Haemophilia (2002), 8, 607–621
Guidelines for the diagnosis and management
of von Willebrand disease in Italy
A. B. FEDERICI, G. CASTAMAN* and P. M. MANNUCCI, FOR THE ITALIAN ASSOCIATION
OF HEMOPHILIA CENTERS (AICE)
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Department of Internal Medicine, IRCCS Maggiore
Hospital, University of Milan, Italy; and *Division of Hematology, S. Bortolo Hospital, Vicenza, Italy
Summary. von Willebrand disease (vWD) is a bleeding disorder caused by quantitative (type 1 and 3) or
qualitative (type 2) defects of von Willebrand factor
(vWF). The molecular basis of type 2And 3 vWD are
now known and those of type 1 vWD are being
understood. Phenotypic diagnosis is based on the
measurements of plasma and platelet vWF, of the
ability of vWF to interact with platelet receptors and
the analysis of the multimeric structure of vWF. Due
to the heterogeneity of vWF defects and the variables
that interfere with vWF levels, a correct diagnosis of
types and subtypes may sometimes be difficult but is
very important for therapy. The aim of treatment is
to correct the dual defects of haemostasis, i.e.
abnormal intrinsic coagulation expressed by low
levels of factor VIII (FVIII) and abnormal platelet
adhesion. Desmopressin is the treatment of choice in
patients with type 1 vWD, who account for approx-
Introduction
von Willebrand disease (vWD) is a bleeding disorder
caused by a quantitative or qualitative defect of von
Willebrand factor (vWF), the high molecular weight
glycoprotein that plays an essential role in the early
phases of haemostasis by promoting platelet adhesion to the subendothelium, and platelet aggregation
under high shear stress conditions [1]. As vWF is also
the carrier of factor VIII (FVIII) in plasma, deficiency
of vWF also results in an impairment of intrinsic
blood coagulation. In the majority of cases, vWD is
congenital, inherited in an autosomal dominant
fashion; an autosomal recessive inheritance is also
Correspondence: Augusto B. Federici, MD, Via Pace 9, 20122.
Milano, Italy.
Tel.: + 39 02 55 03 53 56; fax: + 39 02 55 03 53 47;
e-mail: [email protected]
Accepted 7 June 2002
2002 Blackwell Publishing Ltd
imately 70% of cases, because it corrects FVIII–vWF
levels and the prolonged bleeding time (BT) in the
majority of these patients. In type 3 and in severe
forms of type 1 and 2 vWD patients, desmopressin is
not effective and it is necessary to resort to plasma
concentrates containing FVIII and vWF. Treated
with virucidal methods, these concentrates are effective and safe, but they cannot always correct BT
defect. Platelet concentrates or desmopressin can be
used as adjunctive treatments when poor correction
of BT after plasma concentrate treatment is associated with continued bleeding.
Keywords: congenital von Willebrand disease, desmopressin, factor VIII/ von Willebrand factor concentrates, genetic and molecular diagnosis, von
Willebrand factor.
described in some cases. Patients with vWD may
have a mild, moderate or severe bleeding tendency
since childhood, usually proportional to the degree
of the vWF defect. Patients with a negative family
history and with a recent personal history of bleeding
may have an acquired disease similar to congenital
vWD; this is usually associated with other clinical
conditions and is called acquired von Willebrand
Syndrome (AvWS) [2,3]. In this article, the guidelines
on diagnosis and treatment of vWD approved by the
Members of the Italian Association of Hemophilia
Centers (AICE) are presented.
Biosynthesis, structure and function of vWF
vWF is synthesized in endothelial cells and either
stored in intracellular organelles known as Weibel–
Palade bodies or secreted constitutively. It is also
synthesized in megakaryocytes and stored in platelet
alpha-granules (4). The gene coding for vWF is
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608
A. B. FEDERICI et al.
178 000 bases long, is located on chromosome
12 and contains 52 exons. vWF is synthesized as a
large precursor protein (360 kDa, 2813 amino
acids), which consists of a 22-amino acid signal
peptide, a pro-polypeptide (100 kDa, 741 amino
acids) also known as vWF antigen II, and a mature
subunit (270 kDa, 2050 amino acids). Dimers are
formed in the endoplasmatic reticulum by covalent
dimerization of the subunits at their C-termini.
Multimers are formed in the Golgi apparatus and
the secretory vesicles by covalent multimerization of
these dimers at the N-terminus (Fig. 1). The propeptide of vWF is required for normal multimer
formation and is cleaved off by furin, a dibasic paired
amino acid-cleaving enzyme. vWF is released from
endothelial cells as ultralarge multimers and circulates in the plasma as a series of multimers of very
high molecular weight (500–20 000 kDa). Proteolysis is involved in the generation of these multimers,
and shear stress enhances the susceptibility to proteolytic cleavage. About 1% of plasma vWF contains
the pro-peptide, possibly due to incomplete processing. Recently, it was shown that human plasma
contains a vWF-degrading enzyme, vWF-cleaving
protease (vWF-CP) and that the cleavage site of this
enzyme is located between amino acid residues
842Thr and 843Met in the A2 domain of the vWF
subunit. vWF-CP cleaves ultralarge multimers, normally stored in Weibel–Palade bodies of vascular
endothelium, from which they are secreted luminally
into plasma and abluminally into the subendothelium. Each vWF subunit shows a characteristic pattern
of homologous A, B, C and D domains, which are
independent building blocks in many other proteins.
