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.     

Willebrand Factor and Ristocetin I. MECHANISM OF RISTOCETIN-INDUCED PLATELET AGGREGATION

S ummary . Ristocetin induces aggregation of normal platelet-rich plasma over a wide range of concentrations. Low doses induce a biphasic response of which the first wave is not mediated by ADP and proceeds without the initial platelet shape change. The absence of aggregation in von Willebrand's disease results from the lack of a component, Willebrand factor, which is eluted together with platelets, factor VIII activity and Willebrand (or factor VIII related) antigen in the void volume fraction when platelet-rich plasma is gel filtered (Sepharose 2B). Twice washed normal platelets aggregate to ristocetin whereas four times washed platelets only aggregate when low volumes of normal haemophilia A plasma or serum are added. The interaction of ristocetin with platelets and plasmatic components has been investigated. A mechanism is proposed for ristocetin-induced aggregation.     

Inherited combined deficiency of factor V and factor VIII: report of a case with normal factor VIII antigen and ristocetin-induced platelet aggregation

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.     

Factor VIII/von Willebrand factor binding to von Willebrand''s disease platelets

A form of von Willebrand's disease has been described with enhanced ristocetin-induced platelet aggregation and anodal migration of the factor VIII/von Willebrand factor protein (type IIb). We studied two families with this form of von Willebrand's disease and macrothrombocytopenia. We have found that these platelets bind more of the normal and intermediate-sized multimers of the factor VIII/von Willebrand factor than normal platelets. Analysis of the binding data show an increased affinity of these vWd platelets for the factor VIII/von Willebrand factor. These findings are consistent with an increased number of platelet receptors, which, either by their native topography or migration on the platelet surface, bind factor VIII/von Willebrand factor protein with greater affinity than normal platelets, platelets of other vWd patients, and large platelets of other etiologies.     

Actions of ristocetin on platelets.

The absence of ristocetin-induced platelet aggregation appears to correlate with the platelet defect in von Willebrand's disease, suggesting that this reaction mimics a physiological process. The effect of ristocetin on plasma and on the residual levels of the von Willebrand factor (vWF), Factor VIII procoagulant activity, and Factor VIII-related protein in plasma after aggregation of platelet rich plasma by this agent has been studied in order to further elucidate the mechanism and requirements of this reaction. Ristocetin-induced platelet aggregation causes a consumption of vWF, Factor VIII procoagulant activity, and Factor VIII antigen from the supernatant plasma which is proportional to the number of platelets aggregated. Such a consumption of these factors does not appear to occur after aggregation by other agents. Factor VIII procoagulant activity does not appear necessary for ristocetin-induced platelet aggregation, yet is utilized in this process. These findings support the hypothesis that the molecule associated with Factor VIII procoagulant activity is carried by the molecule necessary for ristocetin-induced platelet aggregation.     

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.     

Transfusion and Gel Filtration Studies in von Willebrand's Disease

A study has been made of the plasma levels of factor-VIII clotting activity and factor-VIII related antigen following transfusion in five patients with von Willebrand's disease and one patient with severe haemophilia. All of the patients were transfused with cryoprecipitate or an AHG concentrate and their plasma levels of factor-VIII clotting activity and factor-VIII related antigen assayed at intervals after transfusion. In the case of the patients with von Willebrand's disease there was the expected post transfusion rise of factor-VIII activity followed by a fall of activity and then a secondary rise of activity. During this same period there was a rapid fall of the factor-VIII related antigen from the high levels attained immediately after transfusion but at about the same time as the secondary increase of factor-VIII clotting activity took place there was a slowing down of the rate of disappearance of factor-VIII related antigen. Indeed in several patients there was a secondary rise of factor-VIII related antigen which coincided with the secondary peak of factor-VIII activity.
Throughout the post-transfusion period all plasma samples containing factor-VIII clotting activity also contained measurable amounts of factor-VIII related antigen.
Gel filtration studies carried out on plasma obtained from the von Willebrand's patients after transfusion failed to show any small molecular weight factor VIII. The possible implications of those findings are discussed.     

