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The molecular basis ofvon Willebrand disease 233
Propeptide VWF mature subunit
2813 a.a.
5
Pseudogene chromosome 22
https://t.me/med1917
Gene organization, synthesis,
andmultimeric structure ofvon
Willebrand factor
The VWF gene (VWF) is located in chromosome 12p13.3,
has a length of approximately 178 kb with 52 exons and transcribes a messenger RNA of about 8.2 kb. Genetic investigation of the VWF is complicated by the existence of a partial
unprocessed pseudogene in chromosome 22q11.2. The pseudogene extends from exons 23–34 and shares 97% of homology with the VWF. VWF is synthesized exclusively in
megakaryocytes and ECs as a precursor of 2813 amino acids
(pre- pro- VWF). It is composed of a 22- amino acid signal
peptide, a 741- amino acid propeptide, and a 2050- amino acid
mature subunit. More than 95% of the pro- VWF sequence
refers to structural domains arranged in the following order:
D2- D′- D3- A1- A2- A3- D4- C1- C2- C3- C4- C5- C6- CK
D1(Figure17.2). The biosynthesis of VWF is a complex process
and several posttranslational modifications are necessary for
the newly synthesized pre- pro- VWF to become the large
hemostatically active glycoprotein called VWF (Figure17.3).
The signal peptide is immediately removed in the endoplasmic reticulum (ER). Throughout the pro- VWF synthesis, the
formation of intrasubunit disulfide bonds of the cysteine residues guided the folding of the different domains. Pro- VWF
dimerization spontaneously takes place by the formation of
intersubunit disulfide bonds between the cysteine residues in
the carboxyl-
terminal cystine knot (CK) domains (tail- to- tail
dimerization). Also, the N- linked glycosylation process of
pro- VWF is initiated in the ER compartment. Once transported to the Golgi apparatus, the pro- VWF dimers go
through structural rearrangements. Due to the slightly acid
pH (6.2) and the increased Ca
++
concentrations, the domains
of each subunit line up from the carboxy- terminal CK up to
the A2 domains in a tight conformation, the so- called dimeric
bouquet structure (Figure17.3). Multimerization is mediated
by the propeptide, through the formation of intersubunit
disulfide bonds at the N- terminal D3 domains of adjacent
dimers (head- to- head multimerization). Meanwhile, the propeptide is cleaved by furin but remains non- covalently
attached to the mature subunit. During the multimerization
process, the dimeric bouquets form a compact spiral tubular
structure, where the D1- D2 and the D′- D3 domains of each
dimer are the wall of this tubular conformation and the A1CK portions protrude outward from the structure itself
(Figure 17.3). A further N- linked glycosylation of VWF
occurs in the Golgi apparatus along with its O- linked glycosylation, sialylation and sulfation. In the Trans- Golgi network, a portion of the newly synthesized VWF is secreted
basolaterally, as low- molecular- weight molecules, promoting
Gene chromosome 12
51 introns 178 kb
1
ʹ
2A*
(IIC)
SP
NH2
Figure17.2 Structure of the von Willebrand factor (VWF) gene, pseudogene, and protein precursor. The schematic structure of the
pre- pro- VWF is reported along with the homologous repeated domain. Locations of intersubunit disulfide bonds involved in dimerization
andmultimerization are shown together with the binding sites for several ligands. Arrows point out the position of von Willebrand disease (VWD)
type 2mutations. Recessive VWD type 2 variants are indicated by an *.
D1 D2 D3
2A*
(IIC)
11 28
2A
2N* 2N* 2B 2M 2M
Dʹ
FVIII
Multimer
(IIE)
GPIbα
collagen
type IV and VI
S-S
23 24
2A
(IIA)
ADAMTS13
type I and III
Collagen
38 52
3ʹ
2A
(IID)
D4 C1 C2 C3 C4 C5 C6 COOHCKA1 A2 A3
GPIIb/IIIa
Dimer
S-S
本书版权归John Wiley & Sons Inc.所有
234 Molecular Hematology
SP
of the pre-pro-VWF
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ER
Biosynthesis
S
S
NH2
Trans-Golgi
network
and WPBs
D1 D2 D3 D4
Dʹ
multimer
S-S
propeptide
VWF mature subunit
N-linked and O-linked
glycosylation
A1
DʹD3
D1D2
D4
A2
A3
C1-C6
CK
S
S
pH-regulated
dimeric bouquet
formation
Helix
Multimer organizes
S
S
into a helical
tubular structure
Secretion
Furin
cleavage
S
S
C1 C2 C3 C4 C5 C6 COOHCKA1 A2 A3
Golgi
Helix
S
S
DʹD3
D1D2
Dimer
S-S
A1
A2
D1D2
Dimerization
of pro-VWF and
N-linked glycosylation
A3
C1-C6
D4
ER
Vascular lumen
CK
S
S
C1-C6
CK
S
S
DʹD3
S
S
A1
A2
A3
D4
DʹD3
Figure17.3 Processing steps of the von Willebrand factor (VWF) biosynthesis, with multimers assembly in tubular helix structures
and their release into the vascular lumen. The Pre- pro- VWF is synthesized in the endoplasmic reticulum (ER), where the signal peptide (SP)
is removed. Pro- VWF dimerization takes place by formation of intersubunit disulfide bonds at the CK domains (tail- to- tail dimerization). The
N- linked glycosylation process of pro- VWF is also initiated in the ER. The slightly acid pH in the Golgi apparatus rearranges the conformation of
pro- VWF dimers, forming the so- called dimeric bouquets. Further N- linked glycosylation of dimers occurs along with their O- linked glycosylation.
