Spontaneous platelet aggregation in type IIB Tampa von Willebrand disease is inhibited by the 52/48-kDa fragment of normal von Willebrand factor, which contains the GPIb binding domain

The association of Type IIB von Willebrand disease (vWD) with chronic persistent thrombocytopenia and spontaneous platelet aggregation has recently been recognized. It has been shown that IIB von Willebrand factor (vWF) can initiate platelet aggregation by binding to the platelet glycoprotein (GP) lb receptor and inducing exposure of the GpIIb/IIIa fibrinogen receptor. In this study we demonstrate the increased binding of Type IIB Tampa vWF with normal platelets when compared with nonthrombocytopenic Type IIB vWF. Studies further demonstrate that spontaneous platelet aggregation initiated by IIB Tampa vWF can be blocked by a 52/48-kDa fragment of normal vWF, which contains the binding domain.     

A new mutation,S1285F,within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.     

Type 2N von Willebrand disease due to Arg91Gln substitution and a cytosine deletion in exon 18 of the von Willebrand factor gene

Two members of a family previously classified as type 1 von Willebrand disease (VWD), showed a quantitative defect in von Willebrand factor (VWF) antigen and ristocetin cofactor activity and an abnormal capacity of VWF to bind FVIII. Sequencing of the VWF gene region coding for the FVIII binding domain revealed the most frequent type 2N mutation: a single nucleotide change (G2811A) in exon 20, resulting in substitution of glutamine (Gln) for arginine (Arg) 91 in the mature VWF protein in one allele. The other allele contained a cytosine deletion (2680delC) in exon 18, introducing a premature stop codon at position 79 (Val79X) which produced a quantitative defect in VWF levels. The Arg91Gln defect is usually not evident in heterozygotes; however, in these patients it was expressed due to the lack of VWF production from the other allele. This is the first report of type 2N VWD in Italy.     

Heterogeneity in type IIB von Willebrand disease: two unrelated cases with no family history and mild abnormalities of ristocetin-induced interaction between von Willebrand factor and platelets

Two patients from two separate families were diagnosed as having type IIB von Willebrand disease, because they had lifelong bleeding tendencies, prolonged bleeding times, no large von Willebrand factor multimers, and low levels of ristocetin cofactor in plasma with heightened ristocetin-induced platelet aggregation. There was no history of bleeding, and no laboratory abnormalities were found in the parents and sibship of either propositi, in contrast with the autosomal dominant pattern of inheritance usually observed in type IIB von Willebrand disease. Abnormalities of ristocetin-induced von Willebrand factor-platelet interactions were less severe than in a patient from a previously reported family with type IIB von Willebrand disease studied in parallel. The peculiar features of these cases provide additional evidence of the existence of heterogeneity within this variant.     

Large experience with a factor VIII binding assay of plasma von Willebrand factor using commercial reagents

The present diagnostic assay for type 2N von Willebrand disease (VWD) is based on the in vitro measurement of the capacity of plasma von Willebrand factor (VWF) to bind exogeneous factor VIII (VWF:FVIIIB). We report a method using only commercially available reagents that is easy to perform. This method has been validated in a cohort of 144 patients with FVIII/VWF ratios < 0.6 using a plasma control mixture representative of intermediate VWF:FVIIIB. In total, 15 patients were diagnosed with markedly decreased VWF:FVIIIB and five patients were shown to have moderately decreased VWF:FVIIIB. Specific type 2N mutations were identified in all these patients.     

von Willebrand disease type IIC with different abnormalities of von Willebrand factor in the same sibship

