Supranormal von Willebrand factor multimers in scleroderma

Platelet adhesion-aggregation reactions play an early and pivotal role in the pathogenesis of systemic sclerosis in scleroderma, but the mechanisms are incompletely understood. We determined whether or not plasma from 11 consecutive patients with scleroderma contained a subset of larger than normal ("supranormal") multimers of von Willebrand factor (vWF) that are potent inducers of platelet aggregation and adhesion. Supranormal multimers were found in all patients on at least one of two different occasions 9 to 12 months apart, whatever the duration and severity of the disease, but in none of the normal controls. Administration of low-dose aspirin (40 mg) to five of the 11 patients for ten days to inhibit the platelet release reaction slightly reduced the amounts of supranormal multimers suggesting that they might originate in part from platelets. Supranormal multimers may contribute to the pathogenesis of systemic sclerosis by inducing platelet aggregation and enhancing adhesion to subendothelium under the conditions of elevated shear stress occurring in the partially occluded vessels of the arterial microcirculation of scleroderma.     

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.     

High-resolution analysis of von Willebrand factor multimeric composition defines a new variant of type I von Willebrand disease with aberrant structure but presence of all size multimers (type IC)

In Type I von Willebrand disease, the whole series of von Willebrand factor (vWF) multimers is present in plasma, but all are decreased in quantity. No structural abnormality of individual multimers has been demonstrated so far in these patients. We now describe five individuals, from two unrelated families, who had this form of the disease and in whom the complex banding pattern of each vWF multimer was markedly abnormal. Inheritance was autosomal dominant and the clinical expression was mild. A bleeding history was elicited in three of the patients and included recurrent epistaxis, menometrorrhagia, and bleeding following tooth extraction. Replacement therapy had never been required. Although vWF levels in plasma were within the normal range in all of them, the ristocetin cofactor activity was decreased in four, and the bleeding time was prolonged in three. Analysis of vWF multimeric structure by agarose gel electrophoresis, including a newly developed high-resolution technique, demonstrated that the main band of each multimer was present, but a second, well-defined band always seen in normal individuals was missing in the patients. Two additional bands had altered mobility and were less well defined than in normal subjects, and a fifth, less intense band was also undetectable in the patients. Treatment with 1-deamino-8-D-arginine vasopressin (DDAVP) was assessed in two patients. It caused the circulating levels of vWF to increase and correct the bleeding time, but did not alter the structural abnormality. This study describes, therefore, a new variant form of Type I von Willebrand disease with aberrant structure of individual repeating multimers and an associated functional abnormality of vWF. In keeping with previously accepted terminology, the designation of Type IC von Willebrand disease has been adopted for this new variant.     

Functional analysis of a type IIB von Willebrand disease missense mutation: increased binding of large von Willebrand factor multimers to platelets.

Type IIB von Willebrand disease is an autosomal dominant bleeding disorder characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma, presumably due to their abnormally increased reactivity with platelets. We and others have recently identified a panel of missense mutations clustered in the platelet glycoprotein Ib binding domain of vWF from patients with type IIB von Willebrand disease. We now report functional analysis of one of the most frequent type IIB missense mutations, Arg-543----Trp (vWF R543W). vWF from a human umbilical vein endothelial cell culture heterozygous for the vWF R543W mutation showed markedly increased binding of large vWF multimers to platelets in the presence of a low dose of ristocetin compared to vWF from a normal control culture. Recombinant vWF containing the vWF R543W mutation expressed in COS-7 cells also demonstrated increased binding of large vWF multimers. Mixed multimers obtained by cotransfection of mutant and wild-type cDNAs showed partial dominance of the vWF R543W mutation. Thus these data demonstrate that the vWF R543W mutation alone is sufficient to confer increased binding of large vWF multimers to platelets in a dominant fashion and that no other factors relating to vWF posttranslational processing or secretion in endothelial cells are required for this effect.     

Reduced von Willebrand factor survival in type Vicenza von Willebrand disease.