The propeptide mediates dimers multimerization by intersubunit disulfide bonds formation at the N- terminal D3 domains (head- to- head
multimerization). The propeptide is cleaved by furin, but remains attached to the mature subunit. In the trans- Golgi network, during the
multimerization process, the dimeric bouquets form compact spiral tubular structures so that the VWF molecules are packaged into ordered
tubules within the Weibel–Palade bodies (WPBs). Upon secretion into the circulation, VWF unfolds from its compact tubular structure as a
result of blood shear flow. Source: Adapted from Springer T.A. (2011). Biology and Physics of von Willebrand Factor Concatamers. Journal of
Thrombosis and Haemostasis 9: 130–143.
its accumulation in the subendothelial matrix. The remaining VWF molecules are packaged into ordered tubules
within specific storage organelles, called Weibel–Palade
bodies (WPBs). The size of VWF multimers in the WPBs
exceeds the size of the high molecular weight multimers
(HMWM) circulating in plasma and are therefore called
ultra- large VWF (ULVWF). The secretion of ULVWF from
WPBs in the vessel lumen occurs either via basal or
S
S
regulated secretory pathways. Upon release into the circulation, the ULVWF persist only for a limited time, because
ADAMTS13 (a disintegrin and metalloprotease with a
thrombospondin type 1motif, member 13) physiologically
cleaves these molecules, reducing their size to HMWM. At
variance from the ECs, ULVWF is stored in the α- granules
in megakaryocytes and platelets, which release their content
only after platelet activation.
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The molecular basis ofvon Willebrand disease 235
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von Willebrand disease andits
classification
The most common symptoms of mild VWD are mucosal
bleeding (epistaxis, gingival bleeding, menorrhagia) and prolonged bleeding after surgical procedures and dental extractions. Hemarthroses and soft- tissue hematomas are rare, but
they occur in severely affected individuals (type 3). VWD
diagnosis is suspected in individuals with the aforementioned
symptoms and a family history of bleeding. Several VWF
assays are used in the diagnosis and classification of VWD.
The initial laboratory assessment includes FVIII coagulant
activity (FVIII:C), VWF antigen (VWF:Ag), and plateletdependent VWF activity (from now on VWF activity), measured historically using the antibiotic ristocetin and formalin
fix or lyophilized platelets (VWF ristocetin cofactor activity,
VWF:RCo). Recently, several new assays, not using platelets
with or without ristocetin, have been developed to measure
the VWF activity. The wide availability of assays allows many
laboratories to evaluate this important VWF function. A disproportionately low VWF activity to VWF:Ag reflects the loss
of HMWM, or a specific VWF GPIbα binding defect, as in the
case of some variants in the A1 domain. Additional tests are
necessary for VWD classification, although some of these are
not yet standardized and are performed only in specialized
laboratories. VWF collagen binding activity (VWF:CB) measures the capacity of VWF to bind collage (typically type I or
type III). A VWF:CB value that is disproportionately low compared with VWF:Ag, reflects the loss of HMWM or can reflect
a specific VWF collagen binding deficiency, as in the case of
mutations in the A3 domain. VWF multimer analysis evaluates the distribution of VWF oligomers in the plasma through
an SDSUsing a non- reducing low- resolutions (1.2% agarose/0.1%
sodium dodecylsulfate) gel, it is possible to appreciate the lack
of high and intermediate- molecular- weight multimers or the
presence of ULVWF. Using a non- reducing intermediateresolutions (1.6% agarose/0.1% sodium dodecylsulfate) gel,
individual VWF oligomers from normal plasma appear as a
triplet, consisting of a main band flanked by two satellite bands,
being the products of ADAMTS13 proteolysis. Measurement
of VWF binding capacity to FVIII (VWF:FVIIIB) is crucial to
diagnose type 2N VWD patients. Ristocetin- induced platelet
agglutination (RIPA) assay evaluates the VWF affinity to
platelet GPIbα. This assay is performed using an aggregometer, measuring patient platelet- rich plasma agglutination at
different concentrations of ristocetin. RIPA test is important
to identify type 2B variants and in particularly to differentiate
type 2B (enhanced RIPA) from type 2A (decreased RIPA). The
evaluation of VWF propeptide (VWFpp) plasma level is useful to establish the VWF clearance rate by determining the
steady- state VWFpp to VWF:Ag ratio. Evaluation of VWF
platelets content, by measuring VWF:Ag and VWF activity,
agarose electrophoresis (western blot technique).
Table17.1 Current classification ofvon Willebrand disease
Type Characteristic
1 Quantitative decrease in VWF with preserved ratios
between VWF:Ag, VWF activity, and FVIII:C;
normal multimer distribution
1C Quantitative decrease in VWF with preserved ratios
between VWF:Ag, VWF activity, and FVIII:C;
increased VWFpp compared with VWF:Ag
2A Decreased platelet-
of high- molecular- weight multimers
2B Increased binding of VWF to GPIbα, often leading to
thrombocytopenia
2M Decreased platelet- dependent VWF activity with
preserved multimer pattern
2N Decreased binding of VWF to FVIII
3 Absence or near absence of VWF
VWF, von Willebrand factor; VWF:Ag, VWF antigen; FVIII:C, Factor
VIII clotting activity; GPIbα, glycoprotein Ibα; VWFpp, propeptide.
