Review

Clinical and Applied

Thrombosis/Hemostasis
Diagnostic Differentiation of von 2017, Vol. 23(6) 518-531
ª The Author(s) 2016

Willebrand Disease Types 1 and 2 Reprints and permission:

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DOI: 10.1177/1076029616647157
by von Willebrand Factor Multimer journals.sagepub.com/home/cat

Analysis and DDAVP Challenge Test

Jan Jacques Michiels, MD, PhD1,2, Petr Smejkal3,4,

Miroslav Penka3,4, Angelika Batorova, MD, PhD5,
Tatiana Pricangova5, Ulrich Budde6, Inge Vangenechten2,8,
and Alain Gadisseur, MD, PhD2,7,8

Abstract
The European Clinical Laboratory and Molecular (ECLM) classification of von Willebrand disease (vWD) is based on the splitting
approach which uses sensitive and specific von Willebrand factor (vWF) assays with regard to the updated molecular data on
structure and function of vWF gene and protein defects. A complete set of FVIII:C and vWF ristocetine cofactor, collagen binding,
and antigen, vWF multimeric analysis in low- and medium-resolution gels, and responses to desmopressin (DDAVP) of FVIII:C and
vWF parameters are mandatory. The ECLM classification distinguishes recessive types 1 and 3 vWD from recessive vWD 2C due
to mutations in the D1 and D2 domains and vWD 2N due to mutations in the D0 -FVIII-binding domain of vWF. The ECLM
classification differentiates between mild vWD type 1 with variable penetrance of bleedings from symptomatic dominant type 1
vWD secretion defect and/or clearance defect with normal vWF multimers versus vWD 1M and 2M with normal or smeary vWF
multimers in low- and medium-resolution gels. High-quality multimeric analysis of vWF in medium-resolution gels based on a
DDAVP challenge test clearly delineates and distinguishes each of the dominant type 2 vWDs 1/2E, 2M, 2B, 2A, and 2D caused by
vWF gene mutations in the D3 multimerization domain, loss or gain-of-function mutations in the glycoprotein Ib receptor A1
domain, gene mutations in the A2 proteolytic domain, and the C-terminal dimerization domain, respectively.

Keywords
von Willebrand disease, von Willebrand factor, ADAMTS13, DDAVP, von Willebrand factor assays, von Willebrand factor
multimers, von Willebrand factor mutations

Introduction
The classification of congenital von Willebrand disease (vWD)
is dominated by the recommendations of the von Willebrand
factor (vWF) Scientific Standardization Committee (vWF-
SSC) of the International Society on Thrombosis and Haemo-
stasis (ISTH).1-5 The ISTH classification of patients with vWD
type 1 is based on quantitative measurement of factor VIII
coagulant activity (FVIII:C) and vWF antigen (vWF:Ag) and
3 insensitive laboratory tests of vWF ristocetine cofactor
(vWF:RCo) activity, ristocetine-induced platelet aggregation
(RIPA), and vWF multimers in low-resolution gel.1 As vWF
multimeric analysis in a low-resolution gel has a low sensitivity
to distinguish the various variant of vWD type 1 and type 2, the
ISTH vWD classification cannot clearly distinguish between
the pronounced variants of type 1 from type 2M or 2E vWD at
vWF levels below 0.20 U/mL.2-5 The ISTH vWD classification
mainly used a ‘‘lumping’’ approach for the classification of
1
Goodheart Institute in Nature Medicine & Health, Blood Coagulation and
Vascular Medicine Center, Rotterdam, The Netherlands
2
Hemostasis Research Unit, Department of Hematology, Antwerp University
Hospital, Belgium
3
Department of Clinical Hematology, University Hospital, Masaryk University,
Brno, Czech Republic
4
Faculty of Medicine, Department of Laboratory Methods, Masaryk University,
Brno, Czech Republic
5
Department of Hemostasis and Thrombosis, National Hemophilia Center,
Medical School of Comenius University, Bratislava, Slovakia
6
Central Laboratory, Asklepios Kliniken, Hamburg, Germany
7
Department of Hematology, Antwerp University Hospital, Antwerp, Belgium
8
Hemostasis Research Unit, Antwerp University Hospital, Antwerp, Belgium
Corresponding Author:
Jan Jacques Michiels, Goodheart Institute in Nature Medicine & Health, Blood
Coagulation and Vascular Medicine Center, Erasmus Tower, Veenmos 13,
Rotterdam, 3069 AT, the Netherlands.
Email: goodheartcenter@upcmail.nl
Michiels et al 519

Table 1. Laboratory Characteristics of the European Clinical Laboratory and Molecular (ECLM) Classification of Von Willebrand Disease
Type 1 Autosomal Recessive Severe Types 1 and 3, 2C and 2N Versus Autosomal Dominant Type 1 Secretion Defect (SD) and Types 2A, 2B, 2E,
and 2D anno 2016.

Type 1: vWF:Ag < 35%, normal vWF:CB/Ag, and vWF: RCo/Ag ratio >0.7. Patients with type 1 having vWF:Ag levels >35% can only be included
with severe bleeding phenotype.
Type 3: autosomal recessive; vWF:Ag and FVIII:C undetectable so-called pseudo-hemophilia first described by Erik von Willebrand.
Severe type 1 autosomal recessive; vWF:Ag and vWF:RCo detectable <5%, high FVIII/vWF ratio in particular after DDAVP.
Type 2C autosomal recessive with increased FVIII:C/vWF:Ag ratio (secretion defect) and loss of large vWF multimers due a multimerization
defect caused by homozygous or double heterozygous mutations in the D1 to D2 of the vWF gene.
Type 2N autosomal recessive with FVIII:c/vWF:Ag ratio <0.5. FVIII–vWF binding defect due to mutations in the D0 FVIII-binding domain of the
vWF gene.
Type 1: autosomal dominant clearance defect (Vicenza type)
Type 2: autosomal dominant with decreased vWF:RCo/vWF:Ag < 0.7 for 2A, 2B, 2E, 2D, and 2M.
Ristocetin-induced platelet aggregometry (RIPA) is decreased in vWD 2M and 2A, and characteristically increased in vWD 2B and 1B
2A: Loss of large MM due to increased vWF proteolysis due to mutations in the A2 domain of the vWF gene.
2B: Increased RIPA (0.8 mg/mL) and thrombocytopenia with vWD type 2 due to gain-of-function mutation in the GpIb receptor in the A1
domain of the vWF gene.
2E: Type 1 or 2 is featured by loss of large multimers due to multimerization defect and increased clearance caused by mutations in the D3
multimerization domain of the vWF gene.
2 M: Decreased vWF:RCo/vWF:Ag ratio (<0.7), normal vWF:CB/vWF:Ag ratio (>0.7), decreased RIPA due to loss of function mutation in the
GPIb receptor in the A1 domain.
2M-CBD: Collagen-binding defect, vWF:RCo/vWF:Ag ratio >0.7 and vWF:CB/vWF: Ag ratio <0.7 due to mutation in the collagen-binding
domain A3 of the vWF gene.
2D: loss of large MM and with odd number of monomers and markedly intervening bands due to loss of function mutation in the C-terminal CK
domain.
Abbreviations: CB, collagen binding; GPIb, glycoprotein Ib; vWD, von Willebrand disease; vWF, von Willebrand factor, MM, multimers.