Type Vicenza variant of von Willebrand disease (VWD) is characterized by a low plasma von Willebrand factor (VWF) level and supranormal VWF multimers. Two candidate mutations, G2470A and G3864A at exons 17 and 27, respectively, of the VWF gene were recently reported to be present in this disorder. Four additional families, originating from northeast Italy, with both mutations of type Vicenza VWD are now described. Like the original type Vicenza subjects, they showed a mild bleeding tendency and a significant decrease in plasma VWF antigen level and ristocetin cofactor activity but normal platelet VWF content. Unlike the original patients, ristocetin-induced platelet aggregation was found to be normal. Larger than normal VWF multimers were also demonstrated in the plasma. Desmopressin (DDAVP) administration increased factor VIII (FVIII) and VWF plasma levels, with the appearance of even larger multimers. However, these forms, and all VWF oligomers, disappeared rapidly from the circulation. The half-life of VWF antigen release and of elimination was significantly shorter than that in healthy counterparts, so that at 4 hours after DDAVP administration, VWF antigen levels were close to baseline. Similar behavior was demonstrated by VWF ristocetin cofactor activity and FVIII. According to these findings, it is presumed that the low plasma VWF levels of type Vicenza VWD are mainly attributed to reduced survival of the VWF molecule, which, on the other hand, is normally synthesized. In addition, because normal VWF-platelet GPIb interaction was observed before or after DDAVP administration, it is proposed that type Vicenza VWD not be considered a 2M subtype.     

von Willebrand disease "Vicenza" with larger-than-normal (supranormal) von Willebrand factor multimers

When normal volunteers or patients with type I von Willebrand disease (VWD) are given desmopressin (DDAVP), a set of larger-than-normal (supranormal) von Willebrand factor (VWF) multimers, similar to those present in VWF-containing cells such as platelets megakaryocytes and endothelial cells, appear transiently in postinfusion plasma. In two kindreds with mild lifelong bleeding symptoms transmitted as an autosomal dominant trait, all ten symptomatic members (but none of the five asymptomatic members) had a supranormal multimeric structure for plasma VWF, apparently identical to that seen for postdesmopressin normal plasma. Plasma factor VIII coagulant activity (VIII:C), VWF antigen (VWF:Ag), ristocetin-induced platelet agglutination, and ristocetin cofactor (RiCof) activity were low. Platelet VWF:Ag and RiCof levels (tested for three patients only) were normal. Bleeding times were normal or slightly prolonged. The patients' platelet multimeric structure was the same as that for normal platelets. After desmopressin infusion the plasma VWF multimeric structure remained supranormal as for preinfusion plasma, with VIII:C VWF:Ag and RiCof increasing markedly over baseline values and disappearing at a normal rate. Examination of the VWF subunit composition from three of these patients indicated that proteolytic processing of their VWF did not differ from normal. This study describes the first variant of VWD with a supranormal multimeric structure.     

Generation and breakdown of soluble ultralarge von Willebrand factor multimers

Ultralarge von Willebrand factor (ULVWF) multimeric strings are rapidly secreted by, and anchored to, stimulated endothelial cells (EC), and are hyperadhesive to platelets until cleavage by ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In ADAMTS-13-deficient familial and autoantibody-mediated thrombotic thrombocytopenic purpura (TTP), there is severely restricted cleavage of EC-anchored ULVWF-platelet strings. The small amount of active enzyme released from their EC cleaves ULVWF strings minimally just above EC surfaces, thus generating soluble ULVWF multimers that are 2.5 to 50 times longer than plasma von Willebrand factor (VWF) forms. Soluble ULVWF multimers (detected in TTP and several other disorders) are also hyperadhesive to platelets and can cause excessive platelet adhesion/aggregation. Without exogenous chemicals or extreme shear stress, soluble ULVWF multimers cannot be cleaved by ADAMTS-13 but can be de-assembled (reduced) in vitro, by a free thiol-containing molecule (>30 kD) present in the cryosupernatant fraction of plasma that is not ADAMTS-13, thrombospondin-1, albumin, cysteine, or glutathione. This reduction may prevent occlusion of the microvasculature by embolic soluble ULVWF multimers (± adherent/aggregated platelets). New inhibitors of platelet adhesion to EC-anchored ULVWF multimeric strings and soluble ULVWF include an aptamer, a nanobody, and N-acetylcysteine.     

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL^?1 per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16–60 years), and duration of follow‐up ranged from 5 to 26 years (mean 11 years). Patients had 2–10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU mL^?1 0.34 IU mL^?1 and 0.75 IU mL^?1. At last follow‐up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU L^?1, 0.56 IU mL^?1 and 0.90 IU mL^?1 (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL^?1 (0.21–0.39, CI 95%, P < 0.0001), 0.20 IU mL^?1 per decade (0.13–0.27, CI 95%, P = 0.0001) and 0.20 IU mL^?1 (0.11–0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age‐related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.     