The pro-peptide contains a D1 and D2 domain. The
mature subunit consists of D¢–D3–A1–A2–A3–D4–
B1–B2–B3–C1–C2 domains, and a C-terminal part
of 151 amino acids that has no internal homology
(Fig. 1). vWF acts as an adhesive glycoprotein and
mediates platelet adhesion to subendothelium
through its binding sites for the platelet receptor
GpIb-a-IX and collagen, and platelet–platelet interactions through its binding site for platelet GpIIb/
IIIa. Additional binding sites are those for heparin
and sulphatides. Apart from its adhesive functions,
vWF serves as a carrier protein for FVIII. By the
noncovalent interaction between the two proteins,
FVIII is protected against binding to membrane
surfaces and to proteolytic attack by a variety of
serine proteases, including activated protein C (5).
Pathophysiology and classification
Inherited vWD has been subdivided into three types,
which reflect its pathophysiology. Type 1 and 3 vWD
reflect, respectively, the partial or virtually complete
deficiency of vWF while type 2 vWD reflects a
qualitative deficiency of vWF. The revised classification introduced in 1994 by Sadler [6], recommends
that type 2 vWD is subdivided into four subtypes
(2A, 2B, 2M, 2N) according to specific details of the
phenotypic features (Table 1). This classification also
addresses the problems of compound heterozygosity
and of disorders related to vWD that are not due to a
defect in the vWF gene, such as Ôplatelet-type/pseudovWDÕ and AvWS.
Fig. 1. Domain structure and processing of von Willebrand factor (vWF). The domain structure (A–D) of prepro-vWF and mature vWF are
shown. In the endoplastic reticulum, carboxy-terminal disulphide-bonded dimers are formed. Disulphide bonds that result in multimerization occur late in the secretory pathway. The two platelet receptors, Gp Ib and aIIb b3, are indicated. Other molecular interactive site
are also depicted.
Haemophilia (2002), 8, 607–621
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DIAGNOSIS AND MANAGEMENT OF VWD IN ITALY
609
Table 1. Classification of vWD.
1
2
3
4
5
6
vWD is caused by mutation of the vWF locus
Type 1 vWD refers to partial quantitative deficiency of vWF
Type 2 vWD refers to qualitative deficiency of vWF
Type 3 vWD refers to virtually complete deficiency of vWF
Type 2A refers to qualitative variants with decreased platelet-dependent function that is associated with the absence of high-molecularweight multimers
Type 2B vWD refers to qualitative variants with increased affinity for platelet GPIb
Type 2M vWD refers to qualitative variants with decreased platelet-dependent function not caused by the absence of high-molecular
weight-multimers
Type 2N vWD refers to qualitative variants with markedly decreased affinity for factor VIII
From an updated version by Sadler [6].
Prevalence
Table 2. Frequency (%) of subtypes of vWD.
vWD is the most frequent inherited bleeding disorder. Nilsson [7] reported a frequency of approximately 125 cases per million in Sweden, twice as
frequent as for haemophilia. Rodeghiero et al. [8],
during a large Italian epidemiological study in
children, found the prevalence to be 0.82%. More
recent studies in different populations confirm a
prevalence of the disease of approximately 1–2%
[9,10]. Among the different vWD types, type 1 is the
most frequent (60–80%); all type 2 vWD variants are
15–30%, while type 3 is diagnosed in 5–10% of
vWD patients [11–15]. For example, in the 1286
vWD patients enrolled into the Italian National
Registry of vWD (ReNaWi), the actual distribution
73% type 1, 21% type 2, and 6% type 3 [16]. The
prevalence of different vWD subtypes as reported in
the literature is shown in Table 2.
Authors
[reference]
No. of
patients
Type 1
Type 2 Type 3
Tuddenham et al. [11]
Lenk et al. [12]
Hoyer et al. [13]
Awidi [14]
Berliner et al. [15]
Federici et al. [16]
134
111
116
65
60
1286
75
76
71
59
62
73
19
12
23
30
9
21
Clinical and phenotypic diagnosis
The diagnostic workup of vWD occurs in three steps:
(i) identification of patients suspected for vWD, on
the basis of the clinical history and results of the
screening tests of haemostasis; (ii) diagnosis of vWD
6
12
6
11
29
6
with indication of its type; and (iii) characterization
of the subtype (Table 3).
Clinical history and screening tests
Clinical history. vWD should be suspected in
patients with a history of mucocutaneous and postoperative bleeding, especially if family history suggests an autosomal pattern of inheritance. The most
common symptoms associated with vWD are epistaxis, bleeding after dental extractions, and menorrhagia. The bleeding tendency, however, is highly
variable and depends on the type and severity of the
disease. In many patients with type 1 or type 2 vWD,
the bleeding tendency may be mild or absent.
In contrast, patients with type 3 vWD have a
Table 3. Clinical and laboratory parameters used for vWD diagnosis.
Patients at risk for vWD
Clinical history: lifelong mucocutaneous and postoperative bleeding. Symptoms are sometimes present in other family members
Screening tests: prolonged bleeding time (maybe normal); normal platelet count; prolonged PTT (may be normal).
Diagnosis and definition of vWD type
vWF antigen
vWF: Ristocetin cofactor activity
Factor VIII
vWF multimeric structure on low resolution gels
Diagnosis of vWD subtype
Ristocetin-induced platelet agglutination (RIPA)
vWF multimeric structure on high resolution gels
Platelet vWF content
Factor VIII binding assay
For the use of these tests see the diagnostic flow-chart reported in Fig. 2.
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Haemophilia (2002), 8, 607–621
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A. B. FEDERICI et al.
moderately severe haemorrhagic tendency; mucosal
bleeding is very frequent and may be life-threatening.
In addition, due to the severe FVIII defect, haematoma and haemarthrosis can occur. Postoperative
haemorrhages are also common, especially in
patients with very low FVIII levels. Data on the
incidence of bleeding symptoms in vWD, as derived
from three published reports [16–18], are summarized in Table 4. Because of the variability of the
clinical history and the relatively low specificity of
the bleeding symptoms, the diagnosis of vWD must
rely also on the results of laboratory tests.