Source: Adapted from James etal. (2021) ASH ISTH NHF WFH 2021
guidelines on the diagnosis of von Willebrand disease. Blood
Advances 5: 280–300.
dependent VWF activity with loss
might be useful to discriminate among different VWD types.
This wide variety of measurements reflects the fact that none
of them is by itself sensitive and specific enough for a VWD
diagnosis. VWD is a highly heterogeneous disease in which
there are quantitative and qualitative abnormalities of VWF,
caused so far, only by mutations at the VWF locus. The classification identifies three basic types (Table17.1). Type 1 is
characterized by the partial quantitative deficiency of VWF.
Quite recently, a new subtype of type 1, type 1C, has been
introduced. Type 2 is characterized by qualitative abnormalities of VWF and is further categorized into subgroups (2A,
2B, 2M, and 2N) depending on the nature of the qualitative
defect (see later). Type 3 is the most severe and rare form of
the disease characterized by the complete absence of VWF in
plasma and platelets. Transmission of types 1 and 2 VWD is
usually dominant, whereas transmission of type 3 is usually
considered to be recessive (Figure17.4A and B).
Genetic defects invon Willebrand
disease
In the last two decades, many molecular defects of the VWF
have been identified in VWD patients with more than 1100
different mutations reported. At first, most of the mutations
were identified in patients with the functional variants (type
2A, 2B, 2M and 2N), then in type 3, and at last, in type 1
VWD. The unique phenotypes of the type 2 variants made it
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236 Molecular Hematology
(A)
Ch12
Ch12 Ch12 Ch12 Ch12
Ch12
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Ch12 Ch12 Ch12 Ch12
Ch12 Ch12 Ch12 Ch12 Ch12 Ch12
Ch12 Ch12 Ch12 Ch12 Ch12 Ch12 Ch12
(B)
Ch12 Ch12
Ch12 Ch12 Ch12 Ch12
Ch12 Ch12 Ch12
Ch12 Ch12 Ch12 Ch12
Ch12 Ch12 Ch12 Ch12 Ch12 Ch12Ch12 Ch12
Ch12 Ch12 Ch12 Ch12 Ch12 Ch12
Figure17.4 (A) A family pedigree with an autosomal dominant segregation, as in von Willebrand disease (VWD) type 1, 2A(IIA), 2A(IID), 2A(IIE),
2B, and 2M. (B) A family pedigree with an autosomal recessive segregation, as in VWD type 2A(IIC), 2N, and 3.
Ch12 Ch12
本书版权归John Wiley & Sons Inc.所有
The molecular basis ofvon Willebrand disease 237
https://t.me/med1917
possible to restrict the genetic analysis to the exons encoding
for specific functional domains, such as the A1 domain for
the 2B and most 2M variants, A2 for the more common variant of 2A and D′- D3 for 2N variants. On the other hand,
characterization of molecular defects in types 1 and 3
requires extensive screening because mutations are scattered
throughout the VWF. In addition, while the identification of
nonsense mutations or small deletions, more common in
type 3, can undoubtedly be considered the cause of the disease, the identification of missense mutations, which are
more common in type 1, requires supplementary studies to
confirm their relationship with the disease. This results in
additional complexity in the molecular characterization of
the most common type of VWD (i.e. type 1). Type 2 is usually caused by the missense mutations, although small inframe deletions or insertions have been reported. Type 3
VWD mostly results from null alleles (nonsense, splice- site
mutations, small deletions, small insertions and, more rarely,
large gene deletions). Nevertheless, numerous missense
mutations have also been identified in this type. In type 1,
the opposite seems to be the case, where the majority of
mutations are missense with a minority of splice- site mutations, small deletion and insertion (sometimes in- frame),
and nonsense mutations. In the last decade, with the diffusion of new molecular techniques such as next- generation
sequencing along with online databases and in silico analysis,
molecular characterization of patients has improved significantly. Multiplex Ligation- dependent Probe Amplification
analysis allows the identification of large deletions or duplications in the whole VWF coding region in a single assessment. NGS enables investigation of the entire VWF coding
region in just a few days, making the molecular characterization of VWD types 1 and 3 as straightforward as type 2. The
use of online databases reporting previously identified VWD
mutations, along with databases containing single nucleotide
variants and their frequency in different populations group,
help to confirm, sustain, or exclude the pathogenicity of an
identified variant. In addition, over the past few years, predicting analytics software for evaluating the negative impact
of candidate missense, synonymous, and splicing variants
has advanced remarkably.
Type 1 VWD
Type 1 is the most common type of VWD, comprising
60–70% of cases and is caused by mild to moderately severe
quantitative deficiency of VWF. It is characterized by proportionately decreased levels of VWF antigen and activities
in plasma, and normal multimeric structure, although some
cases show a slightly decreased level of HMWM. Patients
usually present with mild to moderate bleeding symptoms.
The diagnosis of VWD, especially type 1, is challenging due
to several factors including natural variations in VWF
plasma levels related to the influence of environmental and
genetic factors, such as exercise, thyroid hormone, estrogens,
and ABO blood type. For example, VWF levels are about
25% lower in individuals with blood group O compared to
those with other blood groups. Also, the absence of a clear
cutoff value for VWF levels and a complex VWD diagnostic
test panel further complicate the diagnosis of VWD type 1.