type 2 vWD.1 Dominant vWD type 2E is usually present with a
laboratory vWD phenotype 1, and diagnostic differentiation of
pronounced type 1 vWD from type 2E and type 2M vWD using
the SSC-ISTH criteria remains a persistent problem in routine
daily practice. The ISTH vWD classification is not based on
structural and functional relationship of vWF gene mutations
related to functional domains of the vWF protein, which ham-
per clinicians to gain insights in the etiology and pathophysiol-
ogy of the broad spectrum of type 1 and type 2 vWD.6,7
Clinical, Laboratory, and Molecular Diagnosis
and Classification of vWD 2016
The European clinical laboratory and molecular (ECLM 2016)
classification of vWD following the recommendations of Budde
and Schneppenheim, Michiels et al, and Gadisseur et al is based
on the splitting approach by the use of vWF-specific and -sen-
sitive diagnostic tools in view of new molecular data on structure
and function of vWF gene defects (Table 1; Figure 1A and B).6-11
The ECLM classification is based on a complete set of vWF-
specific measurements including bleeding time (BT), PFA-100
closure time (PFA-CT), FVIII:C, vWF:Ag, vWF:RCo,
vWF:collagen binding (CB), RIPA, the response of vWF, and
FVIII:C to DDAVP. The vWF:RCo/vWF:Ag ratio, vWF:CB/
vWF:Ag ratio, and FVIII:C/vWF:Ag ratio are used by the
ECLM classification for the differential diagnosis of vWD type
1 versus type 2.7,9-11 The normal values of vWF:Ag, vWF:RCo,
and vWF:CB and the ratios for vWF:RCo/vWF:RCo and
vWF:CB/vWF:Ag are around 1 + 0.40 U/mL. The ratio of
FVIII:C binding sites over vWF subunits on a molecular basis
is 1:50 and independent of the size of vWF multimers.
Consequently, the ratio of FVIII:C/vWF:Ag will increase to 2
or above in vWD due to a secretion defect (SD) as a main cause
of quantitative vWF deficiency (eg, vWD type 1 due to muta-
tions in the D4, B1-3, and C1-2 domains; Figure 1B). The ratio
FVIII:C/vWF:Ag remains around 1 in case of rapid clearance of
vWF bound to FVIII:C as the cause of a quantitative vWF defi-
ciency (eg, vWD Vicenza, Figure 1B). High-quality vWF multi-
meric analyses in medium-resolution gels according to the
method of Budde are sensitive to distinguish vWD types 1 and
2 and therefore of key importance to distinguish various variant
of vWD type 1 and vWD type 2. The VWD type 2 phenotypes
are related to mutation defects in the different functional
domains of vWF:recessive vWD 2C are due to mutation in the
D1 and D2 domains, dominant vWD 2E, 2M, 2B, 2A, and 2D are
caused by mutation multimerization domain D3 (vWD 2E),
gain-of-function mutation in the A1 domain (vWD 2M),
mutations in the A2 domain (vWD 2A), loss-of-function
mutations in the A1 domain (vWD 2B), mutations in the CK
dimerization domain (vWD 2D; Figure 1A and B).
Three main categories of vWD can be distinguished: first
autosomal recessive type 3 and severe type 1 vWF:RCo <10U/
dL, second dominant types 1 (vWF:RCo 10-30 U/dL) and 2,
and third a large group of mild vWD type 1 (vWF:RCo 30-50
U/dL) with no or low penetrance of bleeding manifesta-
tions.7,12,13 The mild, moderate to pronounced vWD type 1 is
a quantitative vWF deficiency with equally decreased values of
all vWF parameters and a normal ratio of vWF:RCo/Ag and
vWF:CB/Ag (>0.70) before and after DDAVP (Table 1). von
Willebrand disease type 2 is a qualitative vWF deficiency with
normal, near normal, or decreased levels for FVIII:C and
vWF:Ag, and much lower values for vWF:RCo and vWF:CB
520 Clinical and Applied Thrombosis/Hemostasis 23(6)

Figure 1. A and B, Clustered distribution of von Willebrand disease (vWD) types 1 and 2 with abnormal von Willebrand factor (vWF)
multimers in medium-resolution gels for the classification of types 1 and 2 vWD related to mutations in the D1, D2, D3, A1, A2, D4B1-3, C1-2,
and CK domains of vWF (A and B). In vWD 2A due to mutations in the A2 domain (A), there is a lack of large vWF multimers and the outer
subbands of the individual triplets are markedly pronounced. von Willebrand disease type 2B due to gain-of-function mutations in the A1 domain
cannot be distinguished from vWD 2A because their multimer patterns of pronounced triplet structure of each vWF band are very similar (A). In
vWD 2C due to mutations in the D1 and D2 domains (A), there is a lack of large vWF multimers and absence of triplet structure of the individual
bands. The first band (dimer) and second band (tetramer) are markedly pronounced. In vWD 2E due to mutation in the D3 domain (A and B),
the vWF multimers show lack or relative decrease in large multimers and the absence of the outer subbands of normal triplets, which give the
individual multimers a broader appearance. In vWD 2M due to the loss of functional mutations in the A1 domain (A and B), the vWF multimers
are variable from normal to smeary pattern with some loss or the presence of large multimers. In vWD type 1 due to mutations in the D4–B1-3–
C1-3 domain (A and B), the vWF multimers are normal with some minor smearing pattern (1 m or 1 ms) or with a pronounced smearing pattern
(1 sm). The vWF multimers are normal in vWD 2N and vWD collagen-binding defect (CBD). Type 2N mutations that involve a cysteine (C788R/
Y, Y795C, and C804F) are associated with aberrant multimerization.
Michiels et al 521

Figure 2. Left: Completely normal responses of FVIII: C and von Willebrand factor (vWF) parameters to DDAVP from around 0.4 U/mL to high
levels of 2.5 to 3.6 U/mL followed by normal half-life times of vWF antigen (Ag) and ristocetine cofactor (RCF) and collagen binding (COLL) activities
consistent with the diagnosis of pseudo–von Willebrand disease (pseudo-vWD).2 Right: Restricted responses to DDAVP of vWF ristocetine cofactor
(vWF:RCF) and vWF collagen-binding activity (vWF:CBA) as compared to vWF: Ag and FVIIIC consistent with mild vWD type 1.