Factor VIII-Related Antigen and von Willebrand's Disease

Immunological methods for the detection and assay of factor VIII-related antigen have proved to be valuable tools in the study of factor VIII, haemophilia and von Willebrand's disease. The antibody neutralization tests, with their inherent difficulties and variability, have been largely replaced by the electroimmunoassay based on the method of Laurell. Using this latter method, it has been convincingly shown that whereas normal individuals and haemophiliacs have an antigen which precipitates with the rabbit anti-factor VIII antibody, patients with von Willebrand's disease have a reduced amount of the antigen.
The relationship of the antigen to factor VIII activity is not yet clear; nevertheless the assay of factor VIII-related antigen is proving of some value in the diagnosis of von Willebrand's disease and in the detection of carriers of haemophilia. In von Willebrand's disease the test for the antigen along with the ristocetin test for platelet aggregation has thrown new light on the condition and has helped to define several variants of the disease.     

Synthesis of von Willebrand Factor by Cultured Human Endothelial Cells

Cultured human endothelial cells synthesize and secrete a protein(s) which has Factor VIII antigen but which lacks Factor VIII clot-promoting activity (J. Clin. Invest. 52, 2757-2764, 1973). Von Willebrand factor activity has been identified in medium from cultured human endothelial cells. This activity was demonstrated by the ability to correct the defect in platelet adhesiveness of blood obtained from patients with von Willebrand's disease. This activity also supported ristocetin-induced aggregation of washed normal human platelets. The von Willebrand factor activity from cultured endothelial cells has physicochemical and immunologic properties like those of the von Willebrand factor activity and the Factor VIII antigen present in human plasma and the Factor VIII antigen synthesized by human endothelial cells in vitro. Rabbit antibody to chromatographic fractions containing endothelial cell von Willebrand factor inhibits the platelet retention of normal blood in glass bead columns.     

Platelet function and immunologic parameters in von Willebrand's disease following cryoprecipitate and factor VIII concentrate infusion.

Results of functional and immunologic tests of factor VIII and of platelet function were followed in two patients with severe von Willebrand's disease who were given cryoprecipitate during preparation for surgery. Initial correction of factor VIII coagulant activity was found to persist from 48 to 72 hours. Correction of immunologically measured factor VIII persisted for 48 hours and correlated with the patients' platelet response to ristocetin aggregation. Bleeding times were corrected for only 6 to 8 hours and showed fairly good correlation with the ristocetin aggregation factor, as suggested by Weiss. The administration of commercial factor VIII concentrate corrected all parameters except the bleeding time and the ristocetin aggregation factor measurement.     

Variant von Willebrand's disease and pregnancy

The clinical course and coagulation profile of a pregnant patient with variant von Willebrand's disease were followed from the second trimester through puerperium. The clinical course was characterized by a normal delivery and absence of abnormal bleeding or need for replacement therapy. The coagulation profile demonstrated an increase in factor VIII procoagulant activity, factor-VIII-related antigen, and platelet aggregation activity in response to ristocetin prior to delivery. Postpartum, these factors decreased to prepregnancy values with distinctly different patterns. Factor VIII procoagulant activity continued to rise for 5 days after delivery and then decreased with a half-life of approximately 6 days. Factor-VIII-related antigen began to decrease just prior to delivery, displaying a half-life or approximately 6 days. Ristocetin cofactor activity, however, dropped immediately postpartum and displayed a half-life of approximately 6 hr. The ristocetin cofactor activity was associated with factor-VIII- related antigen, which displayed a significantly smaller molecular weight than does normal factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen. Larger aggregates of factor-VIII- related antigen did not appear during the pregnancy, and ristocetin cofactor activity could not be demonstrated in fragments of less than 0,8 x 10(6).     

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.     

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.     

Platelet release abnormality associated with a variant of von Willebrand's disease.

A family with a platelet release abnormality (PRA) is described. The only son also showed a reduced rate of platelet aggregation in response to ristocetin, markedly reduced levels of von Willebrand's factor (vWf, ristocetin cofactor), and increased mobility of factor VIII-like antigen, features which were suggestive of von Willebrand's disease (vWd). No inhibition of vWf was found in his plasma. Family studies showed no evidence of vWd in the mother. The father's investigations showed a low rate of ristocetin aggregation on one of the two occasions when it was tested and low vWf on two of four occasions. Despite repeated testing, the findings in the father did not conclusively rule out the possibility of mild vWd, and it was impossible to determine whether the vWd in the son was inherited or arose as a mutation. The findings in this family suggest a possible relationship between abnormalities of the factor VIII complex and defective platelet function.     

