ristocetin and Hemophilia-A

Topics: Antibody Formation; Factor VIII; Female; Hemophilia A; Humans; Immunoglobulin G; Male; Ristocetin

Normal human plasma contains a complex of two proteins that are important in hemostasis and coagulation. The factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related protein (von Willebrand factor) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions. While the nature of their interaction and the details of the biochemical structures remain to be determined, the information now available permits a preliminary understanding of the molecular defects in hemophilia and von Willebrand's diseases.

Topics: Animals; Antibody Formation; Blood Coagulation; Blood Coagulation Factors; Calcium; Epitopes; Factor VIII; Hemophilia A; Hemostasis; Humans; Liver; Molecular Biology; Molecular Weight; Rabbits; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Antigens; Blood Coagulation Disorders; Blood Transfusion; Child; Child, Preschool; Factor V Deficiency; Factor VIII; Factor XI Deficiency; Female; Hemophilia A; Hemophilia B; Humans; Male; Pedigree; Ristocetin; Syndrome

Topics: Animals; Blood Coagulation; Blood Proteins; Carrier Proteins; Factor VIII; Female; Hemophilia A; Humans; Methods; Molecular Weight; Platelet Aggregation; Protein Conformation; Ristocetin; Time Factors; von Willebrand Diseases; von Willebrand Factor

Topics: Antigens; Bleeding Time; Blood Coagulation Tests; Blood Platelets; Blood Transfusion; Factor VIII; Female; Genetic Carrier Screening; Hemophilia A; Humans; Immunoelectrophoresis; Platelet Adhesiveness; Radioimmunoassay; Radiometry; Ristocetin; von Willebrand Diseases

Topics: Antibody Specificity; Blood Coagulation Tests; Blood Platelets; Capillary Fragility; Factor VIII; Female; Hemophilia A; Hemorrhage; Humans; Male; Menorrhagia; Plasma; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

We have examined nine patients with presumed von Willebrand's disease who present the spectrum of that disorder. Two had findings that would be accepted generally as diagnostic of von Willebrand's disease, and seven had variations of the usual pattern. The commonest variation was the combination of borderline and variable levels of coagulant Factor VIII, commensurate levels of Factor VIII-related antigen, and low levels of ristocetin-Willebrand factor.

Topics: Adolescent; Adult; Antigens; Blood Coagulation; Blood Platelets; Child; Child, Preschool; Diagnosis, Differential; Factor VIII; Female; Hemophilia A; Humans; Male; Middle Aged; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Autoantibodies; Diagnosis, Differential; Factor VIII; Hemophilia A; Humans; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Animals; Antigens; Cold Temperature; Cross Reactions; Factor VIII; Fibrinolysin; Hemophilia A; Humans; Immune Sera; Molecular Conformation; Platelet Adhesiveness; Rabbits; Ristocetin; Thrombin; von Willebrand Diseases

Topics: Adsorption; Adult; Animals; Blood Coagulation Factors; Blood Platelets; Blood Transfusion; Chromatography; Depression, Chemical; Factor VIII; Female; Glycoproteins; Hemophilia A; Homozygote; Humans; Male; Platelet Aggregation; Receptors, Drug; Ristocetin; Time Factors; von Willebrand Diseases

Von Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

Topics: Antibodies, Monoclonal; Hemophilia A; Humans; Pathologists; Ristocetin; von Willebrand Diseases; von Willebrand Factor

von Willebrand disease (VWD) is the most common inherited bleeding disorder, but variable severity and several classification types mean that diagnosis is often not straightforward. In many countries, the assays are not readily available and/or are not well standardized. The latest methods and the basis of VWD are discussed here, together with information from the international quality assessment programme (IEQAS). Factor XIII deficiency is a rare, but important bleeding disorder, which may be missed or diagnosed late. A discussion and update on this diagnosis is considered in the final section of our review.

