Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody

An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil- T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF- binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF- binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those of the Ab occurring in inhibitor von Willebrand's disease in which vWF inhibitor and binding values were similar, with a strong anamnestic response. The findings indicate that the vWS Ab binds to an epitope on the molecular vWF in such a way that causes only limited inhibition of vWF/ristocetin function and no inhibition of vWF/botrocetin function, suggesting that these two functional domains are at separate sites.     

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.     

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.     

Factor VIII Procoagulant Activity, Factor VIII Related Antigen and von Willebrand Factor in Newborn Cord Blood

The mean level of factor VIII procoagulant acitivity (VIII:C) and factor VIII related antigen (VIIIR:AG) was normal in 100 newborn cord plasmas, whereas that of von Willebrand factor (VIIIR:WF) activity was slightly lower than normal. On crossed immunoelectrophoresis, 20 of 50 newborn infants had an increased anodal mobility of VIIIR:AG. When the cord plasma showing an abnormal electrophoretic pattern was mixed with normal plasma, two precipitation peaks with a broad base were found. Similar mixing experiments with the abnormal cord plasma and plasma from a patient with atypical von Willebrand's disease did not normalize the electrophoretic mobility of VIIIR:AG. Gel filtration of the cord plasma with an abnormal electrophoretic pattern of VIII:AG, showed that the three activities were all detected at the position corresponding to a molecular weight of about 800 000. The results suggest the presence of qualitative abnormalities of the factor VIII molecule in half of full-term newborn cord plasma.     

Acquired von Willebrand's disease in association with Wilm's tumor: regression following treatment

A 9-yr-old female presented with a Wilm's tumor and a coagulopathy consistent with von Willebrand's disease. Factor VIII procoagulant activity (VIII C), factor VIII related antigen (VIIIR:Ag), and von Willebrand factor activity (VIII:vWf) were decreased. There was no evidence for a circulating inhibitor of the factor VIII molecular complex. von Willebrand's antigen II (vW AgII), which is deficient in hereditary von Willebrand's disease, was decreased below detectable levels in this patient. The coagulation studies, VIIIR:Ag, and vW AgII levels returned to normal following therapy of the Wilm's tumor. Wilm's tumor must be included as one of the malignancies associated with acquired von Willebrand's disease. Immunofluorescent studies of the tumor specimen showed normal endothelial staining of VIIIR:Ag by semiquantitative techniques and a lack of specific tumor adsorption of VIIIR:Ag The presence of normal amounts of tissue VIIIR:Ag has not previously been demonstrated in acquired von Willebrand's disease. Since we failed to demonstrate an inhibitor in the plasma in this patient, the etiology of the acquired von Willebrand's disease in this patient appears to differ from other cases of acquired von Willebrand's disease. The finding that vW AgII is decreased in this patient, similar to that reported in hereditary von Willebrand's disease, supports the close association of vW AgII to VIIIR:Ag, even though they are immunologically and biochemically distinct.     

Immunological Evidence that Human Factor VIII is Composed of Two Linked Moieties

The relationship between the three measurable components of the factor VIII complex, procoagulant activity (VIII:C), Ristocetin cofactor (VIIIR:WF) and factor VIII related antigen (VIIR:AG), has been investigated using a solid phase immunoadsorption system in which homologous antibodies specific for VIII:C are insolubilized onto Sepharose beads. The VIII:C component of partially purified factor VIII can be completely separated from the Willebrand factor (VIIIR:WF/VIIIR:AG) by this technique. The loss of VIII:C has no detectable effect on the molecular size, antigenicity or electrophoretic mobility of the original molecule. The Willebrand factor (WF) recovered from these immunoadsorption columns was used to absorb heterologous antisera to factor VIII. A specific heterologous antiserum to VIII:C, which no longer neutralized VIIIR:WF nor precipitated VIIIR:AG, was obtained. Heterologous antisera to WF were prepared which potently neutralized VIIR:WF and precipitated with VIIIR:AG, but also weakly neutralized VIII:C (titre I u/ml). This study is compatible with the theory that VIII:C and VIIIR:WF/VIIIR:AG are two different but linked entities.     