However, the recent joint ASH ISTH NHF WFH 2021 guidelines on VWD diagnosis provided a clarification on type 1
VWD diagnosis. Accordingly, it is recommended classifying
patients with type 1 if VWF level is <30
IU/dL regardless of
bleeding, and for patients with abnormal bleeding if VWF
level is <50 IU/dL. Subjects with VWF levels between 30 and
50 IU/dL without significant bleeding are labeled as “low
VWF” (see later).
Over the last two decades, molecular epidemiological
studies of more than 800index cases with type 1 VWD have
significantly improved our understanding of type 1, particularly its genetic basis. The DNA Sanger sequencing or NGS
approach of the whole coding region and exon/intron
boundaries, along with the promoter region of the VWF, has
allowed the identification of causative variants in about
60–65% of these patients. At variance with type 2 but similar
to type 3 VWD, mutation of type 1 VWD are spread throughout the VWF (Figure17.5). More than 250 different VWF
variants have been reported to be associated with type 1,
most of them are missense variants (64%), followed by
splice- site (15%), promoter sequence variations (5%), small
deletions (5%), nonsense (4%), small insertions (3%), gene
conversions (2%), and large deletion (2%) mutations. Type 1
VWD is usually due to only one affected allele and is inherited in an autosomal- dominant manner; however, approximately 15% of patients have more than one candidate variant,
either homozygous or compound heterozygous. The latter
case often create a “severe” VWD type 1with VWF values
similar to that of type 3. In such circumstances, the distinction between type 3 and type 1might be difficult and the
evaluation of the VWFpp level, detectable in type 1 but not
in type 3 patients, could be helpful to establish the correct
diagnosis. Emerging data from previous studies on type 1
VWD indicate that VWF mutations are more likely to be
detected in cases with lower VWF levels (VWF:Ag <30
IU/
dL). Several large multicenter investigations of type 1 VWD
had shown that 80–90% of patients were found to have VWF
mutations when VWF:Ag was <30 IU/dL. However, only
about 50–60% of patients with VWF:Ag between 30 and
50 IU/dL were found to carry pathogenic VWF variants.
The underlying pathogenic mechanisms causing type 1
VWD can be divided into (i) decreased VWF synthesis, (ii)
impaired VWF storage and secretion, and (iii) accelerated
VWF clearance.
i The decreased VWF synthesis is typically due to heterozygous form of nonsense mutations, frameshift mutations
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238 Molecular Hematology
Percentage (%)
Type 1Type 32A 2B 2M 2N
1 2813
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100
90
80
70
60
50
40
30
20
10
0
SP A1
Figure17.5 von Willebrand factor (VWF) domain distribution of the mutations reported in von Willebrand disease (VWD). Schematic
representation of the pre- pro- VWF domains from amino acids 1–2813 is shown in light blue. More than 1100mutations have been identified in
patients with VWD. While VWF mutations in type 2 VWD are concentrated in certain functional VWF domains, mutations in type 1 and type 3
VWD are distributed throughout all VWF domains.
Dʹ-D3
A2 A3 D4D1-D2 C1-C6 CK
caused by small deletions or insertions and by splice site
mutations, where ultimately leading to a null allele (e.g.
c.2435delC, p.Gln2544*). Of note, some of type 1 VWD variants are shared with type 3. In case of heterozygosity for a
null allele, the production of protein is only from the nonmutated allele; thus, there is an expected reduced synthesis
to 50% of the normal VWF production. Considering the
normal range of VWF level in the general population (i.e.
50–150 IU/dL), heterozygous for a null allele variant in combination with other VWF level modulators like blood group
may create levels of about 25–75 IU/dL, therefore, resulting
in type 1 VWD in some cases (haploinsufficiency), but without VWF levels of diagnosis criteria in a larger proportion of
the cases. More than 30 splicing variants are reported in type
1 VWD that might lead to either exon skipping, cryptic
spliceframe deletions or truncated proteins. Small deletions and
insertions often lead to a frameshift, which generates a premature stop codon usually within a few amino acids (e.g.
p.Pro812Argfs*31).
ii In some type 1 patients, VWF synthesis is normal and the
underlying mechanism could be the impaired VWF storage
and secretion. In this instance, these variants cause substantial structural changes in VWF, resulting in VWF retention in
the ER or Golgi (followed by proteasome degradation of
mutant VWF) and decreased WPBs exocytosis. The
dominant- negative effect of some heterozygous mutations
plays a significant role in VWD etiology. Due to the quaternary structure of VWF mutation expressed in one subunit
site activation, or intron retention, resulting in in-
might affect the wild-
type subunit as well, resulting in an
abnormal VWF (e.g. type 2A, 2B, and 2M). This effect has
also been reported in type 1 VWD, such as p.Cys1149Arg and
Cys1130Phe. In this case, the mutated subunits compromised
the intracellular survival of about 50% of the wild- type subunits, resulting in the reduced quantity of secreted VWF.