A family with von Willebrand disease has been identified in which different members of the same sibship exhibit different abnormalities of von Willebrand factor (vWF). The two most severely affected sibs (bleeding time over 20 min) had abnormalities of vWF similar to those seen in type IIC. The smallest detectable multimer was increased and the triplet structure of individual multimers was replaced with a single band. The largest multimers could not be detected and there were relatively more small multimers than intermediate sized forms. vWF antigen (vWF:Ag) was decreased to 12.5-17% by electroimmunoassay (EIA) and to 3.2-5.5% by immunoradiometric assay (IRMA). In the less severely affected sibling (bleeding time 12.5 min) there was a similar relative increase in the smallest detectable multimer. However, the larger multimers were present and the relative concentration of large to small multimers was similar to normal. The triplet structure was altered in that the relative proportion of satellite bands to the central predominant band was decreased. vWF:Ag concentrations were moderately decreased (40-80% by EIA and 25-35% by IRMA). The father and grandfather showed a vWF multimeric pattern similar to the less severely affected sibling but there was no decrease in vWF:Ag concentration and their bleeding times were normal. These observations suggest that the interplay of several genetic factors is responsible for the expression of von Willebrand disease in this family.     

Measurement of von Willebrand factor-FVIII binding activity in patients with suspected von Willebrand disease type 2N: application of an ELISA-based assay in a reference laboratory

Summary.  Laboratory diagnosis of von Willebrand disease type 2N (VWD2N) is based on costly mutation analysis or in vitro measurement of the ability of plasma von Willebrand factor (VWF) to bind exogenous factor VIII (FVIII); however, the VWF-FVIII binding activity assay is complex and not widely used. Our aim was to assess the utility of the in-house VWF-FVIII binding assay in the investigation of patients with suspected VWD2N. A previously described ELISA-based FVIII binding method was simplified and adapted for the clinical laboratory use by optimizing incubation time, reagent concentrations and assay standardization. The assay was validated using samples from eight individuals with known homozygous or heterozygous VWD2N mutations, and 100 healthy adults. An additional 392 patient samples were tested, including 314 with FVIII activity <50% of normal and 78 received for routine VWF-FVIII binding activity testing. Intra- and inter-assay variations were less than 10% and 17%, respectively, and the limit of quantification was estimated as 0.12. The reference range for healthy adults was 0.73–1.42. VWF:FVIII binding activity was consistent with the genotype in subjects with available genetic data, being low in three individuals with homozygous mutation (<0.12) and intermediate in five heterozygous individuals (0.44–0.61). Screening of the 392 clinical samples identified reduced VWF:FVIII binding in 19 subjects. This assay provides accurate measurement of VWF:FVIII binding activity and successfully identifies homozygous VWD2N patients and heterozygous carriers. Use of this ELISA-based assay may help avoid the need for mutation analysis in patients with unexplained low FVIII activity.     

Heightened proteolysis of the von Willebrand factor subunit in patients with von Willebrand disease hemizygous or homozygous for the C2362F mutation

We studied the proteolytic pattern of the mutant von Willebrand factor (VWF) in four patients with von Willebrand disease (VWD) who were either homozygous or hemizygous for the mutation C2362F. A significant decrease in the native fragment of 225 kDa was evident in all the patients, together with a marked increase in the 176 and 140 kDa fragments, a pattern usually observed in type 2A VWD. The proteolytic pattern measured in four heterozygotes for C2362F was within the normal range, suggesting that the mutant VWF C2362F present in the plasma of these patients may be protected from proteolysis by normal VWF.     

The determination of von Willebrand factor activity by collagen binding assay

A new collagen binding assay has been developed for the determination of the functional activity of human von Willebrand factor based on the following principle: pepsin-digested type III collagen from human placenta was covalently immobilized on a microtitre plate. Binding of collagen to the microtitre plate was carried out in neutral phosphate buffer within 1 h. A collagen concentration of 3 μg mL^–1 was sufficient to achieve optimal coating. After drying, the coated microtitre plates remained stable for months without losing their vWF-binding properties and could be incorporated into a ready-to-use kit. The suitability of various types of collagen for use in this assay was evaluated by simulating the binding of vWF to collagen immobilized on the microtitre plate by using surface plasmon resonance technology with collagen immobilized on a sensor chip. vWF was most effectively bound to pepsin-digested type III collagen from human placenta. The assay thus comprises the following steps: (1) serial dilutions of a vWF reference preparation and vWF-containing samples are prepared and bound to a microtitre plate which is precoated with collagen; (2) vWF is detected with a polyclonal antibody; (3) the substrate reaction is photometrically measured with an ELISA reader. This test is highly specific and sensitive for vWF (detection limit: 10 ng mL^–1). The collagen binding activity measured corresponds to the degree of multimerization of vWF. The high sensitivity and the short time needed to carry it out may make it useful for clinical diagnosis and for the measurement of the functional activity of vWF in factor concentrates. In certain applications it may represent a suitable replacement for the ristocetin cofactor assay.     