A very-high-purity von Willebrand factor preparation containing high-molecular-weight multimers

BACKGROUND AND OBJECTIVES: We investigated large-scale production of very-high-purity von Willebrand factor (vWf) containing high-molecular-weight vWf multimers, using chromatography alone. MATERIALS AND METHODS: Factor VIII (FVIII)-containing vWf was produced by initial separation of vWf from plasma by gel filtration followed by two ion exchange steps. Two virus inactivation steps were incorporated in the process. RESULTS: The process resulted in FVIII-containing vWf preparations with a mean specific activity of 82 U vWf: collagen-binding activity per milligram protein (excluding added albumin) and with almost intact vWf multimer distributions. CONCLUSIONS: Very-high-purity, double virus-inactivated vWf preparations containing high molecular weight multimers were obtained on a production scale.     

Desmopressin and type II B von Willebrand disease

Summary. Type II B von Willebrand disease (vWD) is a rare subtype of vWD characterized by the presence of an abnormal von Willebrand factor (vWF) with enhanced affinity for the platelet membrane receptor glycoprotein Ib. The phenotypic hallmarks of the disease are represented by heightened ristocetin-induced platelet aggregation, occurring at very low ristocetin concentration, and the lack of high-molecular-weight vWF multimers in plasma. When infused with desmopressin, a variable degree of thrombocytopenia usually occurs in these patients, resulting from in vivo platelet aggregation caused by the release of abnormal vWF multimers from endogenous stores. We have reviewed the available literature data concerning the biological and clinical effects of desmopressin in the few cases so far reported. Despite the fear of thrombotic or haemorrhagic events, no significant side-effects have been reported also in the cases (70%) with severe thrombocytopenia. Moreover, the few clinical reports with the use of desmopressin in type II B patients showed a favourable effect in the prevention of bleeding during surgery or dental extraction. Thus, it appears that desmopressin could be safely used in selected clinical situations in patients with type II B vWD.     

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease

To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312-19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD.     

New variant of von Willebrand disease type II with markedly increased levels of von Willebrand factor antigen and dominant mode of inheritance: von Willebrand disease type IIC Miami

A variant of von Willebrand disease (vWD) was identified in six members of a kindred spanning four generations. The proband was a 46-year-old woman with a lifelong history of bleeding, a prolonged bleeding time (> 15 minutes), markedly elevated von Willebrand factor (vWF) antigen (vWF:Ag = 2.09 U/mL), slightly reduced ristocetin cofactor activity, and a plasma vWF multimer pattern similar to that of vWD type IIC. Similar findings were observed in her three children, mother, and brother. In affected family members, platelet and plasma vWF multimer patterns were discrepant with higher molecular weight multimers observed in platelet vWF. Following a 1-Des-amino-8-D-arginine vasopressin (DDAVP) challenge, the proband failed to normalize her bleeding time even though vWF: Ag rose by 70% and higher molecular weight multimers were increased slightly. Genetic studies were consistent with autosomal dominant inheritance of a mutation within the vWF gene. By sequencing of cloned genomic DNA, mutations were excluded in exons 4, 5, 14, and 15, which encode regions of the vWF propeptide proposed to be important in multimer biosynthesis. Mutations also were excluded in exons 28 to 31, which encompass the known mutations that cause vWD types IIA, IIB, and B. This new variant of vWD, characterized by autosomal dominant inheritance, a qualitative defect that resembles vWD type IIC, and increased plasma vWF:Ag, was tentatively designated vWD type IIC Miami.     

von Willebrand's disease characterized by increased ristocetin sensitivity and the presence of all von Willebrand factor multimers in plasma

In eight members of one family, platelets in platelet-rich plasma aggregated at much lower ristocetin concentrations than normal. Ivy bleeding time was variously prolonged, and von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity, and factor VIII coagulant activity were decreased. Most of the affected members had had slight to rather severe bleeding symptoms. Platelet-type von Willebrand's disease (vWD) could be ruled out. All multimers of vWF:Ag were found in plasma as well as platelets. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) to the propositus did not cause thrombocytopenia, and platelet-poor plasma obtained immediately after did not aggregate normal platelets. The molecular defect in this family, inherited as an autosomal dominant, resembles the one in type IIB because of the response to ristocetin but differs from IIB because all vWF:Ag multimers are present in plasma and the response to DDAVP is atypical. We conclude that this family has a new subtype of vWD and propose that structural as well as functional criteria should be used for a proper classification of vWD.     