Several variants have been reported to create reduced VWF
secretion such as p.Arg782Gln, p.Cys1130Phe, p.Cys1149Arg,
p.Ser1285Pro, p.Val1822Gly, and p.Cys2693Tyr. There are
also other variants that disrupt tubule formation and storage
of VWF, resulting in less exocytosis of WPBs and ultimately
less VWF release (e.g. p.Cys2190Tyr, p.Ala1716Pro).
iii The enhanced VWF clearance is confirmed in about 30% of
type 1 VWD cases in several cohorts and the recent joint guidelines designated these patients as type 1C (clearance). Essentially,
missense variants are responsible for the shorter half-
life of
VWF observed in type 1 VWD, mainly located in the VWF D3
domain (e.g. p.Arg1205His/Cys/Ser, p.Cys1130Phe/Gly/Arg,
p.Trp1144Gly, and p.Cys1149Arg). Nevertheless, variants in the
A1, A3, and D4 domains have also been associated with
increased clearance in VWD. This enhanced clearance is characterized by either an elevated VWFpp/VWF:Ag ratio or a rapid
elimination of VWF from plasma following desmopressin
administration. Variant p.Arg1205His (also known as Vicenza)
is always characterized by low levels of VWF:Ag, normal platelet VWF, elevated VWFpp/VWF:Ag ratio (often >10) and
plasma ULVWF. Compared with healthy controls, the half- life
of VWF Vicenza is reduced 4.4- fold after desmopressin administration. In addition, rapid clearance decreases the time during
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The molecular basis ofvon Willebrand disease 239
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which a large, circulating VWF multimer can be cleaved by
ADAMTS13, resulting in partial preservation of ULVWF.
Mutation p.Ser2179Phe is also characterized by a short VWF
half- life after desmopressin, although not to the same extent as
mutation p.Arg1205His. The fast removal of p.Arg1205His was
found to be mainly macrophage- mediated and the macrophage
scavenger receptor SR- AI was recently reported to contribute to
the accelerated removal of mutated VWF for p.Arg1205His and
p.Ser2179Phe.
The most frequently occurring type 1 VWD variant is
p.Tyr1584Cys (located at the A2 domain), which does not
share the same etiology as of those described above. This
variant is associated with VWF:Ag levels of around 40 IU/dL
with an incomplete penetrance and variable bleeding severity. Animal model studies on p.Tyr1584Cys showed a mild
impaired secretion and enhanced ADAMTS13 cleavage.
Apparently, only its co- inheritance with the blood group O
results in type 1 VWD. Of note, there is still some debate
regarding the pathologic role of this variant.
Although in the recent joint guidelines of VWD, “low
VWF” has not been recognized as an official VWD type,
other guidelines, such as those of the National Heart, Lung,
and Blood Institute and the United Kingdom Haemophilia
Centre Doctors Organization, describe these subjects as a
separate category with increased bleeding risks. “low VWF”
(i.e. with VWF plasma levels of 30–50 IU/dL) is, perhaps, the
most frequent type of quantitative VWF deficiency. Based on
two independent studies, only 44% and 40% of low VWF
cases had potential VWF variants. Accordingly, other genes
may be involved in the mild reduction of VWF levels that
have not yet been identified. Indeed, genome- wide association studies have identified more than 10novel loci (in addition to ABO blood group genes and VWF), such as STXBP5
(syntaxin- binding protein 5), CLEC4M (C- type lectin
domain family 4– member M) and STAB2 (Stabilin- 2) that
are associated with variations in VWF plasma levels. Almost
half of people with “low VWF” do not have significant bleeding tendency and the reduced VWF levels do not correlate
with bleeding severity. In contrast with type 1 VWD, they
have an incomplete penetrance. In addition to reduced VWF
synthesis/secretion and enhanced VWF clearance, aberrantly glycosylated VWF, due to exposure to terminal galactose on VWF, was also found to be an underlying mechanism
of low VWF levels.
Type 2 VWD
Type 2 VWD, as a result of qualitative defects of VWF,
accounts for approximately 20–35% of all VWD cases. Type 2
VWD (2A, 2B, 2M, and 2N) etiology is complex and a variety
of VWF functional abnormalities, including multimerization
defects and altered interactions with GPIbα, ADAMTS13,
FVIII, or collagen can be observed.
Due to this distinct etiology of each VWD type 2, the correct diagnosis of these VWD types requires a battery of tests.
Differently from VWD types 1 and 3, the VWF mutations are
localized in some specific functional domains of VWF and
similarly to type 3 VWD, but at the variance of type 1, mutations are identified in almost all patients.
Type 2A
Type 2A is perhaps the most common subtype of
VWD type 2 and it is characterized by reduced
VWF- dependent platelet adhesion and therefore severely
decreased VWF activity/VWF:Ag ratio. Type 2A is due to
reduction or absence of the most biologically active VWF
forms, the high- and intermediate- molecular- weight multimers (Figure17.6A). Almost 180 different type 2A mutations have been ascribed in the literature and the majority
of them are missense with few exceptions of small in- fame
deletions or splice variants. In the current classification of
VWD, several different phenotypes with a common feature
of defective multimerization, are designated as VWD type
2A. However, these distinct phenotypes which have been
described in the previous classification of VWD as IIA, IIC,
IID, and IIE, are due to different molecular etiologies.