Expression of two type 2N von Willebrand disease mutations identified in exon 18 of von Willebrand factor gene

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). The FVIII binding site has been localized within the first 272 amino acid residues of mature VWF, encoded by exons 18-23. Two substitutions in exon 18 of VWF gene, inducing candidate mutations Y795C and C804F were identified in the heterozygous state in two French patients who also displayed the frequent R854Q mutation in exon 20. Expression studies in Cos-7 cells showed that these abnormalities, which implicate cysteine residues, induced secretion, multimerization and FVIII binding defects of corresponding recombinant VWF. Results from transfection experiments with R854Q, performed to reproduce the hybrid VWF present in patient plasma, were in agreement with those obtained for patient's plasma VWF. These findings confirm the importance of the VWF D' domain in FVIII binding. In addition, this work shows that exon 18 should preferentially be sequenced in type 2N VWD patients when the frequent R854Q mutation in exon 20 has been excluded or detected in the heterozygous state.     

Platelet von Willebrand factor determination does not improve the diagnosis of patients with suspected Type 1 von Willebrand disease

Summary.  Establishing a laboratory diagnosis of a bleeding disorder can be challenging for some patients who present with mucocutaneous bleeding symptoms. A common clinical scenario is an older patient with a prior diagnosis of von Willebrand disease (VWD) or a family history of VWD, who now has von Willebrand factor (VWF) values repeatedly within the normal range. Plasma VWF antigen levels have been shown to increase with age. Whether platelet VWF increases with age is unknown. We hypothesized that platelet VWF does not increase with age and low platelet VWF levels, despite normal plasma levels, could be a reason for continued bleeding symptoms in some patients. Therefore, we compared the platelet and plasma VWF antigen and activity as well as the platelet function analyzer (PFA)-100^® closure times in 35 patients with a history of mucocutaneous bleeding symptoms and consistently normal levels of VWF antigen and activity, despite a prior history of a VWD diagnosis and/or a positive family history of VWD. Overall in our patients (bleeders), the platelet VWF values correlated with the plasma values and only three patients had reduced platelet VWF. In the bleeding group, the PFA-100^® results showed an inverse correlation with plasma and platelet values, which was stronger for the plasma values. Therefore, platelet VWF determination was not helpful in the diagnosis of suspected mild type 1 VWD.     

Acquired von Willebrand disease in multiple myeloma secondary to absorption of von Willebrand factor by plasma cells

A case of acquired von Willebrand disease (AvWD) associated with an IgA lambda multiple myeloma is reported. No form of inhibitor could be detected. SDS-agarose gel electrophoresis patterns of von Willebrand factor (vWF) both in plasma and platelet lysates were normal but a decrease in all-sized multimers with a type IA pattern was seen. After 1-deamino-8-D arginine vasopressin (DDAVP) infusion, vWF multimers larger than those seen in the resting state appeared in patient plasma, which were progressively cleared. Indirect immunofluorescence studies with a monoclonal antibody to vWF showed that vWF was selectively absorbed into myelomatous cells. This is the first case of AvWD associated with multiple myeloma resulting from the selective absorption of vWF into abnormal plasma cells. This feature established a new pathophysiological mechanism of AvWD in multiple myeloma and probably in other lymphoproliferative diseases.     

Life-threatening reaction to factor VIII concentrate in a patient with severe von Willebrand disease and alloantibodies to von Willebrand factor

A 16-yr-old girl with severe von Willebrand disease complicated by the development of precipitating alloantibodies to von Willebrand factor (anti-VWF) had a life-threatening anaphylactoid reaction immediately after the infusion of a commercial plasma concentrate of factor VIII/von Willebrand factor. An early post-infusion activation of the complement system was demonstrated by the appearance of C3 split products and by the drop of serum CH50 activity, occurring in parallel with a post-infusion drop in the anti-VWF antibody levels. Immune complexes remained unchanged in the early post-infusion period and rose to a moderate extent only after 24 h. We conclude that biologically active products of the complement system contributed to the onset of this life-threatening reaction which occurred after concentrate infusion.     