Topics: Clinical Laboratory Techniques; Collagen; Factor XIII Deficiency; Hemagglutinins; Hemophilia A; Humans; Platelet Aggregation; Quality Control; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Severe haemophilia is a serious, haemorrhagic disorder of the plasmatic coagulation system. In this study we investigated, whether 'compensatory' activation of the platelet coagulation system occurs in this situation. Platelet function was investigated with aggregation, adhesion and flow cytometric assays. In addition, we performed clot and platelet plug formation tests and determined endogenous thrombin potentials in patients with severe haemophilia A or B; results were compared to those of healthy controls. Platelet aggregation in response to stimulation with ADP, ristocetin and epinephrine was similar in patients and controls; aggregation in response to collagen was reduced significantly in haemophiliacs. Flow cytometric analysis of P-selectin (CD 62P) and CD 63, of the conformationally changed GP IIb/IIIa with PAC 1 and of thrombospondin bound to CD 36 (GP IV) was performed at baseline and post stimulation. Baseline expression of all markers was similar in haemophiliacs and controls. After stimulation of the platelet thrombin receptors with the thrombin receptor activating peptide (TRAP) 6, the surface expression of all markers increased significantly; again, the expression was similar in haemophiliacs and controls. With thrombelastography and PFA 100 analysis, clot formation under low shear and platelet plug formation under high shear is measured. Both test results revealed a significantly reduced clot and platelet plug formation capacity in severe haemophiliacs. Our results did not reveal signs of enhanced platelet preactivation in haemophiliacs, indicating that baseline platelet reactivity in severe haemophilia remains in a neutral state, despite the severely haemorrhagic condition. As expected, both thrombin and clot formation capacities were impaired significantly in severe haemophilia. The reduced response to collagen-based platelet stimulation tests is indicative of a concomitant platelet function defect. This defect probably contributes to the intensity of bleeding events in patients with severe haemophilia.

Topics: Adenosine Diphosphate; Adolescent; Adult; Aged; Blood Coagulation; Blood Platelets; Child; Collagen; Epinephrine; Factor IX; Factor VIII; Flow Cytometry; Hemophilia A; Humans; Male; Middle Aged; Platelet Activation; Platelet Adhesiveness; Reference Values; Ristocetin; Thrombin

The action of platelet aggregation and release reaction on the tendency toward bleeding in patients with hemophilia was studied. Were examined 43 patients with hemophilia A, 14 of them with hemorrhages, 5 patients with hemophilia B, 5 patients with hemophilia A and factor VIII inhibitor and 2 patients with congenital factor VII deficit. Disturbed aggregation with ADP was found in 2 patients and with adrenalin also in 2 patients with hemophilia A without hemorrhages, in 1 patient with hemophilia A with hemorrhages and in 1 patient with hemophilia B. In the remaining patients the examined indices were normal. No data for spontaneous aggregation were found. The results of the study indicate that no connection was established between the tendency toward bleeding in the hemophiliac patients and the platelet aggregation and release reaction. No data for platelet hyperactivation by the hemorrhages were found.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Epinephrine; Factor VII Deficiency; Hemophilia A; Hemophilia B; Humans; Platelet Aggregation; Ristocetin

The authors simultaneously performed the Ivy (IBT) and the Simplate I (SBT) bleeding time in 17 volunteers with classic hemophilia A to determine whether a prolonged Simplate bleeding time was indeed indicative of impaired primary hemostasis, as has been postulated recently, or whether the technic itself accounted for the observed changes. They also assessed platelet function and Factor VIII-related activities on blood drawn that day. The SBT was prolonged in 11 patients, while the IBT was consistently normal. The platelet aggregation studies and the levels of Factor VIII-related antigen (VIII R:Ag) and ristocetin cofactor (VIII R:Rc) were normal, providing no evidence of von Willebrand's disease. The patients with a prolonged SBT were all younger than 20 years of age, bled two to three times more often than those with a normal SBT, and consumed more Factor VIII concentrate. A prolonged SBT with depressed VIII:C therefore is not indicative of von Willebrand's disease but is shared by a substantial proportion of hemophiliacs, who may be a greater risk of bleeding.