The most common form of type 2A (originally designated
as type IIA) is due to mutations in the binding site of
ADAMTS13 on VWF, i.e. A2 domain (Figure17.5). Affected
patients show a lack of the VWF high- and intermediatemolecular- weight multimers, markedly pronounced satellite
bands (Figure17.6B) with a dominant inheritance. Because
some type 2B patients have a similar pattern of multimer loss,
hence the use of RIPA assay or genetic testing is required to
discriminate between these two types. At variance with type
2B, where platelet- rich plasma agglutinates at lower concentrations of ristocetin (<0.7 mg/mL; normal range 0.8–1.2 mg/
mL), in type 2A, a higher concentration of ristocetin is
required to agglutinate the platelet- rich plasma (>1.2 mg/
mL). Two mechanisms have been reported to explain 2A(IIA)
phenotype, subclassified as group1 and group2. VWF subunit with group1 mutations have an improper folding, are
retained in the ER and are subsequently degraded following
quality check (dominant negative mechanism). In this case,
multimers are formed by the interactions between normal
and mutant subunits and thus the large multimers are retained
in the cell. The group2mutations do not interfere with normal VWF secretion, but instead renders the mutant A2
domain subunit more susceptible to proteolytic cleavage by
ADAMTS13 at the peptide bond between Tyr1605 and
Met1606. This enhanced plasma proteolysis results in the
selective loss of the largest VWF multimers, which is typical
of type 2A. However, more recently, invitro expression studies have shown that group1mutations in the A2 domain also
confer increased susceptibility to ADAMTS13 cleavage, as
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240 Molecular Hematology
N
N2M 2A 2B
(A) (B)
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HMW
multimer
Figure17.6 (A) Comparison of plasma von Willebrand factor (VWF) multimeric structure from von Willebrand disease (VWD) patients and normal
subjects in non- reducing low- resolutions gel. Lane 3 (type 2A/IIA) and lane 4 (type 2B) show the lack of high- molecular- weight multimers. Lane 2
(type 2M) shows a normal multimeric pattern. Lanes 1 and 5 show a normal multimeric structure from healthy controls. (B) Comparison of plasma
von Willebrand factor (VWF) multimeric structure from von Willebrand disease (VWD) patients and a normal subject in non- reducing intermediateresolutions gel. Lanes 1 shows a normal multimeric structure from a healthy control with the typical triplet structure, presenting a central heavier
marked band surrounded by two lighter bands, one above and one below (curly bracket). Lane 2 (type 2A/IIA) and lane 3 (type 2B) show
markedly increased triplet structure with enhanced satellite bands due to the increased susceptibility of these variants to ADAMTS13 proteolysis.
Lane 4 (type 2A/IIC) shows the absence of triplet structure (only the main band is present), whereas lane 5 (type 2A/IIE) shows markedly reduced
satellite bands. Lane 6 (type 2A/IID) shows additional bands between the individual VWF oligomers, representing multimers with an odd number
of monomers.
confirmed by the pronounced satellite bands that these
patients showed in their plasma VWF multimers. The results
of molecular dynamics simulations on 3D protein models
showed that several regions of the A2 domain are significantly impaired by group1mutations such as p.Gly1505Arg,
p.Leu1540Pro, and p.Ser1506Leu. It was also found that the
variants p.Leu1561Ile, p.Ile1628Lys, and p.Glu1638Lys reduce
forces involved in the A2 domain unfolding. Group2mutations (e.g. p.Arg1597Trp, p.Glu1638Lys, and p.Gly1505Glu)
were found to create milder A2 domain structural changes
that ultimately effect on the interactions between VWF and
ADAMTS13.
Type 2A(IIE) phenotype is the second most frequent type
2A, caused by mutations in the multimerization site of VWF
(D3 domain, Figure 17.5), and is autosomally dominant
inherited. This variant is typically characterized by decreased
levels, more rarely lack, of HMWM and reduced or absent
satellite bands (Figure17.6B). A large cohort of patients with
VWD type 2A(IIE) from Germany were well characterized
on the basis of their plasma VWF multimeric structure and
most of the mutations identified involve a cysteine residue
(p.Cys1130Arg, p.Cys1130Trp, p.Tyr1146Cys, p.Cys1153Tyr,
p.Cys1173Phe). These mutations often correlated with a
moderate-
to- severe defective VWF secretion and compromised
the multimerization process, leading to the lack/decreased
N2A
(IIA)
2B 2A
(IIC)
2A
(IIE)
2A
(IID)
level of HMWM. In addition, because these mutations are
located at the FVIII binding site of VWF (D3 domain), a
moderately reduced VWF:FVIIIB was found for some of
these variants. The significant proportion of type 2A(IIE)
variants among type 2A patients has also been highlighted by
the investigation of a France VWD cohort (42 type 2A/IIE
cases out of 122 type 2A patients) and more recently, by an
Italian investigation (32 out of 98 type 2A patients). It is
noteworthy that type 2A(IIE) mutation spectrum partly
overlapped with VWD type 1, indicating problems in performing non- standardized multimer analysis or interindividual phenotypic differences despite having the same
VWF genotype. Several VWF variants with a significant
enhanced VWF clearance are reported in type 2A(IIE).
A more rare type 2A phenotype, type 2A(IIC), is due to
mutations in the D1 and D2 domains of propeptide
(Figure 17.5). This variant is characterized by a lack of
HMWM, the absence of the triplet structure, where only the
main band is present (Figure17.6B), and recessive inheritance. The propeptide is required for the formation of VWF
multimers and the sorting of VWF to storage granules. The
type 2A(IIC) variants compromise the multimerization process, therefore the lack of HMWM. The lack of triplet structure in this variant is believed to be a consequence of the
absence of ULVWF, which represents the main substrate for
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The molecular basis ofvon Willebrand disease 241
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ADAMTS13. Only a few mutations have been identified
associated with type 2A(IIC), mainly in the D2 domain (e.g.
p.Gly550Arg, p.Asn528Ser and p.Cys623Trp). In vitro studies of p.Asn528Ser have also shown its role in preventing the
intracellular transport of VWF multimer into the storage
organelles.