Screening tests. The platelet count is usually normal;
mild thrombocytopenia may occur in patients with
type 2B or Platelet-type/pseudo vWD. The bleeding
time (BT) is usually prolonged, but may be normal in
patients with mild forms of vWD such as those with
type 1 and normal platelet content of vWF [19,20].
The prothrombin time (PT) is normal whereas the
partial thromboplastin time (PTT) may be prolonged
to a variable degree, depending on the plasma FVIII
levels.
Diagnosis of vWD and identification of the type
vWF antigen (vWF:Ag), which can be measured in
plasma by electroimmunoassay, immunoradiometric
assay or a variety of enzyme-linked immunoadsorbent assays, is undetectable in type 3 vWD, whereas
it may be low in type 1 and low or normal in type 2
vWD.
The assay for ristocetin cofactor activity
(vWF:RCo) explores the interaction of vWF with
the platelet glycoprotein Ib/IX/V complex and is still
the standard method for measuring vWF activity. It
is based on the property of the antibiotic ristocetin to
agglutinate formalin-fixed normal platelets in the
presence of vWF. In patients with a normal vWF
structure (type 1 vWD), values of vWF:RCo are
similar to those of vWF:Ag. Levels of vWF:RCo
lower than those of vWF:Ag (ratio vWF:RCo/
Ag < 0.7) are characteristic of type 2 vWD (see later,
Fig. 2).
FVIII:C plasma levels are very low (1–5%) in
patients with type 3 vWD. In patients with type 1 or
type 2 vWD, FVIII may be decreased to a variable
extent but is sometimes normal.
vWF can be analysed by agarose gel electrophoresis. Low-resolution agarose gels separate vWF multimers, which are conventionally indicated as high,
intermediate and low molecular weight. In type 1
vWD all multimers are present, whereas in type 2
vWD, high and intermediate multimers are lacking.
Diagnosis of the subtype
For a diagnosis of patients with vWD and their
correct treatment, other assays are necessary to
define specific subtypes.
Ristocetin-induced platelet agglutination (RIPA) is
measured by mixing different concentrations of
ristocetin and patient platelet-rich plasma (PRP) in
an aggregometer. Results are expressed as the concentrations of ristocetin (mg mL)1) able to induce
30% of agglutination. Most vWD types and subtypes
are characterized by hyporesponsiveness to ristocetin. Important exceptions are patients with type 2B
vWD and Platelet-type/pseudo vWD, characterized
by hyper-responsiveness to ristocetin due to a higher
Table 4. Incidence (%) of bleeding symptoms in Italian, Iranian and Scandinavian vWD patients.
Italian vWD (n ¼ 1286)
Bleeding symptoms
Type 1
(n ¼ 944)
Type 2
(n ¼ 268)
Type 3
(n ¼ 74)
Iranian vWD
Type 3
(n ¼ 348)
Scandinavian
vWD patients
(n ¼ 264)
Epistaxis
Menorrhagia
Bleeding after dental extraction
Hematomas
Bleeding from wounds
Gums bleeding
Postoperative bleeding
Postpartum bleeding
Gastrointestinal bleeding
Petechiae
Joint bleeding
Hematuria
C.N.S. bleeding
56
31
31
14
36
30
20
17
5
n.r.
2
2
0.5
63
32
39
19
40
37
23
18
11
n.r.
5
4
2
74
32
53
31
50
48
41
26
18
n.r
42
11
8
77
69
70
n.r
n.r
n.r.
41
15
20
n.r.
37
1
n.r.
62
60
51
49
36
35
28
23
14
11
8
7
n.r.
Updated data from Federici et al. [16] compared with previous report by Silwer [17] and Lak et al. [18]. n.r., not reported.
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DIAGNOSIS AND MANAGEMENT OF VWD IN ITALY
611
Fig. 2. Flow chart for the diagnosis of von Willebrand disease (vWD). Undetectable plasma levels of vWF antigen (vWF: Ag) (a) are
important to identify type 3 vWD. When vWF: Ag is present, the vWF: Ristocetin cofactor activity (vWF:RCo) (b) is important to
distinguish type 1 or 2 vWD. In fact, in the case of a vWF:RCo/Ag ratio > 0.7, type 1 vWD is diagnosed, characterized by factor VIII
(FVIII:C) (c) levels equal to or higher than vWF; in the case of vWF:RCo/Ag ratio < 0.,7 type 2 vWD is diagnosed. To further characterize
type 2 vWD, ristocetin-induced platelet agglutination (RIPA) (d) is required; when RIPA is increased, type 2B vWD is diagnosed. In case of
decreased RIPA, type 2A or 2M are diagnosed according to the absence or the presence of the high molecular weight vWF multimeric (e).
Type 1 vWD can be further characterized by platelet vWF content (f) and, in case of FVIII levels lower than vWF:Ag, diagnosis of type 2N
vWD should be confirmed by factor VIII binding assay (g).
than normal affinity of vWF for, respectively, platelet
GP Ib/IX/V complex and platelet GPIb/IX/V.
vWF multimeric analysis with high resolution
agarose gels allows better identification of type 1
and type 2 vWD subtypes [2,3].
Platelet vWF plays an important role in primary
haemostasis, because it can be released from alpha
granules directly to the site of vascular injury. On the
basis of its measurement, type 1 vWD may be
classified in three subtypes: type 1 Ôplatelet normalÕ,
with a normal content of functionally normal vWF;
type 1 Ôplatelet lowÕ, with low concentrations of
functionally normal vWF; and type 1 Ôplatelet
discordantÕ, with normal concentrations of dysfunctional vWF [19,20].