Topics: Adolescent; Adult; Age Factors; Bleeding Time; Blood Platelets; Child; Factor VIII; Hemophilia A; Hemostasis; Humans; Middle Aged; Partial Thromboplastin Time; Platelet Aggregation; Platelet Function Tests; Prospective Studies; Ristocetin

A 60-year-old Black female presented with a haemorrhagic diathesis and an acquired factor VIII/von Willebrand factor (VIII/vWf) inhibitor. This inhibitor was classified as an IgA immunoglobulin and was active not only against factor VIII coagulant (VIII:C) activity but also against plasma von Willebrand factor (vWf). The purified IgA also interacted with normal platelets to inhibit ristocetin-induced platelet aggregation (RIPA). In contrast, studies with haemophilia A plasma and platelets revealed that the inhibitor did not react significantly with these plasmas or platelets. The significant differences in the inhibition of vWf assay both of the plasma and the platelets of the haemophilia A patients suggests that part of the haemorrhagic diathesis may be related not only to the inhibition of VIII:C but also to interference with platelet function. In addition, these studies suggest that there may be significant differences in the factor VIII-related antigen (VIII R:Ag) on platelets in haemophilia A patients compared to normal.

Topics: Blood Coagulation Factors; Blood Coagulation Tests; Factor VIII; Female; Hemophilia A; Hemorrhagic Disorders; Humans; Immunoglobulin A; Middle Aged; Platelet Aggregation; Ristocetin; von Willebrand Factor

Topics: Antigens; Factor VIII; Hemophilia A; Humans; Methods; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Topics: Adult; Blood Coagulation Factors; Female; Hemophilia A; Humans; Male; Middle Aged; Platelet Count; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A monoclonal antibody to human antihemophilic factor (AHF, factor VIII) was derived from BALB/c mouse spleen cells fused with P3x63Ag8 mouse plasmacytoma cells. This antibody, harvested from culture medium or ascites fluid, reacted with purified AHF and with plasmas with normal subjects or classic hemophiliacs, as measured by enzyme-linked immunosorbent assay (ELISA), but not with plasmas from patients with severe von Willebrand's disease. The antibody possessed only IgG, heavy chains and kappa light chains. It blocked ristocetin-induced platelet agglutination and, to a lesser degree, platelet retention by glass bead columns, but it did not inhibit the procoagulant activity of AHF significantly. An amount of rabbit antiserum against AHF that provided equivalent inhibition of ristocetin-induced platelet agglutination inhibited glass bead retention much more effectively than the mouse monoclonal antibody. This difference was exaggerated in studies of the corresponding Fab fragments. These data suggest that the site or sites on the AHF complex molecule that are associated with ristocetin-induced platelet agglutination differ quantitatively or qualitatively from those associated with enhancement of platelet retention by glass beads. ELISA titers of immunoreactive AHF, using the monoclonal antibody, were closely correlated to those using rabbit antiserum against AHF in normal, hemophilic, and most von Willebrand's disease plasma.

Topics: Antibodies, Monoclonal; Blood Coagulation Factors; Factor VIII; Glass; Hemophilia A; Humans; Platelet Aggregation; Ristocetin; Spleen; von Willebrand Diseases; von Willebrand Factor

From a material of 18 obligate carriers of haemophilia A and 40 healthy females, a discriminant function was created, based on ratio of factor VIII related antigen (electroimmunoassay = EIA) to factor VIII activity (one-stage assay), factor VIII related antigen (radioimmunoassay = RIA) and number of bleeding symptoms. The standard deviation of p-values for carrier- and non-carrier state (less than 0.05) was estimated by a procedure built on the 'jack-knife' method. By combined information of pedigree- and discriminant analysis data, 43 possible carriers were classified as carriers/noncarriers with about 95% confidence. Carriers were significantly older, had more bleeding symptoms, longer APTT, lower factor VIII activity, factor VIII procoagulant antigen, and higher ratio of factor VIII related antigen (EIA) to factor VIII activity (one-stage) and to factor VIII related antigen (RIA) respectively, than classified noncarriers. Individuals with blood group A, B, AB had significantly higher levels of factor VIII related antigen (EIA) and (RIA), and ristocetin cofactor, compared with blood group O. Obligate carriers with severe haemophilia A in their families had more bleeding symptoms than corresponding group with moderate haemophilia.