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.     

Platelet-collagen interactions: increase in rate of adhesion of fixed washed platelets by factor VIII-related antigen

A simple technique using an aggregometer and fixed washed human platelets (FWP) and fibrillar collagen has been used to evaluate the contribution of the two components of the factor VIII (FVIII) complex to platelet-collagen interactions. FWP bound individually to collagen fibrils in suspension, and both the total number of FWP bound and the rate of adhesion increased with increasing collagen concentration. Von Willebrand's disease (vWD) type I or normal plasma immunoadsorbed with anti-factor VIII-related antigen (anti-FVIIIR:Ag) antiserum gave 20% and vWD type IIa gave 50% of the rate of adhesion obtained with normal, hemophilia A, or hemophilia A with inhibitor plasma, but the same percent adhesion was found with all plasmas. The rate of adhesion of both vWD type I and type IIa was corrected by the addition of purified FVIII complex. These results indicated that the FVIIIR:Ag and not the factor VIII coagulant activity (FVIII:C) in normal plasma or purified FVIII complex caused an accelerating effect on the rate at which FWP bound to collagen. Collagen fibrils not only bound FWP, but also adsorbed the FVIII complex with preferential adsorption of the forms of FVIIIR:Ag with the greatest ristocetin cofactor (FVIIIR:RCoF) activity. Saturation of collagen with FWP did not change the adsorption pattern of the FVIII complex. Also anti-FVIIIR:Ag blocked the accelerating effect of the FVIII complex but not the adhesion of FWP. Thus, FWP and FVIIIR:Ag appeared to bind to separate sites on collagen.     

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.     

Undetected factor VIII in a patient with type 3 von Willebrands disease mistaken as severe haemophilia A

Summary.  von Willebrand disease (VWD) type 3 is a rare disorder characterized by absent or <0.1 UmL⁻¹ of ristocetin cofactor (VWF:RCo), and a very low level of factor VIII (FVIII:C). A total absence of FVIII:C has never been reported in type 3 VWD. This case illustrates the effect of severe von Willebrand factor (VWF) deficiency on the factor VIII level.     

The Separation of Willebrand Factor from Factor VIII-Related Antigen

The three activities associated with factor VIII--coagulant (VIII:C), antigenic (VIIIR:Ag), and platelet agglutinating or Willebrand factor (VIIIR:WF)--have been separated by sequential antibody affinity chromatography, utilizing a rabbit antibody to factor VIII and a spontaneous human antibody to VIII:C. Normal plasma differentially lost its factor VIII-related antigen following passage over the rabbit antibody column. Subsequent passage of the VIIIR:Ag-depleted plasma over the human antibody column resulted in the loss of VIII:C activity, with retention of the Willebrand factor activity, antigen being partially recovered from the heterologous antibody column. These experiments demonstrate that it is possible to separate two of the factor VIII activities, VIIIR:Ag and VIIIR:WF, which are usually regarded as properties of a single 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.     

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.     

Optimizing therapy with factor VIII/von Willebrand factor concentrates in von Willebrand disease

In von Willebrand disease, the main goals of treatment are to correct the dual defect of haemostasis caused by a reduced or abnormal von Willebrand factor (vWF), i.e. the prolonged bleeding time (BT) and the deficiency of factor VIII coagulant activity (FVIII:C). The synthetic vasopressin analogue, desmopressin (DDAVP), has reduced the need for transfusions in most of the mild forms of von Willebrand disease but DDAVP is ineffective in type 3 and in other severe cases of types 1 and 2 von Willebrand disease. For many years cryoprecipitate has been the mainstay of replacement therapy but, after the introduction of virucidal methods, concentrates containing FVIII/vWF have been considered much safer than cryoprecipitate and proposed in von Willebrand disease management. FVIII/vWF concentrates have been produced and tested by many authors but there is only one report describing four virus-inactivated FVIII/vWF concentrates evaluated in a cross-over randomized trial. According to these in vitro and pharmacokinetic data, the following information can be derived: (a) no FVIII/vWF concentrate had an intact multimeric structure similar to that of normal plasma or of cryoprecipitate; (b) all FVIII/vWF concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed in the concentrate devoid of FVIII:C; (c) no FVIII/vWF concentrate consistently normalized the BT in a sustained fashion. On the other hand, clinical haemostasis can be achieved in the management of bleeding episodes and of surgery for most of von Willebrand disease cases regardless of whether the BT is corrected; in the few rare cases with mucosal bleeding not controlled by FVIII/vWF concentrates, infusion of DDAVP or platelet concentrates can be administered in addition.     