FVIII binding assay measures the affinity of vWF
for FVIII. In this assay, anti-vWF antibody is coated
on wells of a microtitre plate and test plasma is
added to the well. The FVIII/vWF complex from the
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plasma is bound by the antibody, following which
FVIII is removed from the complex by a high ionic
strength buffer. Excess recombinant FVIII (rFVIII) is
then added and, after removal of unbound rFVIII,
the vWF and bound rFVIII are assayed. This assay
can permit type 2N vWD to be distinguished from
mild to moderate haemophilia A because FVIII is
abnormal in the former. Differential diagnosis of
vWD subtypes can be performed by using the
methods described above.
Diagnosis of type 3 vWD requires only the
demonstration of no detectable values of vWF:Ag.
Type 1 vWD is characterized by a proportionate
reduction of both vWF:Ag and vWF:RCo with a
RCo/Ag ratio > 0.7, while a type 2 vWD is present
when the vWF:RCo/Ag ratio is < 0.7. According to
the RIPA method, type 2B vWD is characterized by
enhanced RIPA (< 0.8 mg mL)1) while RIPA is
always reduced (> 1.2 Mg mL)1) in type 2A and
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A. B. FEDERICI et al.
2M. Multimeric analysis in plasma is necessary to
distinguish type 2A vWD (lack of the largest and
intermediate multimers) from type 2M vWD (all the
multimers are present as in normal plasma). Type 2N
vWD can be suspected in case of discrepant values
between FVIII and vWF:Ag (ratio < 1), and diagnosis
should be confirmed by the specific test of vWF:FVIII
binding capacity (vWF:FVIIIB). In type 1 vWD, the
ratio between FVIII and vWF:Ag is always ‡ 1 and
the severity of type 1 vWD phenotype can usually be
evaluated by performing platelet vWF measurements. A flow chart to be used in the diagnosis of
vWD is reported (Fig. 2).
Additional tests used in the diagnosis of vWD
include the closure time (CT) and assays of vWF
activity based on binding to collagen (vWF:CB). The
evaluation of CT with the PFA-100 (platelet function
analyser) allows rapid and simple determination of
vWF-dependent platelet function at high shear stress.
This system has been demonstrated as being sensitive
and reproducible for the screening of vWD, even
though the CT is normal in type 2N vWD and
remains abnormal in patients with type 3 vWD
following the infusion of FVIII–vWF concentrates.
The assays for vWF:CB are also available, and the
ratio of vWF:CB and vWF:Ag levels appears to be
useful to distinguish type 1 and 2 vWD [21]. Both
assays are still not well standardized and are not
officially approved by the Scientific Standardization
Committee on vWF of the International Society of
Thrombosis and Haemostasis.
Type 1 vWD
Type 1 is the most frequent form of vWD, inherited
as an autosomal dominant trait in most cases;
recessive trasmission of type 1 vWD defects has also
been shown [22,23]. Patients with type 1 vWD are
characterized by mild to moderate bleeding symptoms, normal or variably prolonged BT and low
levels of vWF:Ag, vWF:RCo and FVIII and normal
multimeric structure. Definite diagnosis requires
documentation of all three factors: inheritance, a
history of bleeding, and low levels of normal vWF.
Limitation of these criteria are the broad normal
range for vWF levels and the variation in these levels
over time [24–26]. Patients with type 1 vWD are very
heterogeneous. As mentioned before, based on the
platelet vWF content, three subtypes have been
identified [19,20]. Patients with low levels of platelet
vWF have more prolonged BT and usually more
severe symptoms than those with normal platelet
vWF, because they have also low levels of vWF
stored in endothelial cells [27]. The diagnosis of type
Haemophilia (2002), 8, 607–621
1 vWD may also be complicated by several factors.
ABO blood groups modify vWF levels in plasma
[28]. vWF levels are higher in older individuals and
in patients with diabetes. There is also evidence for
hormonal regulation of vWF levels, which can be
influenced by oestrogen and thyroid hormones and
are significantly increased during pregnancy; vWF
also appears to fluctuate in response to mild exercise
[29]. A dominant type 1 vWD has been identified
with mutated cysteine residues on the D3 domain of
vWF [30].
Type 2 vWD
Type 2A is the most frequent subtype among type 2
vWD [31]. It is inherited mainly with an autosomal
dominant pattern but a recessive pattern is also
described [32]. Patients with 2A vWD are identified
by normal to slightly reduced vWF:Ag levels and
markedly low vWF:RCo, with an abnormal multimeric pattern characterized by loss of the high
molecular weight multimers and increase of the
intensity of low molecular weight multimers. Type
2A vWD are due to specific mutations located within
the A2 domain of vWF subunit, and data obtained by
expression studies show that two mechanisms are
responsible for this defect [33]. One class of mutations, referred to as group 1, causes defective
intracellular transport of vWF and impairs the
assembly, storage and secretion of large vWF multimers in both plasma and platelets. Group 2
mutations do not interfere with vWF assembly or
secretion, but render the multimers more sensitive to
proteolysis in plasma [34]. Another cause of high
molecular weight deficiency in type 2A vWD is a
defective post-translational processing that includes
defects of dimerization at the vWF C-terminus in
the subtype previously described as type IID [35],
and defects of further polymerization of vWF dimers
to multimers in their N-termini. The latter multimeriztion defects can result either from mutations in
the D1 and D2 domains of the vWF propeptide,
which is necessary to catalyse intermolecular disulphide binding at the D3 domain of mature vWF in
the subtype previously indicated as IIC, or from
mutations in the D3 domain itself in the subtypes
indicated as IIE and IIF as well as in type IIC Miami
[35,36].