with decreased vWF:RCo/Ag ratio and vWF:CB/Ag ratio
(<0.70; Table 1). von Willebrand factor multimeric analysis
using low- and medium-resolution gels clearly distinguish
vWD type 2A, 2C, 2E, 2D, and 2M (Figure 1A and B).7,12-14
The responses of FVIII and vWF parameter to intravenous
DDAVP is an essential tool to distinguish pseudo-vWD from
mild type 1 and to separate in a much better manner the various
variants of autosomal dominant types 1 and 2.2,5,6 The inter-
pretation of vWF response curves to DDAVP has significant
therapeutic implications for the different variants of dominant
type 1 and type 2 vWD mutations for both clinicians and
patients with vWD.2,6,15,16 Responses of FVIII:C and vWF
parameters to DDAVP related to the structural and functional
relationship between laboratory phenotype and expression
studies of vWF gene mutations significantly contribute to better
understanding of the etiology and pathobiology of mutant vWF
and characterization of the various types of congenital vWD
(Figure 1A and B).
The Spectrum of vWD Type 1 and Type 2: The
Experience of 1 Center From 1990 to 2010
Between 1992 and 1997, the Rotterdam vWD study group
analyzed 295 index cases of patients coded as vWD irrespec-
tive of blood O or non-O in the Erasmus University Medical
Center, Rotterdam, with a referral region of about 2 million
inhabitants.2 The first group of 128 (46.5%) index patients had
vWF antigen and functional levels of around 40% to 60%, a
very mild bleeding tendency, no family history, normal Ivy
BTs, usually showed normal responses of vWF and FVIII:C
to DDVAP when tested on indication, do not have vWD, and
were diagnosed as pseudo–von Willebrand (Figure 2, left).2-6
The second group of 167 index patients of 94 families were
diagnosed diagnosed as ISTH defined mild vWD type 1 in 65
families; severe dominant vWD type 1 in 10 families (of which
2M in 3); types 2A, 2B, and 2N in 10, 4, and 2 families; and
recessive type 3 in 3 families, respectively.2 The diagnosis of
mild vWD type 1 was based on a personal bleeding history and
decreased values for FVIII:C, vWF:Ag vWF:RCo, and
vWF:CB with normal ratios for vWF:RCo/Ag and vWF:CB/
Ag. Bleeding manifestation in the proband of 24 index family
cases of vWD type 1 was usually mild and occasionally mod-
erate. The Ivy BTs were normal or only slightly prolonged in
only a few cases. The values for vWF:Ag vWF:RCo and
vWF:CB were between 0.20 and 0.60, with normal ratios for
vWF:RCo/Ag (0.62-1.08) in all and also for vWF:CB/Ag in 21
of the 24 cases of mild vWD type 1.
The responses of the vWF parameters to DDAVP in the 24
probands with mild vWD type 1 were normal, showing a 2- to
10-fold increase in vWF:Ag and vWF:RCo. The vWF:RCo/Ag
ratio remained normal (0.65-1.19) after DDAVP in all 24 vWD
type 1 patients. The majority of vWD type 1 patients showed
significantly higher peak values of vWF:CB immediately after
DDAVP, which resulted in significantly higher vWF:CB/Ag
ratios (1.4-2.8) after DDAVP. The group of 24 probands of
vWD type 1 could be separated in a group of 14 probands with
522
Table 2. Dominant vWD Type 1 Secretion Defect (High FVIII/vWF:Ag Ratio) Caused by S2179F Mutation in D4 Domain in 3 Families With
vWD SD (secretion defect).18,19,a
Mutation FVIII:C, % vWF:Ag, % vWF: RCo, %
Family 1 S2179F 33 13 9 2.5
21 7 6
28 12 14
Family 2 S2179F 19 13 12
29 15 12
35 18 10
S2179F 26 6 6
Abbreviations: Ag, antigen; RCo, ristocetine cofactor; vWD, von Willebrand disease; vWF, von Willebrand factor.
a
The high FVIII:C/vWF:Ag ratio is indicative for a secretion defect (SD) similar to the mutations L2207P, C2257S, C2304Y, and R2464C in the D4–B1-3–C1-2
Domain of von Willebrand factor (Figure 1 A and B).7,11,13
normal biological half-life times of the vWF parameters, which
we classified as pseudo-vWD (Figure 2, left) and a group of 10
probands with restricted responses of vWF:RCo as compared to
vWF:Ag (Figure 2, right) followed decreased half-life times of
vWF parameters.2 Three patients with suspected mild vWD
type 1 but completely normal responses to DDAVP (Figure
2, left) underwent major surgery including laparoscopic chole-
cystectomy, uterine extirpation, and orthopedic surgery under
the cover of 1 dose DDAVP (0.3 mg/kg intravenous preopera-
tively and on day 1). No bleedings were noted in the periopera-
tive and postoperative period. In a case of symptomatic mild
vWD type 1 (Figure 2, right) featured by bruises and moderate
menorrhagia since adolescence, we compared the effect of
intravenous (IV) DDAVP, intranasal DDAVP, and 1 dose of
3000 units Humate-P (40 U/kg; Figure 2, right). DDAVP
showed a restricted response of vWF:Ag and vWF:RCo to
values around 1.5 U/mL and normal response of FVIII:C and
vWF:CB to values between 2.0 and 3.0 U/mL, whereas the
responses of DDAVP intranasal were rather poor as compared
to DDAVP IV and Humate-P (Figure 2, right).2 In 2003, Sadler
extended the 2002 concept of Michiels et al2 in distinguishing
pseudo-vWD versus mild, moderate, and severe vWD by the
combined use of vWF multimeric analysis and a DDAVP chal-
lenge test. Sadler calculated that most diagnoses of mild vWD
type 1 are false positive vWD labeled as pseudo-vWD by
Michiels et al17 In the general population, 25% have 1 or 2
mild bleeding (clinically insignificant) and 2.5% low plasma
vWF, indicating that 0.25  0.025 ¼ 0.6% individuals in the
general population have a chance of low vWF and bleeding just
by chance.17 Patients with mild vWD type 1 with levels for
vWF:Ag, vWF:RCo, and vWF:CB between 0.30 and 0.60 U/
mL and normal ratios for vWF:RCo/Ag and vWF:CB/Ag usu-
ally present with no or mild bleeding symptoms, no family
history of bleeding, and normal Ivy BTs, and normal PFA-
CTs on repeated occasions.
Diagnostic Differentiation of Dominant vWD
Type 1 versus 2M versus Vincenza
In 2002, the Rotterdam vWD study group analyzed the clin-
ical and laboratory features and DDAVP responses of domi-
nant vWD type 1 in 10 families (of which 2M in 3), and in
Clinical and Applied Thrombosis/Hemostasis 23(6)
VIII: C/vWF: Ag ratio vWF:Rco/vWF: Ag ratio vWD type References
0.7 Type 1SD Haberichter19
3.0 0.86
2.3 1,2
1.5 0.9 Type 1SD Haberichter19
1.9 0.8
1.9 0.55
4.3 1.0 1 MCMDM-118
vWD type 2A, 2B, and 2N in 10, 4, and 2 families, respec-
tively.2 The FVIII and vWF parameters in 6 probands with
pronounced autosomal dominant vWD and vWF levels
between 0.07 and 0.20 U/mL are presented in Table 2.2 Cor-
rect typing of such pronounced cases of vWD as type 1 versus
2M versus Vicenza by the vWF:RCo/Ag ratio is problematic
because of the low sensitivity of the vWF:RCo assay at
vWF:Ag levels below 0.20 (Table 2). The vWF:CB/Ag ratio
was normal in all 6 probands with pronounced vWD type 1
(Table 2). RIPA is normal in vWD type 1 (probands 1, 2, 3,
and 6) and decreased in vWD 2M (probands 4 and 5; Table 2).
The probands 1 and 2 in Table 2 were diagnosed as pro-
nounced vWD type 1 with a bleeding tendency since early
childhood, normal RIPA, normal to slightly prolonged Ivy
BT, and normal vWF multimeric pattern. In 3 family mem-
bers of proband 3 (mother P1, brother P2, and daughter P3;
Figure 3, left), the response to DDAVP was high for FVIII:C
but decreased for all vWF parameters (Figure 3, left), and the
vWF multimeric pattern in medium-resolution gels is normal
consistent with vWD type 1. Such high ratios of FVIII:C/
vWF:Ag above 3 before and after DDAVP in pronounced
vWD type in the family members of proband 3 (P1 and P2;
Figure 3, left) are indicative of an secretion defect (SD) of
vWF from endothelial cells as the main cause of pronounced
vWD type 1.2
The probands 4 and 5 in Table 2 had overt vWD with mod-
erate bleeding tendency particularly after trauma and surgery
with vWF levels below 0.20 U/dL for all vWF parameters,
normal vWF multimers in low-resolution gel before and after
DDAVP (Figure 3, right), a decreased RIPA and a slightly
prolonged Ivy BT (Table 2). The response to DDAVP of
vWF:Ag and vWF:CB was rather good, but the response of
vWF:RCo to DDAVP was poor (Figure 3, right), indicative
of vWD 2M. The half-life times of FVIII:C, vWF:Ag, and
vWF:CB post-DDAVP were significantly shortened, indicative
of a rapid clearance of the FVIII-vWF complex. The laboratory
diagnosis findings in probands 4 and 5 of these 2 families with
vWD 2M2-6 are typically featured by decreased RIPA; normal
vWF multimers before and after DDAVP; decreased response
of vWF:RCo to DDAVP and good responses of FVIII:C,
vWF:Ag, and vWF:CB to DDAVP followed by rapid clearance
of the FVIII-vWF complex.
Michiels et al 523

