arginine Vasopressin; Electrophoresis, Agar Gel; Heterozygote; Humans; Infusions, Intravenous; Male; Pedigree; Platelet Aggregation; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases; von Willebrand Factor

In Type I von Willebrand disease, the whole series of von Willebrand factor (vWF) multimers is present in plasma, but all are decreased in quantity. No structural abnormality of individual multimers has been demonstrated so far in these patients. We now describe five individuals, from two unrelated families, who had this form of the disease and in whom the complex banding pattern of each vWF multimer was markedly abnormal. Inheritance was autosomal dominant and the clinical expression was mild. A bleeding history was elicited in three of the patients and included recurrent epistaxis, menometrorrhagia, and bleeding following tooth extraction. Replacement therapy had never been required. Although vWF levels in plasma were within the normal range in all of them, the ristocetin cofactor activity was decreased in four, and the bleeding time was prolonged in three. Analysis of vWF multimeric structure by agarose gel electrophoresis, including a newly developed high-resolution technique, demonstrated that the main band of each multimer was present, but a second, well-defined band always seen in normal individuals was missing in the patients. Two additional bands had altered mobility and were less well defined than in normal subjects, and a fifth, less intense band was also undetectable in the patients. Treatment with 1-deamino-8-D-arginine vasopressin (DDAVP) was assessed in two patients. It caused the circulating levels of vWF to increase and correct the bleeding time, but did not alter the structural abnormality. This study describes, therefore, a new variant form of Type I von Willebrand disease with aberrant structure of individual repeating multimers and an associated functional abnormality of vWF. In keeping with previously accepted terminology, the designation of Type IC von Willebrand disease has been adopted for this new variant.

Topics: Adult; Blood Coagulation Tests; Densitometry; Electrophoresis, Agar Gel; Epistaxis; Female; Humans; Lasers; Pedigree; Protein Conformation; Sodium Dodecyl Sulfate; von Willebrand Diseases; von Willebrand Factor

We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D-arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.

Topics: Deamino Arginine Vasopressin; Electrophoresis, Agar Gel; Glycoproteins; Humans; Membrane Proteins; Platelet Aggregation; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases; von Willebrand Factor

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

The purified factor VIII-related protein we have previously characterized from normal cryoprecipitate possesses both procoagulant activity and vWf activity. We have attempted to isolate and characterize this protein from three patients with severe vWd. This protein is absent or markedly diminished in amount in these vWd patients, as judged by gel filtration, polyacrylamide-gel electrophoresis, and immunoprecipitation assays. Likewise, the procoagulant and vWf activities are deficient. As vWf activity is one of the major biologic functions of either the normal or hemophilic factor VIII-related protein, the purified protein should be designated the f VIII/vWf protein.

Topics: Animals; Antigens; Blood Coagulation Tests; Blood Proteins; Chromatography, Gel; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Factor VIII; Female; Humans; Immune Sera; Immunoelectrophoresis; Male; Platelet Aggregation; Rabbits; Ristocetin; Sodium Dodecyl Sulfate; von Willebrand Diseases

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