Type 2B can be identifed because of heightened
response to ristocetin and absence of large multimers
from plasma [37]. The multimeric structure of
platelet vWF and of vWF produced by cultured
endothelial cells is normal [38]. The inheritance
pattern is mainly autosomal dominant, but cases
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DIAGNOSIS AND MANAGEMENT OF VWD IN ITALY
with apparently recessive pattern have also been
described [39]. A large degree of phenotypic heterogeneity has been identified since the original
description. Typical features for 2B vWD are mild
thrombocytopenia with increased mean platelet volume, prolonged BT, low to normal FVIII, low to
normal vWF:Ag, low vWF:RCo and heightened
RIPA. Thrombocytopenia can be more pronounced
during pregnancy [40,41]. In some families, spontaneous platelet aggregation occurs [42,43]. More than
20 different missense mutations and one small
insertion have been identified in type 2B, all located
within the A1 domain of the vWF subunit, and the
abnormal binding to platelet glycoprotein has been
confirmed by expression studies in the majority of
these mutants [33].
Type 2M (ÔMÕ for multimers) includes variants in
which binding to platelets is impaired but the vWF
multimeric distribution is normal. This phenotype
may be produced by mutations that inactivate
specific binding sites for ligands on platelets or
collagens. Laboratory results generally are similar to
those in type 2A, but there are high molecular weight
forms. The type 2M mutations that have been
characterized are located within domain A1 of the
vWF subunit and show reduced binding to platelet
GpIb in studies of expressed mutants [44–47].
Among this subgroup of vWD variants, patients
with type 2M ÔVicenzaÕ have low levels of vWF
antigen but have larger than normal multimers
(supranormal) in plasma, similar to those observed
in plasma after desmopressin infusion and in endothelial cells and platelets [48]. Candidate missense
mutation in type 2M ÔVicenzaÕ have been identified in
domain D3 [49].
Type 2N vWD (ÔNÕ for Normandy) is characterized
by normal levels of vWF:Ag and vWF:Rco, and
normal multimeric structure, but low plasma FVIII
levels. It therefore resembles haemophilia A, but its
inheritance pattern is not X-linked but autosomal
recessive. Low FVIII levels, at variance with haemophilia, are due to decreased plasma half-life of FVIII,
which cannot bind to vWF as a consequence of an
intrinsic abnormality of vWF [50,51]. Type 2N can
be caused by several missense mutations, all localized
to the D¢ and D3 domains of vWF subunit. Coinheritance of a type 2N mutatiom with a type 1
vWD allele may contribute to the variable expressivity of type 1 vWD [52].
Type 3 vWD
Type 3 (severe) vWD is caused by impaired biosynthesis of vWF and is characterized by undetectable
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613
levels of vWF in plasma and platelets. As vWF is also
the carrier of FVIII, plasma levels of FVIII are very
low (1–5%). As a consequence, patients with type 3
vWD have a severe bleeding tendency, characterized
not only by mucocutaneous haemorrhages but also
by haemarthroses and haematomas as observed in
severe haemophilia. The inheritance pattern of type 3
vWD is autosomal recessive and its prevalence is 1–5
per million population [1–3]. Alloantibodies against
vWF may arise in 5–8% of patients treated with
FVIII–vWF concentrates [53,54]; gene deletions predispose to the formation of alloantibodies [55]. The
presence of antibodies has been demonstrated in
vitro by the capacity of patient plasma to inhibit
RIPA of normal PRP in a time-dependent manner
[53]. In vivo, the antibodies are responsible for a
poor clinical response to replacement therapy. In
some patients with high antibody titres, replacement
therapy not only is ineffective but also may trigger
life-threatening anaphylactic reactions [56,57]. The
most common mutations in type 3 vWD are total or
partial deletions, nonsense, splicing and frameshift
mutations, found throughout the 52 exons of the
vWF gene [58–62].
Platelet-type/pseudo vWD
Platelet-type or pseudo vWD is a primary platelet
disorder, characterized by increased affinity of the
platelet GPIb/IX/V complex for normal vWF [63,64].
These patients have clinical and laboratory features
similar to those of type 2B vWD: BT is prolonged,
FVIII and vWF levels are variably reduced, RIPA is
heightened, multimeric analysis reveals a deficiency
of the high molecular weight multimers. The lack of
high molecular weight multimers is probably due to
increased in vivo utilization, secondary to heightened
interaction between vWF (abnormal in 2B and
normal in platelet-type vWD) and its receptor
(normal in 2B and abnormal in platelet-type vWD).
A way to distinguish platelet-type/pseudo vWD from
2B vWD is to add purified vWF to patient PRP in an
aggregometer. In case of platelet-type/pseudo vWF,
normal vWF will induce platelet aggregation, whereas it will not in type 2B vWD.
Acquired von Willebrand Syndrome
AvWS is similar to the congenital disease in terms of
laboratory findings, being characterized by a prolonged BT and low plasma levels of FVIII–vWF [65].
About 270 cases of AvWS have been reported in the
literature since the original description of a case
associated with systemic lupus erythematosus [66].
Haemophilia (2002), 8, 607–621
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A. B. FEDERICI et al.
The condition appears to be associated mainly with
lympho-myeloproliferative disorders, immunological
diseases and tumours. At variance with other
acquired haemostatic defects (i.e. acquired FVIII
deficiency), the presence of an inhibitor to vWF has
been demonstrated in a relatively small number of
cases. Characterization of the reported cases has
consistently shown that vWF is normally synthesized
but is rapidly removed from plasma through four
possible pathogenic mechanisms [67]: (a) specific
autoantibodies; (b) nonspecific antibodies that form
circulating immunocomplexes and favour vWF clearance by Fc-bearing cells of the reticuloendothelial
system; (c) absorption onto malignant cell clones; or
(d) increased proteolytic degradation. The severity of
the bleeding tendency varies from mild to lifethreatening. The data of an lnternational Registry
of the clinical and laboratory parameters of patients
with AvWS, organized on behalf of the Scientific
Subcommittee on vWF of the International Society of
Thrombosis and Haemostasis, have been published
[68].