Topics: ABO Blood-Group System; Aging; Analysis of Variance; Antigens; Blood Coagulation Tests; Factor VIII; Female; Genetic Carrier Screening; Hemophilia A; Humans; Male; Pedigree; Ristocetin; von Willebrand Factor

Topics: Blood Coagulation Factors; Diagnosis, Differential; Hemophilia A; Humans; Platelet Aggregation; Reagent Kits, Diagnostic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

A sensitive and reproducible method for the quantitation of ristocetin-Willebrand factor activity in canine plasma is described. This assay measures the initial velocity of aggregation of suspensions of canine washed platelets in the presence of ristocetin and ristocetin-Willebrand factor. The washed platelets are stable for 4 h and when prepared from the same donor vary little in their day-to-day response to ristocetin. The calculated mean plasma ristocetin-Willebrand factor activity in 37 normal dogs using this method is 98 +/- 26% (mean +/- SD) Ristocetin-Willebrand activity is 106 +/- 25% in dogs with severe hemophilia A and 45 +/- 13% in dogs with moderate forms of von Willebrand's disease.

Topics: Animals; Blood Coagulation Factors; Blood Platelets; Dogs; Hemophilia A; Platelet Aggregation; Ristocetin; von Willebrand Factor

Diagnosis of deficiencies of coagulation factor VIII can be difficult to establish in some cases. The use of the factor VIII-related antigen and the use of the ristocetin cofactor assays have increased the reliability of diagnosis of factor VIII deficiency in patients with hemophilia A or von Willebrand's disease, and in carriers of hemophilia A. The authors re-evaluated samples, from frozen storage, of blood from patients previously diagnosed as having von Willebrand's disease. This diagnosis was based on clinical history, family history, bleeding time, factor VIII procoagulant activity, and response to ristocetin in platelet-aggregation studies. Eleven cases were studied by the review of previously obtained data and the addition of the factor VIII-related antigen and ristocetin-cofactor assays. In two of eleven cases, the diagnosis was changed to possible hemophilia A carrier state.

Topics: Adolescent; Adult; Antigens; Blood Proteins; Child; Child, Preschool; Factor VIII; Female; Hemophilia A; Humans; Male; Methods; Middle Aged; Ristocetin; von Willebrand Diseases

Topics: Adolescent; Aged; Arginine Vasopressin; Blood Coagulation; Blood Donors; Child; Deamino Arginine Vasopressin; Factor VIII; Female; Hemophilia A; Humans; Male; Ristocetin; Tranexamic Acid; von Willebrand Diseases

Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly removed by the reticuloendothelial system.

Topics: Adult; Blood Platelets; Chemical Precipitation; Chromatography, Gel; Cold Temperature; Factor VIII; Half-Life; Hemophilia A; Humans; Immunoelectrophoresis, Two-Dimensional; Iodine Radioisotopes; Male; Molecular Weight; Protein Binding; Ristocetin

Topics: Adolescent; Adult; Aged; Blood Platelets; Blood Transfusion; Chemical Phenomena; Chemistry; Factor VIII; Female; Hemophilia A; Humans; Infant, Newborn; Macromolecular Substances; Male; Middle Aged; Platelet Aggregation; Ristocetin; von Willebrand Diseases; von Willebrand Factor

Commercially available antiserum to factor VIII was used in several tests to determine whether it might serve as a reference between research laboratories involved in investigation of the factor VIII complex and whether the antiserum might be useful in the screening of large populations of patients with hereditary disorders of factor VIII. In Ouchterlony plates, the antiserum gave a single line of identity with concentrated factor VIII, cryoprecipitate, and human plasma. The antiserum was capable of inhibiting the ristocetin response of normal platelets. Testing antigenic factor VIII by the Laurell technic with the commercial antiserum on plasmas from normal and stressed normal controls, patients with von Willebrand's disease, patients with hemophilia A, and obligate carriers of hemophilia A gave diagnostic and reproducible results.

Topics: Adult; Antigens; Factor VIII; Female; Hemophilia A; Humans; Immune Sera; Immunodiffusion; Male; Middle Aged; Physical Exertion; Platelet Aggregation; Ristocetin; von Willebrand Diseases

A patient with inherited combined deficiency of factor V and factor VIII is reported, who demonstrated normal levels of factor VIII antigen and plasma cofactor for ristocetin-induced platelet aggregation. The relationship of this condition to classical hemophilia and von Willebrand's disease is discussed. The data presented suggest that multiple loci on at least 2 chromosomes are necessary for the normal expression of factor VIII activity.

