In vivo dissociation of factor VII (AHF) activity and factor VII-related antigen in von Willebrand's disease.

Using monospecific rabbit antihuman factor VIII antiserum, we have examined the amounts of factor VIII-related antigen and compared these to the levels of factor VIII procoagulant activity in normal subjects and patients with von Willebrand's disease. We have observed that even without transfusion all nine probands with von Willebrand's disease and 20 of their 34 relatives possessed a significantly elevated factor VIII activity/factor VIII-related antigen ratio when compared to that of 55 normal subjects. It is suggested that an elevated factor VIII activity/factor VIII-related antigen ratio may be used for detection of the carriers of von Willebrand's disease.     

Binding of Low-Molecular-Weight Canine Factor VIII Coagulant from von Willebrand Plasma to Canine Factor VIII-Related Antigen

S ummary . Plasmas having no detectable factor VIII-related antigen but moderate factor VIII coagulant were obtained from two unrelated dogs homozygous for von Willebrand's disease and with the severe clinical expression of the disease. When these plasmas were gel-filtered in a buffer at physiologic ionic strength, the factor VIII coagulant eluted in the bed volume as a single well-defined peak. Addition of protease inhibitors, including diisopropylfluorophosphate, did not change the elution pattern. Each plasma was then combined individually with plasmas from six different mutants of canine haemophilia, all of which had normal factor VIII-related antigen but no detectable factor VIII coagulant. The factor VIII coagulant elution profile of these combined plasma resembled that of normal canine plasma. Slightly over half of the recovered factor VIII coagulant coeluted in the void volume with the factor VIII-related antigen; the rest eluted as a second, distinct peak of lower molecular weight. These results demonstrated that part of the factor VIII coagulant of the von Willebrand plasmas had bound to the factor VIII-related antigen of the haemophilic plasmas. This finding supports the theory that factor VIII exists as a macromolecular complex of nonidentical components in normal citrated plasma.     

The Detection of Factor-VIII-like Antigen in Haemophilic Carriers and in Patients with Raised Levels of Biologically Active Factor VIII

S ummary . A rabbit antibody has been prepared against therapcutic factor VIII of intermediate purity. Using this antibody in quantitative immunoelectrophoresis, the results of Zimmerman et al (1971a, b) have been confirmed. Female carriers of haemophilia were classified with greater certainty than by the use of bioassay alone. The factor-VIII-like antigen was present in all haemophilic patients tested, and was reduced or absent in patients with von Willebrand's disease. The antigen was increased in all cases following physical exercise, the infusion of adrenaline and following major gynaecological surgery. Raised values of factor-VIII-like antigen correlated well with increase in biological activity of factor VIII. The method has been applied to whole plasma and the antigen may be quantitated directly without the necessity for initial ethanol fractionation of plasma.     

Simplified immunoradiometric assay for factor VIII coagulant antigen

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.     

Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immuno-depletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain. The immunodepleted plasma contained less than 0.002 u/ml factor VIII coagulation activity (VIII:C) less than 0.0001 u/ml von Willebrand factor antigen and 1-2 g/l fibrinogen, while the levels of other clotting factors were unchanged. This immunodepleted plasma was compared with commercial factor VIII deficient plasma obtained from a severe haemophilia A patient as substrate in the one-stage factor VIII assay. Plasmas obtained from 20 normal subjects and 28 patients with von Willebrand's disease or haemophilia A were assayed for VIII:C using the two substrates. The results were very highly correlated (r = 0.96). The columns have high capacity and can be regenerated at least 10 times. Large-scale production of a substrate for factor VIII assays free of virus contamination is now feasible.     

An immunoradiometric assay for human factor VIII/von Willebrand factor (VIII:vWF) using a monoclonal antibody that defines a functional epitope

A murine monoclonal antibody has been produced (RFF-VIII:R/2) that binds specifically to human factor VIII-related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin-induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF-VIII:R/2 can be used in a one-stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrand's disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally-defined epitope and VIII:vWF levels measured by ristocetin-induced aggregation of washed platelets. This correlation was maintained in those patients with the 'variant' types of vWD who exhibit highly disparate VIII:vWF and VIII:RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF-VIII:R/2 offer a simplified, reproducible means of detecting functionally-active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.     