A rare type 2A variant (phenotype IID) is caused by mutations localized in the dimerization site of VWF, i.e. the
cystine- knot (CK) domain at the carboxy terminal of the
mature subunit (Figure 17.5). Given the critical role of
Cys2771–2773′, Cys2771′–2773, and Cys2811–2811′ intermolecular disulfide bonds, mutations in these amino acids
such as p.Cys2771Tyr, p.Cys2771Ser, and p.Cys2773Arg lead
to VWD type 2A(IID). This type is defined by a decreased
level of HMWM, the presence of uneven bands and dominant inheritance. These variants compromised the pro- VWF
dimerization process. The mutated subunits do not form
dimers, but still take part in the multimerization process.
Once a defective monomer is incorporated at one of the
growing ends of the forming multimers blocks further addition of dimers. Therefore, multimer analysis shows additional bands between the individual VWF oligomers,
representing multimers with an odd number of monomers
(Figure17.6B).
Type 2B
Type 2B VWD is the result of the gain of function mutations
with an increased affinity of VWF A1 domain for platelet
GPIbα (Figure 17.5). The optimal diagnosis of type 2B
requires RIPA assay that in these patients results to be
enhanced (see earlier). Type 2B is usually associated with the
absence of HMWM in VWF circulating plasma (Figure17.6A)
but not in intra- platelets VWF. Therefore, type 2B mutations
do not impair the synthesis or assembly of large VWF multimers, but after secretion, VWF binds spontaneously to
platelets leading to low plasma levels. In addition, the spontaneous interaction of type 2B VWF with GPIbα accelerates the
cleavage of VWF by ADAMTS13, resulting in the further
depletion of the HMWM along with the enhancement of satellite bands (Figure 17.6B). The occasionally occurring
thrombocytopenia in these patients appears to be associated
with severe type 2B variants (e.g. p.Val1316Met). However,
under particular stress conditions also patients with milder
variants might experience it. The reduced platelet count
observed in type 2B patients is ascribed to several mechanisms. It has been shown that the VWF/platelet complexes
are removed from the bloodstream by macrophages in the
spleen and liver. However, the reduced platelet production
seems to be the most relevant cause of thrombocytopenia in
type 2B. The presence of giant platelets and platelets with
altered morphology in these patients suggest a direct effect of
type 2B VWF variants on megakaryocytopoiesis. Using a
mouse model expressing VWF/p.Val1316Met, the role of
cytoskeletal changes in megakaryocytopoiesis has been
investigated and a disturbed regulation of actin remodeling
has been found to impair proplatelet formation in megakaryocytes of type 2B (p.Val1316Met) patients. Furthermore,
as a result of the spontaneous interaction of the mutant
VWF and the GPIbα receptor, using a type 2B mouse model
(VWF/p.Val1316Met) and platelets from type 2B patients
(p.Val1316Met), a reduced activation of integrin αIIbβ3has
been documented. In these type 2B patients, a Protein
kinase C signaling dysfunction results in an acquired thrombocytopathy, which is likely to aggravate the clinical phenotype in these patients. However, it is not clear if such platelet
dysfunction is also present in other severe type 2B variants.
Type 2B is inherited as a dominant trait and mutations
responsible for this subtype are mainly located in the A1
domain (or at the D3- A1junction), which is the binding site
of GPIbα receptor (Figure 17.5). More than 45 different
VWF variants have been associated with type 2B. With a
fewexceptions, all mutations result in amino acid substitutions, and few of them (p.Arg1306Trp, p.Arg1308Cys,
p.Val1316Met, and p.Arg1341Gln) account for about 70% of
the reported cases. In some patients, type 2B has been misdiagnosed and treated as autoimmune thrombocytopenia.
Thrombocytopenia and the absence of the larger multimers
are not constantly present in type 2B phenotype. For
instance, mutation p.Pro1266Leu causes an increased GPIbα
binding with no apparent loss of HMWM and absence of
thrombocytopenia before and after desmopressin, leading
to its earlier classification among type 1 variants (type
1NewYork or type 1Malmo). Patients carrying this mutation usually have mild bleeding symptoms and in some
cases, may have none. Pseudo- VWD also known as platelettype VWD, is a rare platelet disorder due to gain- of- function
mutations in the platelet receptor GPIbα (e.g. p.Gly233Val
and p.Met239Val). These mutations cause platelet GPIbα to
bind spontaneously to VWF, resulting in enhanced RIPA
similar to that of type 2B; hence, a differential diagnosis is
warranted between type 2B and Pseudo-
VWD. The correct
diagnosis can be achieved by either performing platelet/
plasma mixing studies (performing RIPA assay also using
patient plasma with normal platelets and vice versa) or
genetic analysis of VWF and GPIBα genes.
Type 2M
Type 2M VWD refers to qualitative variants with decreased
VWF- dependent platelet adhesion not caused by the absence
of the HMWM (Figure17.6A). There have been more than
80 VWF variants associated with type 2M, mostly missense
mutations with a few exceptions of inframe deletions or gene
conversions. In type 2M, a functional defect is caused by
mutations that compromise VWF binding to platelets or
subendothelium collagen.
