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.     

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.     

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.     

Association of the hemophilia A carrier state and hemorrhagic thrombocytopathy with dilatation of the platelet membrane complex

Associations of hereditary abnormalities of the factor VIII complex and hereditary platelet disorders have previously been reported in 12 families. Another family is reported in which 6 members had a bleeding tendency and thrombocytopathy characterized by impaired platelet aggregation and dilatation of the platelet membrane complex. Apart from the platelet function abnormalities the proband had diminished levels of factor VIII clotting activity (36 U/dl) and factor VIII clotting antigen (31%) while factor VIII-related antigen and ristocetin cofactor were normal. The other affected family members had normal levels of factor VIII:C. Consequently, the proband was defined as a hemophilia A carrier manifesting also hereditary thrombocytopathy.     

Procoagulant Specificity of Factor VIII Inhibitor

Nine haemophilia A patients with an inhibitor to factor VIII procoagulant and eight without an inhibitor were studied for the presence of an inhibitor to von Willebrand factor (vWf) in a quantitative ristocetin-induced platelet aggregation system. The mean vWf, factor VIII related antigen (FVIII Ag) and vWf:FVIII Ag ratio were not significantly different in the two groups (P greater than 0.6). The inhibitor plasmas did not reduce the wWf level in normal plasma after a 2 h incubation. The factor VIII inhibitor is highly specific for the procoagulant function of the factor VIII complex.     

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.     

Antibody nature of circulating inhibitor of plasma von Willebrand factor.

A circulating plasma inhibitor of the "von Willebrand factor" was observed in a multiply tranfused subject with severe von Willebrand's disease. The platelet-active von Willebrand factor is associated with a plasma protein macromolecular complex that is deficient in the disease. The inhibitor appears to be an IgG antibody, kappa type, based on neutralization tests with goat antisera to specific human immunoglobulins. The IgG and inhibitor separated out together in plasma fractions obtained by "salting-out" and chromatographic procedures. Two separate inhibitor neutralization tests for the platelet-active factor, one with human plasma and ristocetin, the other with bovine plasma, gave similar results, based on the macroscopic aggregation time test of fixed human platelets. With cryoprecipitate transfusions the inhibitor was transiently neutralized with the temporary appearance of von Willebrand factor, factor VIII, and factor VIII-like antigen in the plasma. The plasma inhibitor level increased after transfusion, suggesting an anamnestic response. Lower titer inhibitor plasmas neutralized only the platelet activity. Highest titer plasma also neutralized human factor VIII, but only in part; it did not neutralize either bovine factor VIII or the human small active factor VIII fragment. The anti-factor VIII activity of the von Willebrand factor inhibitor may be due to steric hindrance, dependent on the spatial relationship of factor VIII sites on the macromolecular complex.     

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.     

Acquired von Willebrand syndrome due to an inhibitor specific for von Willebrand factor antigens

A patient with acquired von Willebrand syndrome associated with polycythemia rubra vera is described. Her plasma factor VIII procoagulant activity (67 U/dl) and factor VIII-related antigen (117 U/dl) were normal but no von Willebrand factor activity could be detected. Factor VIII crossed immunoelectrophoresis revealed decreased levels of less anodic polymeric forms of factor VIII. Mixture of her plasma or immunoglobulin G (IgG) fraction with normal plasma resulted in complete recovery of factor VIII activity and related antigen but no measurable von Willebrand factor activity, confirming the presence of an unique inhibitor. The limited specificity of this inhibitor to antigenic sites solely on the von Willebrand portion of the factor VIII bimolecular complex is distinct from all previous reports of this syndrome. This unique inhibitor offers a molecular probe to examine the von Willebrand factor: platelet interaction.     