Clinical application of the chromogenic assay of factor VIII in haemophilia A, and different variants of von Willebrand's disease

A chromogenic substrate kit for determination of factor VIII activity (COATEST Factor VIII) was compared to a one-stage clotting assay and the correlation was evaluated in different genetic variants of mild and moderate haemophilia A, in severe haemophilia A and in all known variants of von Willebrand's disease. In all these cases a high correlation between the two methods was obtained. A good correlation was also obtained after intranasal administration of DDAVP (1-desamino-8-D-arginine vasopressin) to patients with von Willebrand's disease. The chromogenic substrate method was performed using a microtray technique.     

Diagnosis of von Willebrand's disease. A comparative study of diagnostic tests on nine families with von Willebrand's disease and its differential diagnosis from hemophilia and thrombocytopathy.

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.     

''Autosomal Haemophilia'': A Variant of von Willebrand''s disease

S ummary . A patient representing the family with autosomal haemophilia A described elsewhere (Hensen et al , 1965) shows 'de novo synthesis' of factor VIII after transfusion with normal and haemophilic cryoprecipitate. Several patients in the family have very low factor VIII-related antigen levels. These findings are consistent with a diagnosis of von Willebrand's disease but the normal or nearly normal bleeding times and the similarity in factor-VIII activity levels in all the members of this family are unusual in this condition.
Thus far, three loci involved in factor-VIII synthesis have been postulated. The locus most frequently occupied by a mutant gene is on the X chromosome and results in haemophilia A, a trait long known to be sex-linked (for instance in ancient Jewish and Arabian literature; see review by Ikkala, 1960). The effects of mutation in an autosomal locus were first described by von Willebrand (1926, 1931). A third locus involved in factor-VIII synthesis was postulated by Hensen et al (1965). The effects of this mutant gene have been particularly difficult to study because the features that led to postulate its existence have been reported in only one family. This report deals with the evidence that this condition, originally described as 'autosomal haemophilia', is only a variant of von Willebrand's disease.     

Factor VIII:C and VIII:CAg Response in Patients with Haemophilia A and von Willebrand's Disease after Administration of Different Factor VIII Concentrates or Plasma

S ummary . Factor VIII procoagulant activity (VIII:C) and factor VIII procoagulant antigen (VIII:CAg) were studied in seven patients with haemophilia A after administration of three different factor VIII concentrates or plasma. The in vivo recovery of VIII:CAg was less than that of VIII:C and the disappearance rate of VIII:CAg was much higher either when concentrates or plasma were given. The half-life of VIII:C was thus about 12 h but of VIII:CAg only about 3 h or less. Six patients with von Willebrand's disease were studied after administration of AHF- Kabi. In contrast to haemophilia A the discrepancy between VIII:C and VIII:CAg disappearance rates was not present in von Willebrand's disease, since both VIII:C and VIII:CAg showed a typical progressive increase. We conclude that factor VIII:C given to haemophilia patients does not behave like native VIII:C, not even when fresh plasma is used. Patients with von Willebrand's disease are capable of forming a normal VIII:C when appropriately stimulated.     

Use of a Simple Visual Assay of Willebrand Factor for Diagnosis and Carrier Identification

A visual assay of factor VIII-related Willebrand factor (VIIIR:WF) is described which utilizes formaldehyde-fixed platelets, end points being read in microflocculation tiles. Four dilutions of a sample can be assessed simultaneously, and the correlation with aggregometric assays is high (r = 0.91). Measurement error is 8.0% for a single assay in triplicate and less than 5% if an assay is repeated three times. The method has been used for 2 years by the coagulation genetics group at Chapel Hill for diagnosing subjects with von Willebrand's disease and assigning genotypes to members of families transmitting this disorder. Its utility in classifying known carriers of haemophilia A has also been examined, both in conjunction with assays of VIII:C and in a three-way test with assays of VIII:C and VIIIR:Ag. As predicted by the Lyon hypothesis, the rate of false negative diagnosis was higher than false positive diagnosis, but the overall rate of misclassification on single plasma samples was 7/51 = 13.7%. The error rate was the same whether discrimination was based upon assays of VIII:C vs. VIIIR:Ag, VIII:C vs. VIIIR:WF, or VIII:C vs. VIIIR:Ag vs VIIIR:WF, the same individuals being misclassified by each method. The observed rate of misclassification was well within the rates reported by others and very similar to our previous experience. We have concluded that this method of assaying VIIIR:WF is highly useful for diagnosing vWd, detecting inhibitors to VIIIR:WF, and examining large numbers of column fractions. It is a useful supplement, although it cannot yet substitute for, assays of VIIIR:Ag in detecting carriers of haemophilia A.     

The combined use of monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haemophilic plasmas tested, VIII:Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII:C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII:Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF:Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII:C/vWF:Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII:Ag and vWF:Ag.     