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242 Molecular Hematology
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In the first case, type 2M is characterized by a reduced
VWF activity/VWF:Ag ratio, despite a normal multimer distribution (hence 2M for multimer). This VWD type is inherited as an autosomal dominant trait and the heterozygous
mutations are identified in the A1 domain (Figure17.5) such
as p.Ser1285Phe, p.Phe1293Leu, p.Arg1315Cys, p.Val1360Ala,
p.Arg1399Cys, and p.Lys1408Leu. These type 2M mutations
either affect the direct binding of VWF to GPIbα (e.g.
p.Ser1285Phe) or enhance the stability of the A1 domain,
thereby reducing the rate of A1 unfolding under the shear
conditions (e.g. p.Gly1324Ala and p.Gly1324Ser). Therefore,
in the RIPA assay, platelet- rich plasma of these patients agglutinate at a concentration of ristocetin >1.2 mg/mL (normal
range 0.8–1.2 mg/mL), similar to type 2A patients. Three frequent type 2 VWD mutations (p.Arg1374Cys, p.Arg1374His,
and p.Arg1315Leu) have been reported often as type 2M
variants; however, their classification as type 2M has been
controversial.
In the second case, type 2M is characterized by a decreased
VWF:CB/VWF:Ag ratio with a normal multimer distribution.
VWF binds collagen types I and III through its A3 domain and
collagen types IV and VI through its A1 domain. Therefore, a
defective collagen binding of VWF A1 or A3 domains can
cause type 2M (Figure17.5). This type is inherited as a dominant trait and the mutations causing VWF collagen binding
defect have been found mainly in the A3 domain (p.Ser1731Thr,
p.Ser1738Ala, p.Trp1745Cys, p.Met1761Lys, p.His1786Asp,
and p.Ser1783Ala) with only few reported variants in A1 domain
(Arg1399His, p.Gln1402Pro, and p.Arg1392_Gln1402del).
Type 2M patients with VWF collagen binding defects have
been reported less frequently than those with VWF defective
GPIbα binding and they seem to have also milder bleeding
symptoms. Two independent investigations on large cohorts of
VWD patients from France and Italy showed that about 22%
and 26% of patients with type 2M were classified as having
VWF collagen- binding defects.
Type 2N VWD
Type 2N VWD refers to qualitative variants characterized by
a decreased affinity of VWF for FVIII. The first description of
this type of variant was that of a patient from Normandy
(hence the 2N). This subtype is characterized by low levels of
FVIII; however, VWF levels are often normal and affected
patients usually have an intact VWF multimeric pattern. This
rare type of VWD may easily be misdiagnosed with hemophilia A or its carrier state, so the differential diagnosis should
be taken into consideration to reduce incorrect diagnoses and
inaccurate treatments. Hemophilia is an X- linked disease,
whereas type 2N VWD is an autosomal recessive disease. The
differential and definite diagnosis of type 2N is straightforward by measuring the capacity of VWF to bind FVIII (i.e.
VWF:FVIIIB). Because the majority of mutations are located
in the N-
terminus of the mature VWF subunit (D′- D3), diagnosis can be established or confirmed by screening for mutations from exons 18–26 (Figure 17.5). Three mutations
(p.Thr791Met, p.Arg816Trp, and p.Arg854Gln) account for
the majority of the identified type 2N variants.
Since the first description of the type 2N missense mutation in the female propositus from Normandy, more than 50
different mutations have been reported in type 2N of which
about 90% of them are missense. Type 2N patients may be
homozygotes for a single type 2N mutation, compound heterozygotes for two distinct 2N mutations, or compound heterozygotes for a type 2N mutation and a quantitative VWF
mutation. Two mutations were reported (p.Arg760Cys and
p.Arg763Gly) to disrupt the furin cleavage site of VWF, thus
leading to the persistence of the propeptide along with the
mature VWF subunit. Aside from having a ULVWF, patients
with these mutations exhibited a laboratory phenotype suggesting steric hindrance to FVIII binding to VWF.
Type 3 VWD
Type 3 is the most rare and severe form of VWD. It accounts
for less than 5% of all VWD cases and is characterized by a
virtually complete deficiency of VWF (<5 IU/dL) in both
plasma and platelets. Due to the deficiency of VWF also FVIII
plasma levels are severely reduced (<10 IU/dL). Therefore,
type 3 patients present a defect in both primary hemostasis
and intrinsic coagulation. As for type 2N, type 3 VWD may be
confused with hemophilia A, sometimes leading to misdiagnosis. At variance from hemophilia A, type 3 VWD is inherited with an autosomal recessive manner. At first, the
characterization of the genetic defects in type 3has been relatively slow, in particularly due to the large coding region (52
exons) of the VWF and the lack of a specific location of the
variants. The introduction of screening methods (e.g. singlestranded polymorphism conformation) along with direct
DNA Sanger sequencing analysis, allowed the identification of
the genetic variants in most patients. To date, in spite of the
low prevalence of the disease, and thanks to the introduction
of NGS technique, about 400 different genetic variants have
been reported among the type 3 patients (Figure17.5). As
expected, a large part of these defects cause null alleles. Among
these, nonsense variants are the most common, followed by
small deletions, splice-
site variants, small duplications, large
deletions, gene conversions, and small insertions. Nevertheless,
about 20% of the variants found in these patients were missense. Among the nonsense variants, a few hotspot mutations
at the arginine codons (p.Arg365*, p.Arg1659*, p.Arg1853*,
and p.Arg2535*) have been found repeatedly in different populations. The cytosine deletion in exon 18 (c.2435delC), identified in the original VWD family from the Åland Islands, was
recently confirmed to be the most common type 3 variant in
Northern Europe. A large deletion, including exons 1–3, was
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