Replacement therapy in platelet-type von Willebrand disease

The response to infusions of cryoprecipitate and factor VIII concentrate was studied in a patient with platelet-type von Willebrand disease (vWD) who showed lack of the large multimers of von Willebrand factor in plasma, increased platelet aggregation with low concentrations of ristocetin, and in vitro platelet aggregation by normal plasma. The cryoprecipitate and factor VIII concentrate to be infused induced platelet aggregation when added to patient platelet-rich plasma at concentrations higher than 0.86 U/ml and 3 U/ml of factor VIII-related antigen (VIIIR:Ag), respectively. Administration of cryoprecipitate (41.9 U VIIIR:Ag/kg body weight) was followed by a shortening of the bleeding time, and hemostasis was achieved during tooth extractions. Factor VIII concentrate (70.2 U VIIIR:Ag/kg) failed to correct the prolonged bleeding time and proved ineffective to control the gum bleeding. No significant diminution of the platelet count was observed following any infusion. These results indicate that cryoprecipitate is hemostatically effective and safe when infused in such a dosage, but factor VIII concentrate is not effective in platelet-type vWD in analogy to what is observed in various types of vWD.     

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.     

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.     

Inherited platelet abnormalities associated with low factor VIII activity in the same family.

Six of eight examined members belonging to two generations of the same (NEG-TUR) family were shown to have functional changes in platelets and/or a moderate decrease of factor VIII activity (FVIII:C) in plasma, with normal values of factor VIII-related antigen (VIII R:AG). Platelet defects (mainly a reduced PF3 availability, present in five patients) and factor VIII decrease were combined differently in individual members. Only two male members with both the PF 3 and FVIII:C defects had moderate haemorrhagic symptoms following traumatic injuries. One of them had also an absent adhesiveness to glass, the other one an absent adhesiveness to collagen and a reduced platelet aggregation by ADP and by collagen. Bleeding time, platelet function tests (in the other members), and routine coagulation tests were within normal range; ristocetin aggregation was also normal in all members. We think that two inherited defects, a mild haemophilia A and a "sui generis" thrombocytopathy, co-exist in this family.     

Exaggeration of defective platelet function by methyldopa in hypothyroidism

Platelet function was analyzed in two hypothyroid patients who were taking methyldopa. Results from these two patients are compared to those obtained in seven others with hypothyroidism on no medications. Bleeding times in the two drug patients were 33 and 26 minutes compared to normal values (4.0 to 8.0 minutes) in the other hypothyroid individuals. In addition, platelet aggregation responses to epinephrine, collagen, and ristocetin were abnormal in these two patients; these were normal in the others with hypothyroidism. Repeat studies were done in the first drug patient when she was euthyroid and still receiving methyldopa. Her bleeding time decreased to 11 minutes, platelet retention increased from 31% to 75%, and abnormal aggregation responses corrected to normal. Factor VIII coagulant activity, VIII antigenic activity, VIII ristocetin cofactor activity, and two-dimensional immunoelectrophoresis of VIII antigen were normal. These findings suggest that methyldopa can potentiate the mild platelet function defect seen in patients with hypothyroidism.     

Von Willebrand disease associated with familial thrombocytopenia and increased ristocetin-induced platelet aggregation

Two cases of von Willebrand disease (vWD) associated with familial thrombocytopenia were reported. The proband (daughter) and her father showed thrombocytopenia with large platelets and decreased von Willebrand factor activity (VIIIR:WF). Factor VIII procoagulant activity (VIII:C) and factor VIII-related antigen (VIIIR:AG) were normal, but both patients revealed an increased ristocetin-induced platelet aggregation and a qualitative abnormality of the factor VIII protein, which was characterized by fast electrophoretic mobility of VIIIR:AG and an abnormal elution of factor VIII-related activities on Sepharose 2B. DDAVP was hemostatically effective even in this thrombocytopenic patient undergoing a dental extraction.     

Heterogeneity of von Willebrand''s disease: Study of 40 Iranian Cases

Forty Iranian patients with von Willebrand's disease were tested for bleeding time, platelet retention to glass beads, ristocetin-induced platelet aggregation, and assay of factor VIII procoagulant activity (VIII:C), Willebrand factor activity (VIIIR:WF), and factor VIII-related antigen (VIIIR:AG) by two methods (Laurell and immunoradiometric assay). In 22 cases from 11 families, levels of VIII:C, VIIIR:WF and VIIIR:AG (Laurell) were below 5% and the immunoradiometric assay showed total lack of VIIIR:AG in all cases (sensitivity of the method 0.01%). In 10 of these families, the parents were related, raising th;e possibility that these patients are homozygous. The occurrence of precipitating antibodies to factor VIII was demonstrated in one of these severe patients. In seven cases from five families the anomaly was less severe, with results of VIII:C between 5 and 17%. In 11 cases from six families VIII:C was normal or moderately decreased, contrasting with lower levels of VIIIR:WF and VIIIR:AG. The presence of an abnormal factor VIII/von Willebrand factor protein was assessed by double-cross immunoelectrophoresis and gel filtration.     