Topics: Adult; Antigens; Factor V Deficiency; Factor VIII; Hemophilia A; Humans; Male; Platelet Aggregation; Ristocetin

Antiserum specific for that part of the factor VIII complex designated ristocetin aggregation factor (VIIIRAF) was prepared by immunizing rabbits with VIIIRAF derived from the plasma of a hemophilic patient with circulating antibody to factor VIII procoagulant activity (VIIIcoag). The rabbit antiserum prevented ristocetin-induced platelet aggregation and platelet retention by glass-bead columns. Although the antiserum also inactivated VIIIcoag in normal plasma, it did not inactivate VIIIcoag which had been dissociated from VIIIRAF by chromatography in 1 M NaCl, thus establishing the antigenic specificity of these two factors. When the VIIIRAF antibody was conjugated with fluorescein isothiocyanate and used to examine platelets from normal subjects and patients with von Willebrand's disease, the normal platelets showed a granular fluorescence similar to that observed with antifibrinogen serum whole von Willebrand platelets showed no fluorescent staining. The normal platelets retained the VIIIRAF granules during 18 hours incubation and 5 washings in artificial medium while the von Willebrand platelets failed to acquire granules after 18 hours in normal plasma. When the unstained platelets from patients with von Willebrand's disease were suspended in normal plasma and then aggregated by the addition of ristocetin, the aggregates not only stained brightly for VIIRAF, but fluorescent granules could be seen on individual platelets in the aggregates. These observations suggest that ristocetin causes the binding of VIIIRAF to platelets as well as platelet-to-platelet adhesion.

Topics: Blood Platelets; Factor VIII; Fluorescent Antibody Technique; Hemophilia A; Humans; Immune Sera; Platelet Aggregation; Receptors, Antigen, B-Cell; Ristocetin; von Willebrand Diseases

Nine probands with von Willebrand's disease, and their family members, totalling 43 people, were examined. Twenty-seven had a history of bleeding; 29 had an increased factor VIII activity:factor VIII related antigen ratio; 24 had a decreased factor VIII related antigen; 23 had a prolonged bleeding time; 19 had a reduced platelet adhesiveness; 16 had a decreased factor VIII activity; and 14 had an abnormal ristocetin-induced platelet aggregation. Eight members with both normal beleeding time and normal factor VIII activity were found to have other abnormal tests: elevated ratio of factor VIII activity to factor VIII related antigen in seven; decreased factor VIII related antigen in four; and reduced platelet adhesiveness in one. Therefore, ratio of factor VIII activity to factor VIII related antigen and factor VIII related antigen are more sensitive and may be used for the detection of heterozygous carriers of von Willebrand's disease. Although patients with thrombocytopathy may have a prolonged bleeding time, decreased platelet adhesiveness and reduced platelet aggregation by ristocetin, their factor VIII activity, factor VIII related antigen and ratio of factor VIII activity to factor VIII related antigen are normal and their abnormal ristocetin test cannot be corrected by the addition of factor VIII concentrate. Hemophilic subjects and hemophilic carriers, who are deficient in factor VIII activity, usually have a normal bleeding time, normal platelet adhesiveness, and normal ristocetin test. In contrast to patients with von Willebrand's disease, their factor VIII related antigen is normal or slightly increased and their ratio of factor VIII activity to factor VIII related antigen is significantly reduced. We conclude that ratio of factor VIII activity to factor VIII related antigen and factor VIII related antigen are not only more sensitive but also more specific for the diagnosis of von Willebrand's disease.

Topics: Antigens; Blood Coagulation Tests; Blood Platelet Disorders; Diagnosis, Differential; Factor VIII; Female; Hemophilia A; Heterozygote; Humans; Male; Pedigree; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

A case of acquired von Willebrand's syndrome (vWs) is described which appeared to be due to antibodies directed against factor VIII clotting activity (FVIIIC), factor VIII-related antigen (FVIIIRAg) and von Willebrand factor. The antibodies directed against FVIIIRAg was demonstrated by the inhibitory effect of a platelet eluate on Ristocetin-induced aggregation of normal platelets. This effect was not shown by the patient's platelet-poor plasma alone, nor could it be demonstrated in platelet eluates from 13 other patients who had antibodies to FVIIIC but in whom there was no evidence of an acquired vWs.