Selective absence of large forms of factor VIII/von Willebrand factor in acquired von Willebrand's syndrome. Response to transfusion.

A previously healthy elderly man with mucocutaneous bleeding was found to have a benign monoclonal IgG gammapathy associated with criteria for severe von Willebrand disease (Factor VIII procoagulant activity, Factor-VIII-related antigen, and ristocetin cofactor activity, less than 10% of normal). Associated qualitative abnormalities of factor VIII/von Willebrand factor were demonstrated by radiocrossed immunoelectrophoresis and immunoradiometric assay. The late clinical onset and negative family history are in favor of an acquired form of vWD. The monoclonal gammapathy and abnormalities of factor VIII/von Willebrand factor have been stable over a 10-yr period. No inhibitor to Factor VIII procoagulant activity, ristocetin cofactor activity, or Factor-VIII-related antigen could be demonstrated. Following transfusion of cryoprecipitate (with a normal cross immunoelectrophoretic pattern), there was a rapid removal of the large forms of Factor.-VIII-related antigen, paralleled by a decay of ristocetin cofactor activity. The transfusion study of this patient with acquired von Willebrand disease type II (variant of von Willebrand disease) serves to emphasize the relationship between polydispersity of Factor VIII/von Willebrand Factor and functional heterogeneity.     

IgA RIPA inhibitor in a case of acquired von Willebrand syndrome

Acquired inhibitor of von Willebrand factor-platelet interaction occurring in a 57 year-old female has been partially characterized. She had no personal or familial bleeding tendencies, but presented a subcutaneous hematoma of recent origin. She was diagnosed as having an acquired von Willebrand syndrome because she had low levels of FVIII complex in plasma, with platelet adhesiveness to glassbeads and RIPA decreased. This inhibitor was classified as an IgA immunoglobulin, and had no activity against any component of FVIII complex. The purified IgA by the chromatographic technology interacted with normal platelets to inhibit RIPA. Following 1-deamino-8-D-arginine vasopressin (DDAVP) infusion, she had higher immediate rise in all components of FVIII complex in plasma, with no rapid decline. Plasma von Willebrand factor (vWF) multimers analyzed by 1.5% SDS-AGE technology revealed to be identical with those of normal plasma. These studies suggest that the abnormality of ristocetin-induced vWF-platelet interaction by IgA RIPA inhibitor and the reduction of all vWF multimers (like type IA von Willebrand disease) may have a relationship with the pathogenesis of bleeding diathesis in this case.     

Pharmacokinetics and hemostatic effect of different factor VIII/von Willebrand factor concentrates in von Willebrand's disease type III

Summary Four different plasma-derived concentrates composed of coagulation factor VIII (FVIII) and von Willebrand factor (vWF) of varying quality (Hemate-P, Behring; Profilate, Alpha; and FVIII-VHP-vWF, C.R.T.S Lille), or almost purified vWF (Facteur Willebrand, C.R.T.S Lille) and one recombinant FVIII concentrate (Recombinate, Baxter) were given, in doses of 30–60 IU VIII:C/kg or 70–110 IU RCof/kg, to five patients with von Willebrand's disease type III, in order to evaluate the role of the vWF in factor FVIII concentrates. All plasma concentrates except Profilate had a multimeric vWF pattern almost similar to that of normal plasma. Bleeding time (b.t.), VIII:C, vWF: Ag, ristocetin cofactor activity, and multimeric pattern of the plasma-vWF were followed for 72 h. Both Duke b.t. and the multimeric pattern in plasma normalized after infusion of Hemate-P, FVIII-VHP-vWF, and Facteur Willebrand and, to a lesser extent, after Profilate. As expected, in response to Recombinate there was no effect on primary hemostasis, and the half-life of FVIII procoagulant activity (VIII:C) was very short. Normalization of the vWF is important not only for improving the primary hemostasis, but also for maintaining the plasma FVIII concentration on a high level, both by reducing the elimination rate of infused FVIII and via a secondary release of endogenous FVIII. If a prompt hemostatic effect is required, we recommend a concentrate containing both FVIII and all vWF multimers, but for prophylactic treatment, pure vWF may be used.     