Figure 3. Left: DDAVP responses in pronounced type 1 von Willebrand disease (vWD) showing a normal response of FVIIIC and restricted
responses to DDAVP of all von Willebrand factor (vWF) parameters in 2 members of 1 family (proband and her brother, proband 3 in Table 2).
The high FVIII/vWF:Ag ratio before and after DDAVP with normal vWF multimers in medium-resolution gels according to Budde14,15 is
consistent with the diagnosis of vWD type 1 secretion defect (SD2). Right: Poor response of vWF:RCo to DDAVP and fairly good responses to
DDAVP of FVIII:C, vWF:Ag, and vWF: CB in a case of pronounced vWD 2M with normal vWF multimers before and after DDAVP (cases 4 and 5
in Table 2) showing normal vWF multimeric pattern in low-resolution gel according to Michiels et al.2 Interestingly the half-life times of FVIII:C,
vWF:Ag, and vWF:CB are shortened in pronounced vWD 2M indicative of an additional rapid clearance defect (CD) of the FVIII-vWF complex.

As shown in Table 2, proband 6 mother and her son had a
well-documented autosomal dominant hemophilia-like bleed-
ing tendency associated with occasional hemarthrosis, recur-
rent hematomas after trauma and muscle bleeding, and
recurrent mucocutaneous bleeding since early childhood.
Laboratory investigations were consistent with severe vWD
type 1 with normal ratios for vWF:RCo/Ag and vWF:CB/Ag,
normal RIPA, and vWF multimers on repeated testing. Multi-
meric analysis of vWF from plasma showed normal vWF mul-
timeric pattern in low- and medium-resolution gels before and
after DDAVP. The response to DDAVP 1 hour post-DVT
showed completely normal responses of FVIII:C, vWF:Ag,
vWF:RCo, and vWF:CB with peak levels 15 minutes post-
DDAVP followed by very short half-life time of FVIII and all
vWF parameters consistent with the rapid clearance of the
FVIII-vWF complex. Such cases with pronounced autosomal
dominant vWD type 1 due to rapid clearance (C) of both FVIII
and vWF parameters have been described in the vWD literature
as vWD type 1 Vicenza (Figure 1B).
Von Willebrand Disease Type 1 With Normal
or Smeary vWF Multimeric Pattern Due to
Mutations in the D4, B1-3, and C1-2 Domains
of vWF
The Canadian and European vWD-1 studies discovered a new
category of vWD type 1 due to mutations in the D4, B1-3, and
C1-2 domains of the vWF gene (Figure 1B).18-22 Data from the
European type 1 vWD study show 2 groups of heterozygous
mutations in the D4, B1-3, and C1-2 domains with either
normal multimers or abnormal multimers.11 The group with
normal vWF multimers L1774S, K1794E*, C2304Y*,
R2313H, G2518S*, Q2544X*, C2693Y, and P2722A has
mild vWD type 1 disease, is autosomal dominant or
recessive, with variable penetrance of bleeding
manifestations.7,11,13 Four of these mutations in the D4, B1-
3, and C1-2 domains with an increased FVIII:C/vWF:Ag ratio
of 2 or more (indicated by an asterisk) had complete penetrance
of bleeding manifestations, consistent with the diagnosis of
vWD type 1 secretion defect (SD). The group with abnormal
multimers of heterozygous mutations in the D4, B1-3, C1-2,
and CK domains V1822G*, L2207P*, C2257S*, C2304Y*,
C2362F*, G2441C*, R2464C*, C2477Y*, C2477S*, and
Q2520P* has mild to moderate vWD type 1 and usually
smeary pattern of abnormal vWF multimers (Figure 1A and
B).7,11,13 The majority of mutations in the D4, B1-3, C1-2,
and CK domain with normal or smeary vWF multimers have
increased FVIII:C/vWF:Ag ratios to around or above 2
(indicated by an asterisk). In vWD type 1 due to mutations in
the D4–B1-3–C1-3 domain (Figure 1B), the vWF multimers
are normal, with minor smearing pattern (1 m or 1 ms), or with
a pronounced smearing pattern (1 sm). The increased FVIII:C/
vWF:Ag ratio and normal vWF:RCo/Ag ratio (Figure 1B) will
predict an SD with restricted responses of vWF to DDAVP
(vWD type 1 SD). Haberichter et al reported 2 unrelated
524
families with the heterozygous S2179F mutation in D4 domain
(Amino acid 1940-2300) featured by moderate vWD type 1,
increased FVIII:C/vWF:Ag ratio, restricted response of vWF to
DDAVP (vWD SD; Table 2).19 The European type 1 vWD
study reported on case of pronounced vWD type 1, normal
vWF multimers but high FVIII:C/vWF:Ag ratio indicative of
an SD (Table 2).18
Prospective Evaluation of vWF Responses to
DDAVP in vWD Type 1 and Type 2
Between 1998 and 2008, the Rotterdam vWD study group
prospectively evaluated the responses of platelet function
analysis closure times (PFA-CTs) vWF:Ag, vWF:CB, and
FVIII before and 1 hour after DDAVP (0.3 mg/kg) in patients
with vWD type 1 (n ¼ 70).16 Pretreatment values of FVIII:C
and vWF parameters in 70 patients labeled as vWD type 1
ranged from severe, moderate, and mild vWF deficiency to
normal values. The PFA-CTs were normal or in the upper
region of normal (>300 seconds) in the majority and prolonged
(>300 seconds) in only a few cases of vWD type 1. The patients
with vWD type 1 treated with DDAVP demonstrate a good
dose–response with correction of the PFA-CTs in all patients
with vWD type 1. Normalization of the PFA-CTs (< 150 sec)
was reached at functional vWF:RCo and vWF:CB levels of
0.75 U/mL and above. Based on the response of vWF:RCo to
DDAVP, patients with vWD type 1 were categorized into 2
groups. First, those with restricted responses of vWF:RCo , that
is, up to a maximum of 0.50 and 1.2 U/mL (Figure 2, right).2
Second those with maximal values of vWF:RCo to above 1.5 to
4.5 U/mL indicating complete normal responses to DDAVP in
patients (individuals) who in fact do not have vWF deficiency
(pseudo-vWD; Figure 2, left).2,16
Between 1998 and 2008, the Rotterdam vWD study group
evaluated the responses of PFA-CT, vWF parameters, and
FVIII:C before and after DDAVP in 24 patients with vWD type