Genetic diagnosis and family studies
The aims of genetic diagnosis are to determine the
causative defects of vWF when phenotypic diagnosis
is certain, and make a definite diagnosis of vWD
when its is uncertain. By definition, vWD results
from mutations in the vWF gene. There are two main
approaches to the genetic diagnosis of vWD: direct
mutation detection and linkage analysis. At the
moment, neither represents a first-line investigation
for diagnosis. In the absence of a characterized
mutation within a family, linkage analysis permits a
defective vWF gene to be tracked, thereby corroborating phenotypic diagnosis. Screening for common
genetic mutations can be obtained, mainly in type 2
vWD variants where clusters of mutations have been
identified. In most type 1 and 3 vWD cases, direct
mutation detection requires the sequence of the
entire vWF gene. In these cases, gene tracking by
analysis of restriction fragment length polymorphisms (RFLPs) or variable number of tandem
repeats (VNTR) sequences of the vWF gene is useful
for family studies. An on-line database of vWD
mutations and polymorphisms is maintained by
Ginsburg and colleagues and is accessible at http://
mmg2.im.med.umich.edu [69,70]. Intron 40 of the
vWF gene contains several VNTR sequences within a
region of repetitive DNA. Analysis of these VNTRs is
very informative and useful in gene-tracking studies
in vWD [71]. These techniques have applications in
the prenatal diagnosis of severe vWD, in carrier
Haemophilia (2002), 8, 607–621
identification in recessive forms of vWD, and in type
1 vWD families when the diagnosis is phenotypically
uncertain.
Prenatal diagnosis in vWD
The procedures available for prenatal diagnosis of
vWD are identical to those developed and used for
the prenatal diagnosis of haemophilia [72]. The
preferred method is to obtain fetal tissue for DNA
analysis by chorionic villus sampling after 10–
12 weeks gestation. This procedure has become
accepted in many centres worldwide as it can provide
diagnosis in the first trimester. Alternative methods
include amniocentesis or ultrasound-guided fetal
blood sampling. Blood samples may be analysed for
vWF:Ag as well for DNA markers. As most type 1
and 2 vWD are mildly affected, prenatal diagnosis is
required mainly for the rare families with type 3
vWD in whom the threat of bleeding is similar to
that of haemophilia A. In the few reported cases of
prenatal diagnosis of vWD, the procedure was
performed in a situation where a severely affected
child already present within the family, so that the
fetus was deemed to be at risk [71,73].
Management of patients with vWD
In patients with vWD the goal of therapy is to correct
the dual defect of haemostasis, i.e. the abnormal
platelet adhesion and abnormal intrinsic coagulation
due to low FVIII levels. There are two treatments of
choice in vWD, i.e. desmopressin and transfusional
therapy with blood products. Other forms of treatment can be considered as adjunctive or alternative
to these [1–3,74,75].
Desmopressin
Desmopressin (1-deamino-8-d-arginine vasopressin;
DDAVP) is a synthetic analogue of vasopressin
originally designed for the treatment of diabetes
insipidus. DDAVP increases FVIII and vWF plasma
concentrations without important side-effects when
administered to healthy volunteers or patients with
mild haemophilia and vWD [76]. The obvious
advantages of DDAVP is that it is relatively
inexpensive and carries no risk of transmitting
blood-borne viruses. DDAVP is usually administered
intravenously at a dose of 0.3 lg kg)1 diluted in
50 mL saline, infused over 30 min. This treatment
increases plasma FVIII–vWF 3–5 times above the
basal levels within 30 min. In general, high FVIII–
vWF concentrations last for 6–8 h. As the responses
2002 Blackwell Publishing Ltd
DIAGNOSIS AND MANAGEMENT OF VWD IN ITALY
in a given patient are consistent on different occasions, a test dose of DDAVP administered at the time
of diagnosis helps to establish the individual response
patterns. Infusions can be repeated every 12–24 h
depending on the type and severity of the bleeding
episode [77,78]. However, most patients treated
repeatedly with DDAVP become less responsive to
therapy [79]. The drug is also available in concentrated forms for subcutaneous and intranasal administration, which can be convenient for home
treatment. The protocol of DDAVP test infusion
with the clinical and laboratory parameters to be
used to evaluate the biological response in each
patient are summarized in Table 5; the definition of
response to DDAVP is also reported together with
the list of DDAVP products commercially available
in Italy.
Side-effects of DDAVP are usually mild tachycardia, headache, flushing: these symptoms are attributed to the vasomotor effects of the drug and can
often be attenuated by slowing the rate of infusion.
Hyponatraemia and volume overload due to the
antidiuretic effects of DDAVP are relatively rare. A
few cases have been described, mostly in young
children who received closely repeated infusions
[80]. Even though no thrombotic episodes have been
reported in vWD patients treated with DDAVP, this
drug should be used with caution in elderly patients
with atherosclerotic disease, because a few cases of
myocardial infarction and stroke have occurred in
haemophiliacs and uraemic patients given DDAVP
[81,82].
DDAVP is most effective in patients with type 1
vWD, especially those who have normal vWF in
storage sites (type 1, platelet normal). In these
patients FVIII, vWF and BT are usually corrected
615
within 30 min and remain normal for 6–8 h. In other
vWD subtypes, responsiveness to DDAVP is variable. A poor and short-lasting response is observed in
type 1, platelet low [20]. In type 2A, FVIII levels are
usually increased by DDAVP but BT is shortened in
only a minority of cases. DDAVP is contraindicated
in type 2B because of the transient appearance of
thrombocytopenia [83]. However, there have been
reports on the clinical usefulness of DDAVP in some
2B cases [84,85]. In type 2N, relatively high levels of
FVIII are observed following DDAVP, but released
FVIII circulates for a shorter time period in patient
plasma because the stabilizing effect of vWF is
impaired [86]. Patients with type 3 vWD are usually
unresponsive to DDAVP.