Desmopressin for the treatment of haemophilia

Summary. The synthetic vasopressin analogue (1–deamino‐8‐d ‐arginine‐vasopressin) increases plasma concentration of factor VIII and von Willebrand factor in normal subjects and patients with mild haemophilia A and von Willebrand disease. Since its first clinical use in 1977, desmopressin has become the treatment of choice for patients with haemophilia A and factor VIII coagulant activity (FVIII:C) > 5% and has spared several patients the risk of acquiring blood‐borne viral infections due to the use of non‐virally inactivated plasma‐derived FVIII concentrates. An average two to sixfolds FVIII:C increase is typically observed in most patients and return to baseline occurs usually within 8 hours. Several clinical studies have demonstrated the clinical efficacy and safety of desmopressin and the availability of concentrated formulation for subcutaneous injection and of a nasal spray has paved the way to home‐treatment. However, overall it appears that haemophilic children may have a lower rate of biologic response compared to adults and a minority of adult patients are not able to attain clinically useful FVIII:C levels post‐desmopressin administration. Thus, in every patient with haemophilia A likely to be treated or candidate to an elective invasive procedure, a test‐infusion/injection should be carried out to assess the future usefulness of the compound.     

Desmopressin for the treatment of haemophilia

Summary.  The synthetic vasopressin analogue (1–deamino-8- d -arginine-vasopressin) increases plasma concentration of factor VIII and von Willebrand factor in normal subjects and patients with mild haemophilia A and von Willebrand disease. Since its first clinical use in 1977, desmopressin has become the treatment of choice for patients with haemophilia A and factor VIII coagulant activity (FVIII:C) > 5% and has spared several patients the risk of acquiring blood-borne viral infections due to the use of non-virally inactivated plasma-derived FVIII concentrates. An average two to sixfolds FVIII:C increase is typically observed in most patients and return to baseline occurs usually within 8 hours. Several clinical studies have demonstrated the clinical efficacy and safety of desmopressin and the availability of concentrated formulation for subcutaneous injection and of a nasal spray has paved the way to home-treatment. However, overall it appears that haemophilic children may have a lower rate of biologic response compared to adults and a minority of adult patients are not able to attain clinically useful FVIII:C levels post-desmopressin administration. Thus, in every patient with haemophilia A likely to be treated or candidate to an elective invasive procedure, a test-infusion/injection should be carried out to assess the future usefulness of the compound.     

Profound alterations of the multimeric structure of von Willebrand factor in a patient with malignant lymphoma

Laboratory investigation of an acquired haemorrhagic diathesis in a 63-year-old man with malignant lymphoma revealed the classical haemostatic defects found in von Willebrand's disease (vWD). In addition, SDS-agarose gel electrophoresis demonstrated alterations of the von Willebrand factor (vWF) multimeric structure. A profound defect of large and intermediate size multimers was observed which was different from those seen in variants of congenital vWD. In vitro, weak inhibitory activity against factor VIII procoagulant activity and ristocetin cofactor activity was present in the patient's plasma. When patient's plasma was incubated with normal plasma, followed by centrifugation, vWF antigen (vWF:Ag) was precipitated. In vivo, after transfusion of cryoprecipitate, there was rapid plasma clearance of vWF:Ag and ristocetin cofactor and of FVIII coagulant activities.