Haemostatic changes in long-distance runners and their relevance to the prevention of ischaemic heart disease

Haemostatic changes may explain the paradoxical observations that regular exercise helps to prevent ischaemic heart disease but the risk of myocardial infarction and sudden death is actually increased during exercise. This study measured relevant haemostatic variables in 100 athletes before and after races of 10-26.2 miles duration and compared resting levels in athletes with 25 non-exercising controls. Prothrombin time, kaolin cephalin clotting time, fibrinogen, factor VII, factor VIII clotting (one and two stage), von Willebrand factor antigen, euglobulin clot lysis time, fibrin degradation products, full blood count, mean platelet volume, and platelet aggregation to collagen, adrenalin and adenosine diphosphate were measured. The immediate post-race results showed the familiar rise in platelet count and factor VIII clotting but there was no evidence of consumption or thrombin modification of factor VIII clotting. Platelet aggregation to adrenalin was reduced after the race and fibrinolysis was increased (P less than 0.05). The athletes at rest showed no significant differences from controls in their coagulation factor levels but showed increased fibrinolytic activity and reduced platelet aggregation to adrenalin (P less than 0.05). These results suggest a hypocoagulable rather than a hypercoagulable state during running and are consistent with the epidemiological evidence that such exercise is beneficial in the prevention of ischaemic heart disease.     

Effect of somatostatin on platelet aggregation and plasma factor VIII level in normal man.

As impairment in coagulation and platelet function has been reported following acute administration of somatostatin, a reevaluation of the effects of the cyclic and linear form of the peptide was undertaken. In vitro somatostatin had no effect by itself on platelet aggregation and no effect on ADP- or ristocetin-induced aggregation. In vivo ten healthy men infused with cyclic or linear somatostatin (loading dose of 250 microgram and three hour-infusion with 1,500 microgram) and five control subjects were investigated during the infusion and for three hours after the end of the infusion. With this dose, sufficient in man for maintaining plasma growth hormone and insulin at fasting levels under arginine stimulation, no abnormalities in platelet count, in ADP-induced platelet aggregation and in plasma VIII von Willebrand factor, factor VIII procoagulant activity and factor VIII related antigen levels were observed.     

Inhibition of human platelet aggregation by the proteolytic effect of streptokinase. Role of the human factor-VIII-related protein.

Defective ADP-induced aggregation was observed in in vitro streptokinases(SK)-treated normal platelet-rich plasma. Classic haemophilia and normal platelet poor plasma (PPP) treated with SK inhibit the aggregation of washed platelets; plasmin-treated normal human serum also shows an inhibitory effect on platelet aggregation. However, SK-treated von Willebrand plasmas do not inhibit the aggregation of washed platelets. This confirms the fact that the anti-aggregating effect is mainly linked to the digested factor VIII) but not to the digested fibrinogen. Defective ristocetin-induced platelet aggregation has also been observed in SK-treated plasmas. The presence of normal PPP does not modify the inhibition of the ADP-induced aggregation of washed platelets in SK-treated PPP. However, it does correct the ristocetin-induced aggregation. These results suggest that the inhibition of the ADP-induced aggregation is caused by the factor VIII degradation products, while the inhibition of the ristocetin-induced aggregation appears because of a defective von Willebrand activity of the factor VIII molecule.     

Additional variant of type I von Willebrand disease

We introduce a family with a von Willebrand subgroup that has not been described before. All of the eight subjects examined had normal levels of factor VIII coagulant activity (FVIII:C), a moderate reduction in the level of von Willebrand factor antigen (VWF:Ag), which resulted in a high FVIII:C/VWF:Ag ratio, and normal crossed immunoelectrophoresis, with normal multimeric pattern. The ristocetin cofactor in plasma and platelets was very low. Bleeding time was prolonged in two subjects without clearcut linkage to laboratory findings. In addition, an abnormality of platelet aggregation in response to ADP was observed: a decreased initial response was followed by marked disaggregation.