2 (n ¼ 14: 8 type 2A, 2 mild type 2, and 4 type 2M).16 The PFA-
CTs were prolonged (<300 seconds) in all patients with vWD
type 2 except in 2. None of the patients with severe vWD type
2A showed a transient correction of the PFA-CTs 1 hour after
DDAVP. In contrast, patients with vWD type 2M or mild-type
2A (N ¼ 6) showed a transient correction of PFA-CTs to near
normal or normal values (<150 seconds) 1 hour post DDAVP.
Normal responses of PFA-CT to DDAVP is dependent on the
transient correction of functional plasma vWF:CB and the
vWF:CB/Ag ratio together with correction of plasma vWF mul-
timer distribution with the reappearance of large multimers.
Diagnostic Differentiation of Dominant vWD
2E Versus 2M Versus 2A
Patients with vWD type 2E caused due to vWF mutations in the
D3 domain have laboratory features of dominant vWD type 1
with a type 2E vWF multimeric pattern in low- and medium-
resolution gels (Figure 1A and B).7,13 Schneppenheim described
22 different mutations in the D3 domain of the vWF gene as the
Clinical and Applied Thrombosis/Hemostasis 23(6)
cause of vWD 2E or vWD 2E phenotype (Table 3). Most of these
22 mutations in the D3 domain of vWF affect cysteine residues,
17 of them being novel, all are clustering in the vWF D3 domain
multimerization domain.23 An intracellular retention of most
mutants and/or a defect of multimerization seem to be the main
pathogenic molecular mechanisms, and the sensitivity to
ADAMTS13 proteolysis of mutant vWF was not different from
wild-type vWF in a static assay.23 Among this cohort of 22 vWD
type 2E patients with mutations in the D3 domain, we contrib-
uted to 2 novel mutations W1120S (Table 3; Figure 4, left) and
C1190R with documented dominant vWD type 1/2E.23 The type
2E vWF multimeric pattern is characterized by a lack or relative
decrease in large multimers and the absence of the outer subband
of the normal triplet structure consistent with a multimerization
defect in low- and medium-resolution gels (Figure 4, left).13,23
The responses of vWF parameters to DDAVP in vWD 2E are
featured by restricted increase in vWF:RCo as compared to
FVIII:C and vWF:Ag (Figure 4, left). Such transient increase
in functional vWF parameters is associated with transient cor-
rection of vWF multimers with reappearance of large multimers,
and transient correction of Ivy BT and PFA-CTs for a few hours.
In contrast vWD 2M typically shows a poor response of
vWF:RCo to DDAVP despite normal vWF multimers before
and after DDAVP, and good responses of FVIII:C, vWF:Ag,
and vWF:CB followed by shortened half-life time indicating
rapid clearance defect of the FVIII:C-vWF complex in addition
to the loss of vWF:RCo function in the A1 domain in vWD 2M
(Figure 4, right).2,5,6,14
In a collaborative basic research, we identified a novel
mutation V1499E with a high penetrance in a large Dutch
family with autosomal dominant type 2A vWD, which was
detected independently in 3 hospitals and academic coagula-
tion laboratories (Table 4).24 Three affected family members
(II-1, III-1, and III-7) had been diagnosed more than 10 years
ago (Table 4). The mutation V1499E was independently dis-
covered in 2 vWF research laboratories. Proteolysis of recom-
binant mutant V1499E vWF by ADAMTS13 was increased
consistent with consistent with vWD type 2A.24 In vWD 2A,
there is a lack of large multimers in low-resolution gels accord-
ing to the methods. The loss of large vWF multimers with
markedly pronounced triplet structure of the outer subbands
due to increased proteolysis was only seen in medium-
resolution gels but not in low-resolution gels (Figure 5). The
loss of large multimers together with increased triplet subbands
representing proteolytic vWF fragments originating from
increased cleavage of vWF at the Y1605–M1606 bond is due
to mutations in the A2 domain of vWF (Figure 1A).15,16,24-28
Original observations in this family with moderate dominant
vWD type 2A group 2 caused by the V1499E mutation2,6,24 and
in 3 families with severe vWD type 2A group 1 (Table 4)25 are
completely in line with the observed heterogeneity of moderate
type 2A versus severe type 2A vWD with regard to bleeding
symptoms, laboratory phenotypes, and typical responses to
DDAVP. Patients with moderate vWD 2A group 2 including
the V1499E mutant are identified by normal or subnormal
values for FVIII:C and vWF:Ag, vWF:RCo, that is, around
Michiels et al 525

Table 3. 2010 Update on Von Willebrand Disease (vWD) Type 2E Due to Missense Mutations in the D3 Multimerization Domain of the Von
Willebrand Factor (vWF; Exons 22, 23, 24, 25, 6, and 27) and Related Laboratory Phenotype Type 2 or Type 1 (bold) According to vWF:RCo/Ag
or vWF: CB/Ag Ratios in 19 Patients With a vWD type 2E Multimeric Pattern.23,a

Exon Mutation vWF:Ag, U/mL vWF:CB, U/mL vWF:RCo vWF:CB/Ag vWF:RCo

22 V956A 0.31 0.37 0.25 1.19 0.81

22 R976C 1.29 1.04 0.81
25 C1091R 1.20 1.20 1.00
25 A1105D 0.14 0.13 0.11 0.93 0.79
25 W1120S 0.34 0.19 0.21 0.56 0.62
25 R1121M 0.23 0.20 0.87
25 C1126F 0.18 0.07 0.36
26 C1130R 0.17 0.10 0.59
26 C1130W 0.22 0.08 0.36
26 Y1146C 0.20 0.16 0.07 0.79 0.35
26 C1149Y 0.17 0.05 0.07 0.30 0.42
26 C1153Y 0.89 0.55 0.40 0.62 0.45
26 C1169W 0.11 0.06 0.52
26 G1172C 0.25 0.14 0.15 0.56 0.60
26 C1173R 0.19 0.08 0.10 0.42 0.53
26 C1173F 0.35 0.23 0.32 0.65 0.93
27 C1190R 0.41 0.24 0.14 0.59 0.34
27 C1190Y 0.94 0.18 0.19
27 D1195Y 0.43 0.38 0.88
Abbreviations: aa, amino acid change; Ag, antigen; CB, collagen binding; RCo, ristocetine cofactor.a Please note that vWD patients with a type 2E vWF multimeric
pattern due to a mutation in the D3 domain may have a laboratory type 1 vWD with a normal vWF:RCo/Ag or vWF:CB/Ag ratio (given as boldface).