Von Willebrand disease--type 2N (case report)

Isolated reduced coagulation activity of FVIII may be a manifestation of haemophilia A, carriership of haemophilia A, haemophilia A in a woman, acquired haemophilia A and type 2N of von Willebrand's disease. The authors were concerned with the cause of isolated reduction of the coagulation activity of factor VIII (19 IU/dl) in a 40-year-old woman with a history of excessive haemorrhage of the type of mild haemophilia A with a negative family history. The personal history, family history and laboratory examination suggested type (variant) 2N of von Willebrand's disease. For indirect evidence the authors used a therapeutic study where they investigated the effect of administration of a concentrate of coagulation factors VIII/von Willebrand's factor (1/2), 28 IU factor VIII/kg body weight, on the coagulation activity of factor VIII. They recorded a half-life prolonged to 53 hours as compared with controls where the half-life was less than 12 hours. The therapeutic study confirmed sufficient coagulation activity of factor VIII, the utilization of which improved as a result of administration of von Willebrand's factor. This investigation confirmed indirectly as the cause of reduced coagulation activity of factor VIII in the examined patient the assumed type (variant) 2N of von Willebrand's disease.     

Evaluation of an automated screening assay for von Willebrand disease type 2N

Evaluating the factor VIII (FVIII) binding activity of von Willebrand factor (VWF) is an important step in the diagnostic work-up of families affected by apparent mild haemophilia A. In von Willebrand's disease (VWD) type 2N (Normandy), mutations at the N-terminal end of the mature VWF subunit gene prevent the binding of FVIII. Individuals heterozygous for type 2N VWD are generally asymptomatic. Homozygotes and compound heterozygotes present with a clinical picture which mimics haemophilia A, with a markedly reduced FVIII : C activity and VWF within the normal range, but instead of exhibiting X-linked inheritance they show an autosomal recessive inheritance pattern. The distinction between haemophilia A and VWD type 2N has important implications for therapy and genetic counselling. We present a highly specific enzyme-linked immunosorbent assay screening method for the Normandy variant, which measures VWF : FVIII binding activity in parallel with VWF antigen, using monoclonal capture and detection antibodies. The assay is fully automated using a robotic microtitre plate processor, requiring minimal user intervention and providing the capacity to screen large numbers of patients.     

A new variant of dominant type II von Willebrand's disease with aberrant multimeric pattern of factor VIII-related antigen (type IID)

A new type II variant form of von Willebrand's disease has been recognized in a mother and daughter who have bleeding manifestations typical of von Willebrand's disease. Laboratory findings include consistently prolonged bleeding times, with normal levels of factor VIII procoagulant and antigen, but decreased ristocetin cofactor activity. Electrophoresis in SDS 1.5% agarose gel and reaction with 125I-labeled anti-factor VIII-related antigen rabbit IgG, followed by autoradiography, revealed that both plasma and platelets lack the large multimers of factor VIII-related antigen. In 2.5% gel, the propositus plasma lacked the normal "triplet" pattern. In 3.0% gel, a 5-band pattern was observed in normal, type IIA, and type IIB plasma, whereas type IIC plasma revealed a 2-band pattern. The patient's plasma revealed a 4-band pattern distinctly different from normal or other type II variants. We suggest that this new variant be labeled type IID, until a more appropriate nomenclature is developed.     

Macromolecular factor VIII complex: functional and structural heterogeneity observed in von Willebrand swine with transfusion.

The physiologic activities concerned with hemostasis and associated with the Factor VIII macromolecular complex were investigated in swine with von Willebrand's disease after infusion of cryoprecipitate, a lyophilized Factor VIII concentrate, or porcine serum. Immediately after each infusion the various activities antihemophilic factor, von Willebrand platelet aggregating factor, and Factor VIII-related antigen, were elevated in approximate proportion to dose and the bleeding time was shortened.There was a late secondary rise in antihemophilic factor. During the period after infusion, there was a differential fall-off of the various activities, with the bleeding time effect lost first, followed by the von Willebrand platelet aggregating factor and then by the Factor VIII-related antigen. The plasma from swine with von Willebrand's disease late after infusion contained high levels of antihemophilic factor without other detectable activities of the complex. Antihemophilic factor, free of the other components, obtained from plasma from swine with von Willebrand's disease either before or late after infusion eluted from agarose gel columns both as high and lower molecular weight material, unlike normal antihemophilic factor, which had a high molecular weight. In contrast, on ultracentrifugation the antihemophilic factor in these plasma sedimented slowly, even though chromatographically the plasmas contained both high and low molecular weight factor. All of the Factor VIII complex activities in normal porcine plasma sedimented rapidly. These studies demonstrate the heterogeneity of the Factor VIII complex and the apparent dependence of its chromatographic and sedimentation behavior on the functional activities associated with the complex.     

