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.     

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.     

Von Willebrand factor testing ratios in the diagnosis and subtyping of von Willebrand disease

Von Willebrand disease (VWD) is a common bleeding disorder of platelet adhesion with six currently recognized subtypes. Laboratory diagnosis consists of an initial test panel including antigen, activity and factor VIII measurements, sometimes followed by further specialized testing. VWF activity/antigen testing ratios help to differentiate type 1 and type 2 disease, which is important for selection of proper therapy. Recommended ratio cutoffs differ by guideline, ranging from 0.5 to 0.7, with 0.7 commonly recommended. The ratio cutoff used affects the sensitivity and specificity for type 2 diagnosis. Variability in VWD due to underlying mutations and patient factors, as well as variability in VWF tests, impact the accuracy of ratios for VWD subtyping. This review discusses the use of activity/antigen ratios in the diagnosis and subtyping of VWD with a focus on technical aspects of the tests.     

Inverse relationship between plasma von Willebrand factor and soluble P selectin in patients with type 1 but not type 2 von Willebrand disease

P selectin is a component of endothelial Wiebel-Palade body membrane and platelet alpha granule membrane. Patients with von Willebrand disease (VWD) have low plasma von Willebrand factor (VWF). To examine the relationship between plasma P selectin and VWF, both were measured by ELISA in 38 patients with VWD and 40 controls. The patient had lower VWF (P < 0.001) but similar P selectin levels (P = 0.458). In 24 type 1 VWD patients, VWF and P selectin correlated inversely (P = 0.005) but in 14 type 2 VWD patients there was no correlation (P = 0.997). These data imply major differences in VWF/P-selectin regulation between types 1 and 2 VWD.     

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.     

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.     

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.     

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.     

Postpartum von Willebrand factor levels in women with and without von Willebrand disease and implications for prophylaxis

The aim of this study was to elucidate the fall in von Willebrand factor (VWF) and factor VIII activity (FVIII) after childbirth in women with and without von Willebrand disease (VWD). VWF:RCo, VWF:Ag, and FVIII were obtained in the third trimester of pregnancy, on admission for childbirth, and 10 times postpartum. Specimens were processed within 4 h and analysed centrally. Means were calculated at each time point. Forty women (40 pregnancies) without VWD and 32 women (35 pregnancies) with VWD were enrolled. 15/32 with VWD were treated (30% of those with type 1 and all of those with type 2) in 17 pregnancies. Treatments prior to delivery consisted of desmopressin (2/17), VWF concentrate (15/17) and after delivery VWF concentrate (16/17). Duration of treatment was 0–21 days (median 6). VWF levels peaked at 250% of baseline – 4 h postpartum in women with VWD and 12 h postpartum in women without VWD. Thereafter, VWF levels fell rapidly, approached baseline at 1 week and reached baseline at 3 weeks. Except immediately postpartum, when the levels among treated cases were higher, levels among women with VWD appeared to parallel, but were lower than those among women without VWD. Levels were lowest among those who received treatment. VWF levels fall rapidly after childbirth. Except immediately postpartum, current treatment strategies do not raise VWF levels to the levels of women without VWD or even to the levels of women with milder, untreated VWD. Consequently, women with VWD may be at risk of postpartum haemorrhage despite treatment.     

Von Willebrand factor propeptide makes it easy to identify the shorter Von Willebrand factor survival in patients with type 1 and type Vicenza von Willebrand disease

Reduced von Willebrand factor (VWF) half-life has been suggested as a new pathogenic mechanism in von Willebrand disease (VWD). The usefulness of VWF propeptide (VWFpp) in exploring VWF half-life was assessed in 22 type 1 and 14 type Vicenza VWD patients, and in 30 normal subjects, by comparing the findings on post-Desmopressin (DDAVP) VWF t(1/2) elimination (t(1/2el)). The VWFpp/VWF antigen ratio (VWFpp ratio) was dramatically increased in type Vicenza VWD (13.02 +/- 0.49) when compared to normal subjects (1.45 +/- 0.06), whereas it appeared to be normal in all type 1 VWD patients (1.56 +/- 0.7), except for the four carrying the C1130F mutation (4.69 +/- 0.67). A very short VWF t(1/2el) was found in type Vicenza VWD (1.3 +/- 0.2 h), while all type 1 VWD patients had a t(1/2el) similar to that of the controls (11.6 +/- 1.4 and 15.4 +/- 2.5 h respectively), except for the four patients carrying the C1130F mutation, who had a significantly shorter VWF survival (4.1 +/- 0.2 h). A significant inverse correlation emerged between VWFpp ratio and VWF t(1/2el) in both VWD patients and normal subjects. The VWFpp ratio thus seemed very useful for distinguishing between type 1 VWD cases with a normal and a reduced VWF survival, as well as for identifying type Vicenza VWD.     

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.     

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.     

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.     