Other nontrasfusional therapies for vWD
Two other types of nontransfusional therapies are
used in the management of vWD, i.e. antifibrinolytic
amino acids and oestrogens. Antifibrinolytic amino
acids are synthetic drugs that interfere with the lysis
of newly formed clots by saturating the binding sites
on plasminogen, thereby preventing its attachment to
fibrin and making plasminogen unavailable within
the forming clot. Epsilon aminocaproic acid
(50 mg kg)1 four times a day) and tranexamic acid
(25 mg kg)1 three times a day) are the most frequently used antifibrinolytic amino acids. Both medications can be administered orally, intravenously or
topically and are useful alone or as adjuncts in the
management of oral cavity bleeding, epistaxis, gastrointestinal bleeding and menorrhagia. As drugs
that inhibit the fibrinolytic system, they carry a
potential risk of thrombosis in patients with
an underlying prothrombotic state. They are also
Table 5. Products containing Desmopressin commercially available in Italy.
Product (company)
Emosint(Kedrion)
Volume
per ampoule
Number of
ampoules per pack
4 lg
0.5 mL
10
20 lg
40 lg
4 lg
1.0 mL
1.0 mL
1.0 mL
10
10
10
Preparation
Comments
Concentrated ampoules
can also be administered
subcutaneously
Minirin DDAVP
(Ferring/Valeas)
Infusion test with desmopressin (DDAVP)
Infusion protocol: administer over 30 min 0.3 lg kg)1 of DDAVP in 50 mL of saline. The same dosage can be administered also
subcutaneously.
Clinical and laboratory parameters: factor VIII/vWF activities must be measured before and 0.5, 1, 2 and 4 h after the injection of
DDAVP; bleeding time must be performed at least before and after 2 h.
Definition of responsiveness: vWD patients should be considered responsive to DDAVP when after 2 h they showed increases of baseline
levels of FVIII:C and vWF:RCo of at least three-fold, with levels of at least 30 U dL)1 and bleeding time of 12 min or less.
2002 Blackwell Publishing Ltd
Haemophilia (2002), 8, 607–621
616
A. B. FEDERICI et al.
contraindicated in the management of urinary tract
bleeding. Oestrogens increase plasma vWF levels, but
the response is variable and unpredictable, so that
they are not widely used for therapeutic purposes. It
is common clinical experience that the continued use
of oral contraceptives is very useful in reducing the
severity of menorrhagia in women with vWD, even
in those with type 3, despite the fact that FVIII–vWF
levels are not modified.
Tranfusional therapies
Transfusional therapy with blood products containing FVIII–vWF is at the moment the treatment of
choice in patients unresponsive to DDAVP. Early
studies indicated that cryoprecipitate administered
every 12–24 h normalized plasma FVIII levels,
shortened BT, and stopped or prevented clinical
bleeding in vWD [87]. Based on these observations,
cryoprecipitate has been the mainstay of vWD
therapy for many years. However, a recent analysis
of previously published reports has pointed out that
BT is not always corrected following cryoprecipitate
[88]. Virucidal methods cannot be applied to cryoprecipitate, therefore this product carries a small but
definite risk of transmitting blood-borne infections.
Therefore, virus-inactivated concentrates, originally
developed for the treatment of haemophilia A, play
an important role in the current management of
vWD patients unresponsive to DDAVP. Concentrates obtained by immunoaffinity chromatography
on monoclonal antibodies (FVIII > 2000 IU mg)1)
contain very small amounts of vWF and are therefore
unsuitable for vWD management. Recently, a concentrate particularly rich in vWF and with a very low
content of FVIII has also been produced [89]. This
concentrate was clinically effective when tested in a
small group of type 3 vWD cases [90]. The very low
FVIII content in this product necessitates the infusion
of purified FVIII concentrate to ensure haemostatic
levels of this factor in the treatment of acute bleeding
episodes and for emergency surgery. After 6 h,
infused vWF triggers the endogenous synthesis of
FVIII, so that no further infusion of FVIII-containing
concentrates is necessary. Efficacy and safety of this
concentrate are now under evaluation in a larger
number of patients with different vWD types [91].
The dosages of concentrates recommended for the
control of bleeding episodes are summarized in
Table 6. The characteristics of products containing
FVIII–vWF commercially available in Italy until
December 2001 are summarized in Table 7. As
commercially available intermediate and high-purity
FVIII–vWF concentrates contain large amounts of
FVIII and vWF, high postinfusion levels of these
moieties are consistently obtained [92–95]. Furthermore, there is a sustained rise in FVIII, higher than
predicted from the doses infused, lasting for up to
24 h. This pattern is due to the stabilizing effect of
exogenous vWF on endogenous FVIII, which is
synthesized at a normal rate in these patients [96].
The cumulation of exogenous FVIII infused with
concentrates rich in this moiety, together with that
endogenously synthesized and stabilized by infused
vWF, is associated with very high FVIII levels when
multiple infusions are given at frequent intervals for
severe bleeding episodes or to cover major surgery.
There is some concern that sustained high levels of
FVIII may increase the risk of postoperative deep
vein thrombosis [97].
FVIII–vWF concentrates are not always effective in
correcting BT [98]. There are probably multiple
reasons for the inconsistent effects on BT. So far, no
concentrate contains a completely functional vWF,
as tested in vitro by evaluating the multimeric pattern
and using several functional assays, because vWF
proteolysis occurs during purification due to the
action of platelet and leucocyte proteases contaminating the plasma used for fractionation [99].
Despite their limited and inconsistent effect on BT,
FVIII–vWF concentrates are successfully used for the
treatment of vWD patients unresponsive to DDAVP,
especially for soft-tissue and postoperative bleeding
[52]. When BT remains prolonged and bleeding
Table 6. Doses of factor VIII–vWF concentrates recommended in vWD patients unresponsive to DDAVP.