0.30 U/mL; normal RIPA at ristocetin concentrations of 1.2 or
1.75 mg/mL (Table 4); and show a complete but transient cor-
rection of large vWF multimers, BT, FVIII:C, and vWF para-
meters a few hours after DDAVP (Figure 5).25 Patients with
severe vWD 2A group 1 have low values for vWF:Ag, very low
or undetectable level for vWF:RCo and vWF:CB, decreased or
zero RIPA at high concentration of ristocetin 1.75 or 2.0 mg/mL
(Table 4) and show a minor or poor response to DDAVP of
functional vWF:RCo and vWF:CB with no correction of vWF
multimers and BT directly after DDAVP.15,25
Von Willebrand Disease 2A Groups 2 and 1
Due to Mutations in the A2 Domain of vWF
The pertinent findings in patients with vWD type 2A include
prolonged BT, consistently low vWF:RCo/Ag ratio and
vWF:CB/Ag ratio, absence of large vWF multimers, pro-
nounced triplet structure of individual bands, and increased
vWF degradation products due to increased proteolysis (Figure
1; Table 5). von Willebrand disease type 2A results from mis-
sense mutations in the A2 domain, exon 28, of the vWF gene
(C1485Y, L1503P, G1505E, S1506L, F1514C, 1524del,
V1539E, L1540E, L1540P, S1543F, Q1556R, L1562P,
G1579R, L1580V, R1583W, R1597W, R1597G, R1597Q,
V1604F, V1607D, G1609R, S1613P, D1614G, I1628T,
G1629R, E1638K, L1639P, P1648S, V1665E, and G1672R).25
The absence of high vWF multimers and increased triplet
structure is the consequence of increased proteolysis of large
vWF multimers (Figures 5 and 6). Structural changes within
the A2 domain can produce 2 different characteristic
phenotypes of type 2A vWD.25 Expression studies demon-
strated that the single missense mutations V1607D, S1506L,
L1540P, and G1505R resulted in poor or no secretion of high
molecular weight due to impaired transport of vWF multimers
between the endoplasmic reticulum and the Golgi complex (the
so-called vWD 2A group 1 defect) with very likely intracellular
proteolysis of large vWF multimers.26-28 Expression studies
demonstrated that at least 5 missense mutations in the A2
domain R1597W, G1505E, E1638K, I1628T, and L1503Q
result in normal secretion of high-molecular-weight multimers
similar to the wild-type multimers, indicating that subsequent
loss-of-large vWF multimers is caused by hypersensitivity to
ADMAMTS13 induced increased proteolysis in plasma (the
so-called vWD 2A group 2 defect).25-28 Interestingly, platelet
lysates demonstrated decrease in large vWF multimers for
G1505L and S1506L mutants of vWD 2A group 1, but a nor-
mal pattern for the G1505E and R1597W mutants of vWD 2A
group 2. This simply means that vWF of severe vWD 2A group
2 is already proteolysed in endothelial cells before secretion,
whereas vWF in mild to moderate vWD 2A group 2 is secreted
as large multimers that after secretion from endothelial are
proteolysed due to hypersensitivity to ADAMTS13.25
Hassenpflug et al investigated the impact of mutations in the
A2 domain of vWF commonly found in patients with vWD
type 2A on ADAMTS13-dependent proteolysis of vWF.29
They used recombinant human ADAMTS13 (rhuADAMTS13)
to digest recombinant full-length recombinant vWF (rhuvWF)
and a vWF fragment spanning the vWF A1 through A3
domains, harboring 12 different vWD type 2A mutations
(G1505E, G1505R, S1506L, M1528V, R1569del, R1597W,
526 Clinical and Applied Thrombosis/Hemostasis 23(6)

Figure 4. Left: von Willebrand disease (vWD) 2E: Transient correction of PFA-100 closure time and restricted increase of von Willebrand
factor (vWF) parameters from around 0.20-0.40 U/mL to around 1.0 U/mL in a case of vWD type 2E (mutation W1120S). The vWF multimeric
pattern is characterized by a lack or relative decrease in large multimers and the absence of the outer subband of the normal triplet structure.
Please note that both low- and medium-resolution gels according to Budde show a typical vWF 2E multimeric pattern.17,18 Right: von Willebrand
disease 2M: poor response of vWF:RCo to DDAVP, normal vWF multimers before and after DDAVP, and good responses of FVIII:C, vWF:Ag,
and vWF:CB followed by shortened half-life time indicating rapid clearance defect of the FVIII-vWF complex in addition to the loss of vWF:RCo
function in a typical case of vWD 2M with normal vWF multimers before and after DDAVP17 in medium-resolution gel, according to Budde.17,18

Table 4. Laboratory Features of Dominant vWD Type 2A in a Large Dutch Family With the Presence or Absence of the V1499E Mutation in 11
and 5 Family Members, Respectively.a

Patient Gender FVIII:C vWF:Ag vWF:pp pp/Ag vWF:RCo Rco/Ag RIPA V1499E

II-1 M 63 59 76 1.3 <15 0.30 N Yes

III-1 M 41 33 <15 0.46 N Yes
III-4 F 36 33 40 1.3 <15 0.46 N Yes
III-6 F 38-57 31 51 1.5 21 0.77 Yes
III-7 F 40-55 22 44 2.0 16 0.73 Yes
III-9 F 38 24 38 1.9 <15 0.40 Yes
IV-2 F 21 17 0.85 Yes
IV-8 M 50 51 43 1.5 16 0.31 Yes
IV-2 F 21 17 0.81 Yes
IV-11 F 60 27 56 2.1 <15 0.56 Yes
IV-12 M 24 20 35 1.6 Yes
Normal
III-2 F 102 112 109 1.0 127 1.13 No
IV-1 M 102 122 99 0.8 148 1.21 No
IV-6 M 54 67 72 1.1 70 1.04 No
IV-9 M 78 60 72 1.2 79 1.31 No
IV-10 M 51 63 77 1.2 79 1.25 No
Abbreviations: Ag, antigen; RCo ristocetine cofactor; F, female; M, male; RIPA, ristocetine-induced platelet aggregation; vWF, von Willebrand factor.
a
Affected family members were difficult to identify owing to the variable vWF levels when the use of insensitive vWF assays and low-resolution vWF multimeric
analysis according to the ISTH classification is applied.24
Michiels et al 527

Figure 5. Left: Normal responses to DDAVP of FVIII:C and von Willebrand factor antigen (vWF:Ag) and restricted but transiently rather good
responses of functional vWF:RCo and vWF:CB to about 1 U/mL with transient correction of Ivy bleeding times and reappearance of large vWF
multimers in moderate vWD type 2A (mutation V1499E) followed by short half-life times of vWF:RCo and vWF:CB due to increased proteolysis
of vWF multimers. von Willebrand disease (vWD) type 2A mutation V1499E is featured by the loss of large vWF multimers and increase in
intermediate and small vWF multimers in low-resolution gels.2,25 Right: Please note that the vWF multimers in low-resolution gels, according to
the methods of Michiels et al (left) and Budde showing loss of large and increase of small vWF multimers, whereas the vWF multimeric pattern in
medium-resolution gel according to Budde reveals the typical triplet structure diagnostic of vWD type 2A.17,18