Role for platelet von Willebrand factor in supporting platelet-vessel wall interactions in von Willebrand disease

Twelve infusions of plasma concentrates of von Willebrand factor (vWF) were given to four patients with severe (type III) von Willebrand disease (vWD). Their prolonged bleeding times were either completely or partially corrected after five infusions and had not changed after the remaining seven. In contrast, the low platelet coverage of the subendothelial surface of rabbit aorta perfused with normal washed platelets and red cells resuspended in preinfusion patient plasma was completely or partially corrected in ten instances by replacing preinfusion plasma with postinfusion plasma and remained unchanged in two. Postinfusion improvement in surface coverage was greater than that in bleeding time, suggesting that vWF from normal platelets is needed to support optimal platelet-vessel wall interactions in vWD. This possibility was further explored through other perfusion experiments. The subendothelial surface covered by platelets from an untreated patient with type III vWD (containing no measurable vWF) or from a type IIA vWD patient (containing dysfunctional vWF) resuspended in normal plasma was much smaller than that covered by normal platelets resuspended in normal plasma. These results establish that platelet vWF is important in supporting platelet-vessel wall interactions in vWD and also provide experimental support in favour of the therapeutic transfusion of normal platelets in addition to vWF concentrates to correct the bleeding time in vWD patients.     

Diagnosis of von Willebrand disease

Summary. von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). vWF is synthesized by endothelial cells and megakaryocytes and circulates in plasma as a multimeric high molecular weight glycoprotein. vWF plays a major role in the early phases of ostasis by promoting platelet-vessel wall and plateletplatelet interactions under high shear conditions. It is also the carrier of coagulation factor VIII (FVIII) in plasma. A deficiency of vWF results in impairment of both primary and secondary phases of ostasis. Therefore, patients with vWD manifest bleeding symptoms that are typical of defects of primary ostasis (mucocutaneous haemorrhages) but, in case of severe deficiency of vWF, there are also haemarthroses and haematomas, which are typical of those seen with coagulation defects. Several types and subtypes of vWD have been described with a high degree of heterogeneity. The diagnosis is based on measurements of plasma and platelet vWF, the ability of vWF to interact with its platelet receptor and the analysis of the multimeric composition of vWF. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate treatment of patients with vWD.     

Screening for von Willebrand disease: contribution of an automated assay for von Willebrand factor activity

Measuring von Willebrand factor (VWF) activity is essential to the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. The HemosIL VWF activity (VWF:AC) is a fully automated assay, recently proposed as a good alternative to VWF:RCo for VWD diagnosis. This study was undertaken to assess this new method. First, the analytical performance of VWF:AC on an automated coagulo-meter (ACLTop) was determined, and then this new method was compared with VWF:RCo and the platelet function analyzer (PFA100) for 160 patients referred for VWD screening. The VWF:AC achieved acceptable precision with within-run and between-run coefficients of variation ranging from 2.3% to 14.1%, and linearity from 10% to 100%. Despite some marked differences between VWF:AC and VWF:RCo for 10 plasmas tested, their agreement for VWD diagnosis was good. The VWF:AC had sensitivity similar to that of PFA100 (close to 100%), but better specificity (97.7% vs. 66% or 60%, depending on the cartridge used). The good analytical performance, and the sensitivity and specificity of VWF:AC to detect VWF deficiency renders it a suitable method for VWD screening. Our findings support VWF:AC use for the diagnostic work-up of VWD, paying close attention to concomitant clinical signs and bleeding score, as recommended for VWD.     

Clinical manifestations and complications of childbirth and replacement therapy in 385 Iranian patients with type 3 von Willebrand disease

Type 3 is the most severe form of von Willebrand disease (VWD) transmitted as an autosomal recessive trait. We collected data on the clinical manifestations of type 3 VWD by examining 385 patients from 300 Iranian kindreds, who were compared with 100 age-matched patients with severe haemophilia A. Joint and muscle bleeding was less frequent than in haemophiliacs, perhaps because factor VIII levels were in general higher (median value 4% vs. 1% or less). Mucosal tract haemorrhages such as epistaxis and menorrhagia were the most prevalent symptoms in VWD. Post-circumcision and oral cavity bleeding occurred frequently when prophylactic replacement therapy was not carried out or was inadequate. The course of pregnancy was usually uneventful, but increased bleeding occurred at parturition when affected women were treated with replacement therapy for less than 3-4 d. Ten of 385 (2.6%) of these multitransfused patients developed an alloantibody to VWF and 55% are chronically infected with the hepatitis C virus.     

New developments in von Willebrand disease

Von Willebrand disease (VWD) constitutes the most common inherited human bleeding disorder. It is associated with a mucocutaneous bleeding phenotype that can significantly impact upon quality of life. Despite its prevalence and associated morbidity, the diagnosis and subclassification of VWD continue to pose significant clinical challenges. This is in part attributable to the fact that plasma von Willebrand factor (VWF) levels vary over a wide range in the normal population, together with the multiple different physiological functions played by VWF in vivo. Over recent years, substantial progress has been achieved in elucidating the biological roles of VWF. Significant advances have also been made into defining the pathophysiological mechanisms underpinning both quantitative and qualitative VWD. In particular, several new laboratory assays have been developed that enable more precise assessment of specific aspects of VWF activity. In the present review, we discuss these recent developments in the field of VWD diagnosis, and consider how these advances can impact upon clinical diagnostic algorithms for use in routine clinical practice. In addition, we review some important recent advances pertaining to the various treatment options available for managing patients with VWD.