Solid-Phase Immunoradiometric Assay of Factor-VIII Protein

A solid phase immunoradiometric assay for factor-VIII protein has been developed using ¹²⁵I-labelled rabbit antibody made against highly purified factor VIII. Antibody specific for high molecular weight factor-VIII protein was isolated using an immunoadsorbent consisting of highly purified factor VIII bound to diazotized m-aminobenzyl (oxymethyl)-cellulose. The purified anti-factor VIII antibody was labelled with ¹²⁵I while bound to the immunoadsorbent and then eluted at pH 2.9. Dilutions of plasma samples for assay were incubated for 48 h in anti-factor VIII antibody-coated tubes. The tubes were washed to remove unbound proteins and the ¹²⁵I-labelled, purified antibody was added. After 48 h incubation the tubes were washed to remove unbound antibody and counted. The concentration of immunoreactive factor-VIII protein in pooled normal human plasma was determined to be 8 μg/ml. The minimum amount of factor VIII measured by the assay is less than 0.16 ng. Factor-VIII protein was present in normal concentration in haemophilia A plasma, and in reduced concentration in von Willebrand's disease plasma. This method has the advantages of improved sensitivity, specificity, efficiency and economy as compared with previous factor-VIII immunoassays.     

Factor VII and fibrinolytic response to deamino-8-D-argenine vasopressin in normal subjects and dissociate response in some patients with haemophilia and von Willebrand''s disease

S ummary Deamino-8- d -argenine vasopressin (DDAVP) was given by intravenous infusion to normal subjects, haemophiliacs and patients with von Willebrand's disease (vWd) and the factor VIII and plasminogen activator response was studied. In normal subjects and most patients with mild haemophilia and mild (intermediate) von Willebrand's disease there was an increase in plasminogen activator and all factor VIII related activities. In patients with mild vWd the prolonged bleeding time was shortened by DDAVP despite only a modest rise in factor VIII related Ristocetin cofactor activity (VIIIR:RiCoF). Sub-groups of patients have been characterized in whom atypical responses were observed. In two brothers with clinically severe haemophilia, but with 5–6 u/dl procoagulant factor VIII (VIIIC), there was an increase in VIIIC but no rise of the corresponding antigen, suggesting increased release of an antigenically abnormal poorly functioning molecule. A patient with intermediate vWd was studied in whom neither DDAVP, adrenaline infusion, nor venous occlusion resulted in an increase in either plasminogen activator or factor VIII related antigen (VIIIRAg), although there was a significant increase in VIIIC. In a further patient with severe vWd, DDAVP failed to elicit any plasminogen activator or VIII response. The results obtained from these two patients suggested that in some individuals the presumed endothelial cell abnormality in vWd may be more extensive than a defect in VIIIRAg synthesis. Sub-groups of patients have been identified for whom treatment with factor VIII concentrates would be more appropriate than DDAVP prior to minor surgery.     

Circulating immune complexes in haemophilia and von Willebrand's disease

Sera from 63 patients with haemophilia A, 21 with haemophilia B and 29 with von Willebrand's disease were screened for the presence of circulating immune complexes (CICs), serological markers of hepatitis A and B virus, autoantibodies and factor VIII or factor IX inhibitors. CICs were detected by the 125J Clq binding assay (ClqBA), the solid phase conglutinin assay (KgBSP) and the solid phase Clq assay (ClqSP). The incidence of CICs detected by the ClqBA and the ClqSP methods in haemophiliacs and in von Willebrand patients was higher than that observed in normal subjects, while the prevalence of CICs detected by the KgBSP method was not. The presence of CICs was not correlated with patient age, severity of disease, presence of hepatitis B virus serological markers, abnormal liver function tests or factor VIII inhibitors. A significant connection was demonstrated between CICs detected by the ClqBA method and replacement therapy when the dose administered over 1 year was over 20 000 U of factor VIII or IX concentrates. The high proportion of CICs in von Willebrand's disease, not connected with the replacement therapy or the presence of serological markers of hepatitis virus, is in agreement with the possibility that immune complexes may be related to the disease itself and independent, at least in part, of exogenous agents.     

IgA inhibitor to factor VIII/von Willebrand factor

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.