Topics: Aged; Antibodies; Antigens; Blood Coagulation Tests; Chemical Precipitation; Cryoglobulins; Factor VIII; Female; Hemophilia A; Humans; Male; Middle Aged; Plasmapheresis; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Animals; Antibodies; Antibody Formation; Antibody Specificity; Antigen-Antibody Reactions; Cattle; Cross Reactions; Epitopes; Factor VIII; Genes; Hemophilia A; Histocompatibility Antigens; Humans; Immune Tolerance; Immunoglobulin Fragments; Immunoglobulin Heavy Chains; Immunologic Techniques; Male; Plasmapheresis; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Species Specificity; Swine; Transfusion Reaction

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

Topics: Adult; Blood Platelets; Cell Aggregation; Factor VIII; Female; Hemophilia A; Humans; Male; Middle Aged; Platelet Adhesiveness; Ristocetin; Time Factors; von Willebrand Diseases

The Factor VIII/von Willebrand factor protein was characterized in two unrelated patients with von Willebrand's disease in whom procoagulant and Factor VIII/von Willebrand factor antigen levels were normal. In both patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis. In one patient the Factor VIII/von Willebrand factor protein eluted from Sepharose 4B in a position and distribution identical to normal with normal levels of procoagulant activity and antigen. However, the partially purified Factor VIII/von Willebrand factor protein had markedly reduced von Willebrand factor activity in a ristocetin assay. In the second patient the peak of Factor VIII/von Willebrand factor protein, antigen, and procoagulant activity eluted from a Sepharose 4B column with an estimated molecular weight of approximately half that of normal. This protein had no von Willebrand factor activity. In both patients the reduced Factor VIII/von Willebrand factor protein subunit was indistinguishable from normal on polyacrylamide gel electrophoresis. These studies indicate that in some patients with von Willebrand's disease there is a qualitative defect of the Factor VII/von Willebrand factor protein; the total amount of protein, antigen, and procoagulant activity are normal while the von Willebrand factor activity is deficient.

Topics: Antibodies; Antigens; Blood Coagulation Tests; Chemical Precipitation; Chromatography, Gel; Chymotrypsin; Coagulation Protein Disorders; Counterimmunoelectrophoresis; Electrophoresis, Polyacrylamide Gel; Female; Fibrinogen; Hemophilia A; Humans; Male; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Platelet aggregation by various inductors was studied in citrated and heparinized plasma of the following groups of subjects: Normal, hemophilia A, combined factor V and factor VIII deficiency, v. Willeprand's disease and congenital afibrinognemia. The results may be summarized as follows: A-platelet aggregation in citrated plasm 1) platelet aggregation by common inductors ADP, adrenalin and collagen was normal in all groups of subjects but for the patients with congential afibrinogenemia in whom adrenalin induced aggregation was absent or markedly refuced whereas ADP and collagen gave slightly reduced or near normal aggregation curves. 2) platelet aggregation by ristocetin was normal in all groups of subjects but for v. Willebrand's disease in which it was absent. B-platelet aggregation in heparized plasma. 1) platelet aggregation by common inductors resulted to be normal in all groups of subjects except in congenital afibrinogenemia. In this latter case the pattern was still mildly defective but here was an increased aggregation as compared to citrated plasma. These findings have been interpretemmon inductors. 2) platelet aggregation by ristocetin resulted to be absent in all groups of subjects investigated. The possible mechanism of action of the inhibitory effect exercised py heparin with regard to restocetin is discussed.

Topics: Adenosine Diphosphate; Afibrinogenemia; Collagen; Epinephrine; Factor V Deficiency; Hemophilia A; Hemorrhagic Disorders; Heparin; Humans; Platelet Adhesiveness; Ristocetin; von Willebrand Diseases

Antibodies against factor VIII collected from six patients were studied for their effect on factor-VIII coagulant activity, Willebrand factor activity (WF) and factor-VIII-related antigen. The results demonstrate differences between individual antibodies; some acting primarily as anti factor VIII, others inhibiting both after VIII and WF. Differences in effect between plasma and serum indicate that the process of coagulation alters the interaction of these antibodies.