Table 5. Laboratory Features of Severe Dominant vWD 2A Group 1 in 3 Unrelated Cases and Moderate vWD of Moderate vWD 2A Group 2
in 3 Related Cases of a large Family With the Mutation V1499E.24,25

vWF: (U/mL)

Case/mutation Sex BT Minutes BT after DDAVP Response to DDAVP VIII:C, U/mL Ag RCo CB RIPA

Three cases with severe vWD 2A Group 1.25

1 Unknown F >15 3-9 Poor 0.93 0.45 0.13 0.05 Decreased
2 Unknown F >15 4-8 Poor 0.38 0.15 <0.10 <0.05 Absent
3 S1506L F >15 >15 Poor 0.66 0.41 0.15 0.05 Absent
Family with moderate vWD 2A group 2 (mutation V1499E).25
II-1 Table 5 M 4-10 <4 Transient 0.92 0.56 0.28 0.23 Normal
III-4 Table 5 F 6-10 <4 Transient – 0.42 0.15 0.18 Normal
III-1 Table 5 M 3-6 <4 Transient – 1.08 0.38 0.52 Normal
All 3 V1499E Figure 5
Normal <4 <4 Normal >0.60 >0.60 >0.60 >0.60 Normal
Abbreviations: Ag, antigen; BT, bleeding time; CB, collagen binding; F, female; RCo, ristocetine cofactor; M, male; RIPA, ristocetine-induced platelet aggregation;
vWD, von Willebrand disease; vWF, von Willebrand factor.

V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K).29 mutations G1505E, M1528V, G1609R, I1628T, G1629E,
With the exception of G1505E and I1628T, all mutations in the G1631D, and E1638K were similar in expression level with
vWF A2 domain showed increased specific proteolysis of vWF normal multimer distribution consistent with group 2 vWD 2A.
independent of the expression level. RhuvWF harboring the Due to the lack of proteolytic activity and shear stress in the
528 Clinical and Applied Thrombosis/Hemostasis 23(6)

Figure 6. Responses to DDAVP of von Willebrand factor (vWF) multimeric pattern induced the transient reappearance of large vWF
multimers in the von Willebrand disease (vWD) 2A type 2 caused by the mutations R1597W and G1629 in the A2 domain, indicating normal
intracellular synthesis and secretion of vWF. Responses of vWF multimers to DDAVP did not induce reappearance of large vWF multimers in
vWD 2A group 1, indicating impaired assembling transport and proteolysis of intracellular vWF multimers caused by the mutations S1506L and
V1665E in the A2 domain of the vWF gene. Adapted from Federici et al.15

culture medium both rhuvWFwild-type (WT) and rhuvWF
mutants group 2 are fully multimerized in these experimental
in vitro studies.29 The rhuvWF mutants were hypersensitive to
ADAMTS13-dependent proteolysis in vitro. The vWD 2M
mutation C1272S resulted in resistance of the recombinant
vWF fragment to ADAMTS13-dependent proteolysis. The
rhuvWF mutants G1505R, S1506L, R1568del, and V1607D
resulted in decreased in vitro expression of high- and
intermediate-molecular-weight multimers of vWF,16 consistent
with a group 1 mechanism.25-29 Proteolytic hypersusceptibility
to ADAMTS13-mediated proteolysis of mutant vWF group 2
in vitro closely correlated with the in vivo phenotype in patients
with vWD 2A. Increased vWF susceptibility for ADAMTS13
is a constitutive property of classical vWD type 2A, thus
explaining the pronounced proteolytic fragments and loss of
HMWM seen in multimer analysis in patients with vWD.25-28
Federici et al nicely showed that patients with severe vWD
type 2A group 1 due to the mutations S1506L and V1665E
showed poor responses of functional vWF parameters to
DDAVP and no reappearance of the large multimers (Figure
6), with persistence of a strongly prolonged BT, whereas
patients with mild to moderate vWD 2A group 2 due to the
mutations R1597W and G1629R responded better to DDAVP
with transient increase in large multimers (Figure 6) associated
with transient correction of BT and vWF:RCo to low normal
values for 1 hour (Figure 6).15 Severe vWD type 2A group 1
shows a typical proteolytic pattern with pronounced triplet
before and after DDAVP and in platelet, which strongly suggest
that proteolysis of large multimers occurs already intracellu-
lar.15 von Willebrand disease type 2A group 2 shows transient
correction of vWF multimers after DDAVP and normal vWF
multimers in platelet, indicating that mutant vWF after secretion
by endothelial cells in group 2 vWD type 2A is rapidly proteo-
lysed in plasma due to increased sensitivity to ADAMTS13.15,25
von Willebrand Disease 2M Due to Loss of
Function in the A1 Domain of vWF
Autosomal dominant vWD 2M has been misclassified in the
literature as vWD 2A, 1B, severe type 1, and 2 Unclassified
(2U).14 Dominant vWD type 2M and 2U are variable expres-
sions of 1 distinct disease entity caused by loss-of-function
Michiels et al 529

Table 6. Laboratory Features and Response to DDAVP of Von Willebrand Factor (vWF:Ag, RCo, CB) Parameters in 6 Probands (P) and
Additional 3 Affected Family Members, Who Presented With Pronounced Autosomal Dominant Von Willebrand Disease (vWD) Type 1
Secretion Defect (SD), 2M, and vWD type 1 clearance defect (type 1 CD ¼ Vicenza). With Normal vWF Multimers in Low- and Medium-
Resolution Gels.2,a

P, U/mL FVIII: C, U/mL : Ag, U/mL : RCo, U/mL : CB ratio Ag/RCo ratio Ag/CB, response RIPA type DDAVP vWD

1 0.19 0.09 <0.10 0.12 1.0 1.3 N Decreased Type 1 SD

2 0.56 0.17 0.16 0.13 0.9 0.8 N Decreased Type 1 SD
3.0 0.67 0.19 0.14 0.21 0.73 1.1 N Decreased Type 1 SD
3.1 1.26 0.18 0.23 0.16 1.3 0.9 N Decreased Type 1 SD
3.2 0.82 0.21 0.17 0.27 0.8 1.3 N Decreased Type 1 SD
4 0.52 0.23 <0.20 0.16 ? 0.7 Decreased Abnormal Type 2M
5 0.41 0.19 <0.10 0.13 0.5 0.7 Decreased Abnormal Type 2M
6.0 0.15 0.07 <0.10 0.07 ? 1.0 N Abnormal Vincenza
6.Son 0.19 0/08 <0.10 0.06 ? 0.8 N Abnormal Vincenza
Abbreviations: Ag, antigen; CB, collagen binding; RIPA, ristocetine-induced platelet aggregation; RCo, ristocetine cofactor.
a
Diagnostic differentiation between vWD SD versus 2M 1 CD ¼ Vicenza can only be made by a DDAVP challenge test.