Assay and Characterization of the Factor in Porcine and Bovine Plasma which Aggregates Human Platelets

S ummary . A sensitive and objective bioassay has been developed to measure the factor present in porcine or bovine plasma which aggregates human blood platelets. The technique is based on direct measurement of platelet aggregation using an electronic particle counter.
Using this assay it has been established that platelet aggregating activity is present in both bovine and porcine plasma and in therapeutic concentrates of factor VIII prepared from these materials. Platelet aggregating activity was found to be a function of the factor-VIII-related antigen concentration and independent of factor-VIII coagulant activity. The activity is absent in the plasma from pigs with von Willebrand's disease. It is concluded that factor-VIII-related antigen and the platelet aggregating factor are closely related and may represent one protein species.     

Characterization of the defect of the factor VIII/von Willebrand factor protein in von Willebrand's disease

The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N-acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.     

Acquired von Willebrand Syndrome with Inhibitors Both to Factor VIII Clotting Activity and Ristocetin-Induced Platelet Aggregation

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.     

Collagen-factor VIII/von Willebrand factor protein interaction

Factor VIII/von Willebrand factor (FVIII/vWF) protein interaction with collagen was studied by incubating plasma or purified FVIII/vWF with purified type I fibrillar collagen. Collagen adsorbed FVIII/vWF activities in a similar time and concentration-dependent manner from normal plasma, plasmas from classical and variant type von Willebrand's disease (vWD), and from purified FVIII/vWF. Incubation with denatured collagen or fibrin, produced in the presence or absence of fibronectin, showed no adsorption of FVIII/vWF. Examination of the multimeric structure of the remaining unadsorbed FVIII/vWF protein by agarose gel electrophoresis and autoradiography showed that the largest multimers had been adsorbed to the collagen. Studies of the adsorbed FVIII/vWF protein when eluted from collagen showed that it complemented the alterations in multimeric structure observed in the supernatants following collagen exposure. The multimeric structure of normal plasma following collagen adsorption resembled that of unadsorbed type IIb plasma; however, the collagen-adsorbed normal plasma did not produce enhanced ristocetin-induced platelet aggregation ( RIPA ). This phenomenon, therefore, must not be due solely to absence of large multimers from type IIb FVIII/vWF protein. The adsorbed multimers of FVIII/vWF protein may act as a subendothelial collagen-platelet bridge to promote primary hemostasis.     

Immunologic studies on human factor VIII (anti-hemophilic factor A, AHF) components produced by low-ionic-strength dialysis.

Since dialysis of human factor VIII against buffers of low ionic strength yielded two distinct components, and since the factor VIII fraction isolated from normal plasma showed von Willebrand factor activity as defined by the corrective effect on abnormal platelet retention and ristocetin aggregation in von Willebrand's disease, the present studies were performed to determine if the correcting activities could be attributed to one or both of the components. Dialysis of factor VIII against buffers of low ionic strength led, however, to a decrease in factor VIII procoagulant activity and the reduction of the correcting activities, which suggested that the intact aggregate was required for procoagulant activity and for von Willebrand factor activity. In this respect dialysis of factor VIII at low ionic strength differed from dissociation at high salt concentrations. The two low ionic strength components were identified by the use of a rabbit antiserum against factor VIII, and could be distinguished on the basis of specific antigenic structures. Dialysis of factor VIII at low ionic strength led to a decrease in antigenic determinants closely related to factor VIII function. Specific antibodies to the low ionic strength components inhibited factor VIII activity in normal plasma, but the residual factor VIII was higher than that after inhibition with antibodies against intact factor VIII. Both antibodies interfered with von Willebrand factor activity.     

An atypical von Willebrand's disease with hyperreactivity of platelet aggregation

Two family members (daughter and mother) with a bleeding disorder showed prolonged bleeding time and activated partial thromboplastin time associated with decreased plasma levels of factor VIII procoagulant activity, factor VIII-related antigen, and factor VIII-ristocetin cofactor activity. The ristocetin-induced platelet agglutination (RIPA) was enhanced, ADP-, collagen- and Ca ionophore-induced platelet aggregation were also increased at low concentrations of these compounds. In the mother, spontaneous platelet aggregation (SPA) was also observed. Contrary to type II B von Willebrand's disease (vWd), pseudo-vWd and platelet-type vWd, the patients did not show any increased binding of factor VIII/vWf to platelets in the presence of ristocetin. The RIPA in normal controls were inhibited by addition of antifactor VIII antiserum to the platelet-rich plasma, but not in cases 1 and 2. In this atypical vWd, therefore, the hyperreactivity of platelet aggregation may be due to an intrinsic abnormality of the platelets, but not to any enhanced interaction between plasma factor VIII and the platelets.