Topics: Factor VIII; Hemophilia A; Humans; Isoantibodies; Neutralization Tests; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Factor

Topics: Absorption; Animals; Antibodies; Blood Platelets; Factor VIII; Female; Fibrinogen; Fluorescent Antibody Technique; Goats; Hemophilia A; Humans; Immunoelectrophoresis; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Serotonin; Serum Albumin; von Willebrand Diseases

A bleeding diathesis is described which is phenotypically indistinguishable from hemophilia A and which has been transmitted as a dominant trait in three generations of women in a North Carolina kindred. The abnormal phenotype is characterized by clinical mildness and slightly abnormal clotting time, prothrombin consumption, and partial thromboplastin time. Bleeding time, platelet count, clot retraction, tourniquet test, and prothrombin time are normal. Concentration of factors I, II, V, VII, IX, X, and XII are normal, while factor VIII activity is reduced to 2%-5% of control values. De novo synthesis of factor VIII does not occur after transfusion; factor VIII-related antigen is normal; patients' plasmas aggregate platelets normally in the presence of ristocetin, and a typical protein pattern is seen when a chymotryptic digest of cryoprecipitate of the proband is examined by SDS-polyacrylamide gel electrophoresis. Six possible genetic explanations are entertained. Balanced X-autosomal translocation of hemophilia A heterozygotes has been excluded by cytogenetic analysis of metaphase chromosomes. Classes von Willebrand's disease (vWd) is probably excluded on the basis of the laboratory data, and extreme lyonization of hemophilia A heterozygotes on probabilistic grounds. The genetic possibilities which cannot be excluded include a previously unrecognized variant mutation at the vWd locus, a dominant mutation at the hemophilia A locus on the X chromosome, and dominant mutation at a hypothetical fourth locus involved in factor VIII synthesis and control.

Topics: Animals; Blood Coagulation Tests; Blood Transfusion; Cells, Cultured; Chromatography, Gel; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Factor VIII; Female; Genes, Dominant; Hematuria; Hemophilia A; Humans; Immune Sera; Karyotyping; Lymphocytes; Pedigree; Phenotype; Plasmapheresis; Platelet Aggregation; Rabbits; Radioimmunoassay; Ristocetin

Topics: Adenosine Diphosphate; Albumins; Aspirin; Carbon Radioisotopes; Chromatography, Gel; Dose-Response Relationship, Drug; Factor VIII; Fibrinogen; gamma-Globulins; Hemophilia A; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Serotonin; von Willebrand Diseases

Topics: Arginine; Blood Coagulation Tests; Blood Platelets; Factor VIII; Hemophilia A; Humans; Ristocetin; Vasopressins; von Willebrand Diseases

Topics: Antigens; Densitometry; Factor VIII; Hemophilia A; Humans; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; von Willebrand Diseases

Topics: Animals; Antibodies; Chemical Precipitation; Dialysis; Factor VIII; Hemophilia A; Humans; Immunoglobulin G; Injections, Subcutaneous; Methods; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Ristocetin; von Willebrand Diseases

Topics: Adenosine Diphosphate; Adult; Animals; Antigen-Antibody Reactions; Binding Sites; Blood Coagulation Tests; Collagen; Epitopes; Factor VIII; Female; Hemophilia A; Humans; Platelet Adhesiveness; Rabbits; Ristocetin; von Willebrand Diseases

Factor VIII corrects both the clotting defect in hemophilia A and an abnormality of platelet aggregation in von Willebrand's disease. These two activities of factor VIII (antihemophilic factor and von Willebrand factor) are both detected in the void volume when human plasma or cryoprecipitate is chromatographed on Bio-Gel 5M under conditions of isotonic salt concentration. In contrast, antihemophilic factor procoagulant activity is detected with proteins of lower molecular weight when the chromatography is performed with a buffer containing 0.8M NaCl. In this way, the two activities of factor VIII can be dissociated. It remains to be determined whether these components are separate molecules associated as a complex of high molecular weight in plasma or whether they are subunits of a complex macromolecule.