mutations in the A1 domain of the vWF gene. von Willebrand
Disease 2M is identified by a poor response of vWF:RCo to
DDAVP; normal vWF multimers before and after DDAVP;
and good responses of FVIII, vWF:Ag, and vWF:CB followed
by shortened half-life time indicating rapid clearance defect of
the FVIII:C-vWF complex in addition to the loss of vWF:RCo
function in vWD 2M (Figures 3 and 4).5,6,10
The A1 domain (aa 1260-1479) is structurally delineated by
a disulfide bridge between Cys1272 and Cys1458. X-ray dif-
fraction studies of the A1 crystal structure revealed a globular
shape comprising a central core consisting of 6 hydrophobic a-
strands surrounded by 6 amphipathic a-helices.30,31 The anal-
ysis of naturally occurring loss-of-function mutations (vWD
2M), together with mutagenesis and glycoprotein Ib (GPIb) a
peptide docking studies, has identified a central front groove on
the A1 domain next to strand a3, as part of the binding site for
GPIba. von Willebrand disease type 2M mutations is charac-
terized by loss of function for vWF, while maintaining a nor-
mal multimerization pattern, primarily clustering around the
vWF interaction site with its platelet receptor GPIba. Two
main clusters vWD 2M with loss-of-function mutations in the
A1 domain are located in amino acid (aa) region 1272 to 1302
and in aa1359 to 1437. Three vWD 2M loss-of-function muta-
tions in aa 1315, aa1324, and aa1462 to 1467 can be identified
as isolated from 2B vWD gain-of-function mutations. Several
mutations in exon 28 of the A1 domain in patients with vWD
type 2M have been described, including L1382P, D1277-
E78delinsnsl, R1342C, G1415D, I1416N.13,32 The mutations
S1285P L1307 in the EU study very likely belong to the 2M
vWD category.18,33 von Willebrand disease cases of compound
heterozygous for R924Q-R1315C, P2145S-R1315C, P1266L-
R1315C, and L1481fs-Y1584C in the European vWD type 1
study33 show predominant features of vWD type 2M. The
ISTH registry of vWD has included the R1374C, R1374H,
R1374S, R1374L, R1379C, K1405del, and P1462A mutations
as unclassified (U) vWD group. L1382 was labeled as 2U or
2A, and F1369I and R1315C as 2M or 2U.31 The mutations
L1282R, S1285F, L1296P, D1302G, G1324S, G1324A,
R1392-Q1402del, E1352K, K1362T, P1475S, and P2781S
were labeled as vWD 2M.31
von Willebrand Disease 2B Due to Gain-of-
Function Mutation in the A1 Domain of vWF
von Willebrand disease type 2B cannot be distinguished from
2A by multimer analysis alone since their multimeric patterns
are very similar (Figure 1A).8-10 The combined use of FVIII:C,
vWF:Ag, vWF:RCo and vWF:CB, RIPA, and vWF multimeric
analysis in low- and medium-resolution gels is of critical
importance to differentiate pronounced vWD 1SD from 2E and
2M and to distinguish vWD 2A from vWD 2B (Table 6). The
vWF:Ag detects all multimeric forms of vWF equally with no
differential sensitivities to species with different molecular
weights including large, intermediate, and small multimers.34
The vWF:RCo assay detects all large and some of the inter-
mediate vWF multimers in all variants of type 2 vWD and in
vWF concentrates.34 The vWF:CB assay better detects the
hemostatically potent large vWF multimers than the vWF:RCo
assay.16,34 Adcock et al retrospectively studied 497 cases in
whom results for vWF multimeric analysis in low-resolution
gels vWF:Ag, vWF:RCo, and vWF:CB were available.35 The
distribution of vWF:RCo/Ag ratios for types 2A and 2B
showed that those classified as type 2A the mean vWF:RCo/
Ag ratio was 0.48 þ 0.040 and for those samples classified as
type 2B, the mean vWF:RCo/Ag ratio was 0.35 þ 0.023.35 von
Willebrand disease type 2A and type 2B with loss-of-large
vWF multimers show that for those classified as type 2A, the
mean vWF:CB/Ag ratio was 0.21 þ 0.019 and for those clas-
sified as type 2B, the ratio vWF:CB/Ag was 0.36 þ 0.025.35
The vWF:CB/Ag ratios were more pronouncedly lower in type
2A as compared to type 2B vWD. The vWF:RCo/Ag ratio was
more pronouncedly lower in type 2B as compared to type 2A
vWD. The sensitivity and specificity of the vWF:RCo and
vWF:CB assays for the diagnosis of vWD types 2A and 2B are
100% in addition to the loss-of-large vWF multimers in a low-
and medium-resolution gel (Figure 5).35
530
von Willebrand disease type 2B is caused by a gain-of-
function mutation of the GPIb receptor in the A1 domain of
vWF.8-11 The crystal structure of a gain-of-function A1 domain
mutant R1306Q in complex with the amino-terminal domain of
GPIba (also containing a gain-of-function mutation) confirmed
that the frontal part of A1 constitutes the contact area for
GPIba.30,33,36 Two distinct areas of tight interaction were
revealed, the first and most extensive contact site being located
near the top of A1, the second involving residues near the
bottom face of A1.36 A main cluster of vWD 2B gain-of-
function mutations (increased binding of vWF-A1 and plate-
let-GPIba) is located between aa1304 and aa1341 with 2
exceptions aa1315 and aa1324. Two minor clusters of vWD
2B mutations are located at aa1268-1272 and aa1460-1462.36
The key criterion for the diagnosis of vWD 2B is the loss-of-
large vWF multimers (Figure 1A: Table 1) due to increased
proteolysis caused by increased interaction of platelets mutated
vWF with a gain of function in the A1 domain as reflected by
increased RIPA.2,6,25,36 von Willebrand disease 2B is caused
by hyperreactive mutant vWF with increased affinity for the
platelet receptor glycoprotein Ib-a in vivo followed by
increased proteolysis of large vWF multimers.34,35 In that pro-
cess of increased vWF-GpIb-platelet interaction of mutant
vWF in vWD 2B clump as soon as the mutant vWF enters the
circulation. Clumps of mutant vWF platelets are cleared from
the circulation and thereby upon DDAVP or stress complicated
by moderate to severe thrombocythopenia.36 In a cohort of 67
vWD 2B patients from 38 unrelated families, Federici et al
evaluated the clinical and molecular predictors of thrombocy-
topenia and the risk of bleeding. Thrombocytopenia was found
in 30% at baseline and in 57% after stress conditions in only
those with vWD 2B who did meet the ISTH criteria.36 Platelet
counts were always normal in 16 (24%) patients from 5 fam-
ilies carrying the P1266L or R1308L mutation, who usually
have a type 1 variant of mild vWD 2B with normal
vWF:RCo/Ag ratios of 0.9 and 0.8, respectively (vWD type
1B).36 In fact, à patients with classical vWD type 2B usually
present with moderate to severe clinical phenotype. Patients
with vWD 2B having the mutations (P1266L, Malmo, or New
York vWD 1B phenotype) do have a mild bleeding illness with
normal ratios of vWF:RCo/vWF:Ag consistent with a labora-
tory vWD type 1B phenotype with increased RIPA.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
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