Fibrinogen and von Willebrand factor in type II diabetes mellitus

A hypercoagulable state may contribute to the formation of early vascular lesions in diabetes. The von Willebrand factor is required for the attachment of platelets to the subendothelium; fibrinogen is required for platelet aggregation. This study was designed to assess in type II diabetic patients plasma levels of fibrinogen and von Willebrand factor to see if these variables are associated with platelet aggregation responses to adenosine diphosphate (ADP). Fibrinogen and the von Willebrand factor were significantly increased in diabetics but only fibrinogen was significantly related to platelet aggregation for ADP. Strict metabolic control does not reduce the increased concentrations of these two proteins. Hyperfibrinogenaemia was related to the presence of macrovascular disease. Therefore measurements of plasma fibrinogen could be added to the cardiovascular risk factor profile of diabetic patients. Intervention studies are also needed to reduce the increased incidence of thrombotic diseases in patients with diabetes mellitus.     

Pharmacokinetics of von Willebrand factor and factor VIII coagulant activity in patients with von Willebrand disease type 3 and type 2

Nine patients with von Willebrand disease type 3, six with type 2B, one with type 2A, and one patient with type 1/2N were infused with one dose of ≈50 or 100 IU ristocetin cofactor activity (RCoF) per kg body weight of von Willebrand factor (vWF) (Human), a product with a very low content of factor VIII (FVIII). Blood samples were collected over 96 h. The data for RCoF and vWF antigen (vWF:Ag) were fitted to a 1-compartment model decay. The data for FVIII:C were fitted to a model with a linear time 'synthesis' term and a 1-compartment decay. Results in von Willebrand disease type 3 patients (nine patients; 10 infusions) indicated a volume of distribution of 39.9 and 39.8 mL kg⁻¹ for RCoF and vWF:Ag, respectively. The FVIII:C rate of synthesis was 6.4 U dL⁻¹ h⁻¹ (range: 4.4–8.8). The decay rates for FVIII:C, RCoF, and vWF:Ag were 0.041 (h⁻¹) [ t _1/2: 16.9 h]; 0.061 (h⁻¹) [ t _1/2: 11.3 h] and 0.006 (h⁻¹) [ t _1/2: 12.4 h], respectively. In patients with von Willebrand disease type 2 ( n  = 8) the RCoF mean volume of distribution was 46 mL kg⁻¹. The factor VIIIC mean rate of synthesis was 5.5 U dL⁻¹h⁻¹ and the decay rate 0.043 (h⁻¹) [ t _1/2: 16.1 h]. The rate of decay for RCoF and vWF:Ag were 0.050 (h⁻¹) [ t _1/2: 13.9 h] and 0.044 (h⁻¹) [ t _1/2: 15.7 h], respectively.     

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.     

Significant linkage and non-linkage of type 1 von Willebrand disease to the von Willebrand factor gene

Significant linkage of types 2A and 2B von Willebrand disease (VWD) to the von Willebrand factor (VWF) gene have been reported, as well as mutations in the VWF gene. However, data for the partial quantitative variant are less consistent. An inconsistency of association between the type 1 VWD phenotype and genotype has been reported recently. We undertook linkage analysis of 12 families with definite or possible type 1 VWD patients. One family with classic type 1 VWD had a high lod score (Z = 5.28, theta = 0.00). A total lod score of 10.68 was obtained for the four families with fully penetrant disease. In two families linkage was rejected, while three families did not show conclusive evidence of linkage. This study corroborates ABO blood group influence, especially in patients with mild deficiencies and/or incomplete penetrance. Indirect genetic analysis may be an option for diagnosing asymptomatic or presymptomatic type 1 VWD carriers, particularly in families showing higher penetrance. The study indicates defects of the VWF locus are to be expected in more than half of the families studied. However, as defects at different loci may be the cause of this phenotype, the results of the segregation analyses should be interpreted with caution, especially in studies involving small families, or mild expressions of the disorder or incomplete penetrance.     

Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete "on" or "off" conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.