Topics: Animals; Blood Coagulation; Chromatography; Epitopes; Factor VIII; Hemophilia A; Humans; Isotonic Solutions; Molecular Weight; Osmolar Concentration; Platelet Adhesiveness; Rabbits; Radioimmunoassay; Ristocetin; Sodium Chloride; Thrombin; von Willebrand Diseases

The antibiotic ristocetin, in concentrations of 1.0-1.5 mg/ml, aggregated normal platelets in citrated platelet-rich plasma by a mechanism in which the release reaction played only a minor role. Platelet aggregation by ristocetin in a concentration of 1.2 mg/ml was absent or markedly decreased in 10 patients with von Willebrand's disease. Lesser degrees of abnormality were obtained with a concentration of 1.5 mg/ml. The magnitude of the defect in ristocetin-induced platelet aggregation correlated well with the degree of abnormality of the bleeding time and the levels of antihemophilic factor (AHF, VIII(AHF)) procoagulant activity. In all patients, the defect in ristocetin-induced platelet aggregation was corrected in vitro by normal plasma. Correction was also obtained with a fraction of normal cryoprecipitate that eluted in the void volume with VIII(AHF) after chromatography on a gel that excludes molecules larger than 5 x 10(6). A similar fraction, devoid of VIII(AHF) activity, obtained from patients with von Willebrand's disease had no corrective effect, but fractions obtained from patients with hemophilia were just as effective as those obtained from normal subjects. The correction activity of plasma and partially purified factor VIII was inhibited by a rabbit antibody to human factor VIII but not by a human antibody against VIII(AHF) procoagulant activity. The studies provide further evidence that patients with von Willebrand's disease are deficient in a plasma factor that is necessary for normal platelet function. The activity of this factor appears to be associated with factor VIII but is unrelated to VIII(AHF) procoagulant activity.

Topics: Adenosine; Adenosine Diphosphate; Antibodies; Blood Coagulation Tests; Blood Platelet Disorders; Blood Platelets; Carbon Radioisotopes; Chromatography; Collagen; Cyanides; Edetic Acid; Factor VIII; Female; Glucose; Hemophilia A; Heparin; Humans; In Vitro Techniques; Male; Platelet Adhesiveness; Purpura, Thrombocytopenic; Ristocetin; Serotonin; von Willebrand Diseases

In a previous paper, we showed that the abnormality of ristocetin-induced platelet aggregation in platelet-rich plasma in 10 patients with von Willebrand's disease could be corrected by a factor in normal plasma that was present in the same fractions as factor VIII procoagulant activity (antihemophilic factor, AHF, VIII(AHF)) when prepared by chromatography on Bio-Gel 5 M (Bio-Rad Laboratories, Richmond, Calif.). This observation suggests that patients with this disorder are deficient in a plasma factor, associated with the factor VIII molecule, that is necessary for normal platelet function. In the present paper, we describe, an assay for this factor, the von Willebrand factor (VIII(VWF)), based on the observation that a log-log relationship exists between the amount of ristocetin-induced aggregation of washed, normal platelets and the concentration of normal plasma present in the test system. We assayed the activity of VIII(VWF) as well as antihemophilic factor procoagulant activity (VIII(AHF)) and factor VIII antigen (VIII(AGN)) in 15 patients with von Willebrand's disease and 20 normal subjects. A highly significant correlation (r approximately 0.80) between VIII(VWF) and both VIII(AHF) was found in normal subjects and in patients with von Willebrand's disease. This finding, in addition to the observation that agarose gel chromatography fractions that have VIII(AHF) procoagulant activity also have VIII(VWF) activity, strongly suggests that the von Willebrand factor is associated with the factor VIII molecule. VIII(VWF) in normal plasma was not inhibited by human anti-VIII, and VIII(VWF) levels were normal in hemophilic plasma. Thus, the VIII(VWF) site on the factor VIII molecule appears to be different from that determining VIII(AHF). Finally, the activity of VIII(VWF) appeared to correlate better with the bleeding time than either VIII(AHF) or VIII(AGN). This suggests that VIII(VWF) assayed in this study may be the "anti-bleeding factor" that is deficient in von Willebrand's disease. These findings are consistent with a decreased synthesis of the factor VIII molecule in von Willebrand's disease and suggest the possibility of additional abnormalities of the site on the molecule that determines the activity of VIII(VWF).

Topics: Adult; Animals; Antibodies; Antigens; Blood Platelets; Chromatography; Factor VIII; Female; Hemophilia A; Humans; In Vitro Techniques; Male; Middle Aged; Platelet Adhesiveness; Rabbits; Ristocetin; von Willebrand Diseases