Type of bleeding
Dose (IU kg)1)
Number of infusions
Objective
Major surgery
50
Once daily or alternate daily
Minor surgery
30
Once daily or alternate daily
Dental extractions
Spontaneous or
post-traumatic bleeding
20
20
Single
Single
Maintain FVIII
complete
Maintain FVIII
complete
Maintain FVIII
Maintain FVIII
> 50 IU dL)1 until healing is
> 30 IU dL)1 until healing is
> 30 IU dL)1 for up to 6 h
> 30 IU dL)1
FVIII, factor VIII.
Haemophilia (2002), 8, 607–621
2002 Blackwell Publishing Ltd
DIAGNOSIS AND MANAGEMENT OF VWD IN ITALY
617
Table 7. Characteristics of products containing FVIII–vWF commercially available in Italy.
Product
manufacturer
Purification
method
Viral inactivation
method
Specific activity*
(U mg)1 protein)
vWF:RCo/
Ag (ratio)
vWF:RCo/
FVIII (ratio)
Additional
proteins
Emoclot D.I.
(Kedrion)
Fanhdi (Grifols)
Ion exchange
chromatography
Affinity
chromatography
(heparin)
Multiple
precipitation
Ion exchange
chromatography
Solv/det + 30 min
at 100 C
Solv/det + 2 h at
80 C
‡ 80
0.61
1.16
Albumin–
> 100
0.83
1.48
Albumin+
40 ± 6
0.96
2.54
Albumin+
100 ± 50
0.47
1.10
Albumin +
Haemate P
(Aventis-Behring)
Immunate (Baxter)
Pasteurization:
10 h at 60 C
Det + vapour heat
10 h at 60,
1 h at 80
* Specific activity measured as FVIII before adding albumin as stabiliser.
vWF:RCo values are not available in the technical description of all concentrates: therefore only the mean values calculated by Producers
on different concentrate stocks could be reported.
Solv/det, solvent/detergent.
persists despite replacement therapy, other therapeutic options are available. DDAVP, given after cryoprecipitate, further shortened or normalized BT in
patients with type 3 vWD in whom cryoprecipitate
failed to correct it [100]. Platelet concentrates (given
before or after cryoprecipitate, at doses of 4–
5 · 1011 platelets) achieved similar effects in patients
unresponsive to cryoprecipitate alone, both in terms
of BT correction and bleeding control [101]. These
data emphasize the important role of platelet vWF in
establishing and maintaining primary haemostasis.
In summary, the therapeutic approaches to vWD
patients according to vWD subtypes are reported in
Table 8.
Treatment of vWD during pregnancy
and delivery
During pregnancy, vWF and FVIII levels tend to rise
in type 1 and 2 vWD but this rise does not occur until
Table 8. Management of different types and subtypes of vWD.
vWD type
Treatment
of choice
Alternative and
adjunctive
Type 1
DDAVP
Antifibrinolytics,
oestrogens
Type 2A
FVIII–vWF
concentrates
FVIII–vWF
concentrates
FVIII–vWF
concentrates
DDAVP
FVIII–vWF
concentrates
Recombinant F VIII
Type 2B
Type 2M
Type 2N
Type 3
Type 3 with
alloantibodies
2002 Blackwell Publishing Ltd
DDAVP, platelet
concentrates
the 10–11th weeks of gestation. No significant
changes occur in patients with type 3 vWD. As
improvements in vWF and FVIII levels during pregnancy are variable, patients should be monitored
during pregnancy and for several weeks after delivery
when levels fall rapidly and may cause late bleeding
[60]. In type 1 vWD, FVIII levels are the best
predictor of the risk of bleeding at delivery. The risk
of bleeding is minimal when FVIII is > 50 U dL)1 but
can be significant when it is lower than 20 U dL)1
[102]. Careful surgical haemostasis along with
effective uterine contraction usually compensates
for prolonged BT. In type 3 vWD, characterized by
prolonged BT and low FVIII levels, replacement
therapy with concentrates is necessary. Patients with
type 2B vWD may develop or aggravate thrombocytopenia during pregnancy [40,41], but it is not clear
whether thrombocytopenia exacerbates clinical
bleeding.
Treatment of patients with alloand auto-antibodies to vWF
For the rare patients with type 3 vWD who develop
anti-vWF alloantibodies after multiple transfusions,
the infusion of vWF concentrates not only is ineffective, it may cause postinfusion anaphylaxis due to the
formation of immune complexes [53–57]. These
reactions may be life-threatening [56,57]. To overcome this reaction, a patient undergoing emergency
abdominal surgery was treated with recombinant
FVIII, because this product, being completely devoid
of vWF, did not cause anaphylactic reactions. Due to
the very short half-life of FVIII devoid of its vWF
carrier, recombinant FVIII had to be administered by
continuous intravenous infusion, at very large doses
Haemophilia (2002), 8, 607–621
618
A. B. FEDERICI et al.
sufficient to maintain FVIII levels above 50 U dL)1
for 10 days following surgery [57].
Future directions
Several aspects of the diagnosis and management of
vWD must be tackled in the near future. Firstly,
better characterization of type 1 vWD, the most
frequent form of vWD is required. This can be
achieved also with the contribution of molecular
biology techniques, which can clarify the basic
mechanisms of this type of vWD. Secondly, a more
precise identification of vWD patients who are
responsive to DDAVP. Thirdly, a better evaluation
of the pharmacokinetics of FVIII:C following FVIII–
vWF concentrate in vWD to establish whether or not
there are indications for pure FVIII–vWF concentrates devoid of FVIII:C. Most of these questions will
be answered when the results of several ongoing
international studies become available in the next
few years.
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