Measurement of von Willebrand factor binding to a recombinant fragment of glycoprotein Ibalpha in an enzyme-linked immunosorbent assay-based method: performances in patients with type 2B von Willebrand disease

Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIb alpha) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIb alpha. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0.2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1.8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.     

Multimeric pattern discrepancy between platelet and plasma von Willebrand factor in type IIC von Willebrand disease

von Willebrand factor (vWF) from platelet lysate and plasma, collected in the presence of protease inhibitors, was studied in two patients with type IIC von Willebrand disease (vWD). Platelet and plasma vWF showed the smallest multimer increased, but the latter had a repeating single band whereas the former had a repeating "doublet." This platelet-plasma discrepancy observed for the first time in these patients suggests that the repeating "doublet" or single band described in other type IIC patients represent minor subgroups of type IIC vWD.     

Studies of multimerin in patients with von Willebrand disease and platelet von Willebrand factor deficiency

In normal platelet α-granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron-lucent zone. Because the platelet stores of VWF are deficient in 'platelet low' type 1 and type 3 von Willebrand disease (VWD), we investigated their electron-lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal α-granular multimerin and factor V, and normal α-granular fibrinogen, thrombospondin-1, fibronectin, osteonectin and P-selectin. In type 3 VWD platelets, α-granular electron-lucent zones lacking VWF-associated tubules were identified and multimerin was found in its normal α-granular location. These findings indicate that the formation of the electron-lucent zone and the sorting of multimerin to this region occur independent of VWF. The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of 'platelet low' type 1 VWD.     

Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions

Interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case-control studies indicated that two GPIb alpha polymorphisms, (145)Thr/Met and/or variable number (1-4) tandem repeats of 13 amino-acid sequences, are associated with arterial thrombosis. The (145)Met-allele and the 3R- or 4R-allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIb alpha fragments and tested them in vitro. The dissociation constants of ristocetin-induced (125)I-labelled VWF binding to two forms of soluble recombinant GPIb alpha [(1)His-(302)Ala, either (145)Thr (145T) or (145)Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full-length GPIb alpha beta/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized-VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.     

The role of platelet von Willebrand factor in platelet adhesion and thrombus formation: a study of 34 patients with various subtypes of type I von Willebrand disease

Summary In order to investigate the respective role of plasma and platelet von Willebrand factor (vWF) in mediating platelet adhesion and thrombus formation, we performed ex vivo perfusion studies with native blood from patients with various subtypes of type I von Willebrand disease (vWD). We studied 34 patients with type I vWD (19 'platelet normal', five 'platelet low', two 'platelet discordant', eight 'Vicenza'). Parallel studies were carried out on nine patients with severe vWD (type III). At high shear rate (2600 s^-1) we found that the defect in platelet-vessel wall interactions in patients having a normal platelet vWF content ('platelet normal' and 'Vicenza') involved thrombus formation, whereas platelet adhesion was normal. At this high shear rate, platelet adhesion and thrombus volume were significantly decreased in patients with subtypes 'platelet low' and 'platelet discordant', i.e. when platelet vWF is either low or dysfunctional. These results indicate that platelet vWF may substitute for plasma vWF to promote platelet adhesion. emphasizing the important role of platelet vWF. They also confirm the role of vWF in thrombus formation at high shear rate because an abnormal thrombus volume was observed in all patients. even when platelet adhesion was normal.     

Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N-terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin- or ristocetin-stimulated platelets, ¹²⁵I-plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5-fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin-stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.     

Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease

Dutch Kooiker dogs with hereditary von Willebrand disease have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous haemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. We used this canine model of von Willebrand disease to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 h in one dog and 22.1 h in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 h, and LMW-rvWF, 9 h. The in vivo recovery of vWF:Ag after administration of rvWF was 59%, 64% and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78%, 110% and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in FVIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.