The Diagnosis, Evaluation and Management of von
Willebrand Disease
Scientific Overview
Discovery and Identification of VWD/VWF
The patient who led to the discovery of a hereditary
bleeding disorder that we now call VWD was a 5-year-old girl who lived on the
Åland Islands and was brought to Deaconess Hospital in Helsinki, Finland,
in 1924 to be seen by Dr. Erik von Willebrand.10 He ultimately assessed 66 members of
her family and reported in 1926 that this was a previously undescribed bleeding
disorder that differed from hemophilia and exhibited (1) mucocutaneous
bleeding, (2) autosomal inheritance rather than being linked to the X
chromosome, (3) prolonged bleeding times by the Duke method (ear lobe bleeding
time), and (4) normal clotting time. Not only did he recognize the autosomal
inheritance pattern, but he recognized that bleeding symptoms were greater in
children and in women of childbearing age. He subsequently found that blood
transfusions were useful not only to correct the anemia but also to control
bleeding.
In the 1950s, it became clear that a "plasma factor,"
antihemophilic factor (FVIII), was decreased in these persons and that Cohn
fraction I-0 could correct both the plasma deficiency of FVIII and the
prolonged bleeding time. For the first time, the factor causing the long
bleeding time was called "von Willebrand factor." As cryoprecipitate and
commercial FVIII concentrates were developed, it was recognized that both VWF
and "antihemophilic factor" (FVIII) purified together.
When immunoassays were developed, persons who had VWD
(in contrast to those who had hemophilia A) were found to have reduced "factor
VIII-related antigen" (FVIIIR:Ag), which we now refer to as VWF:Ag.
Characterization of the proteins revealed that FVIII was the clotting protein
deficient in hemophilia A, and VWF was a separate "FVIII carrier protein" that
resulted in the cofractionation of both proteins in commercial concentrates.
Furthermore, a deficiency of VWF resulted in increased FVIII clearance because
of the reduced carrier protein, VWF. Since the 1980s, molecular and cellular
studies have defined hemophilia A and VWD more precisely. Persons who had VWD
had a normal FVIII gene on the X chromosome, and some were found to have an
abnormal VWF gene on chromosome 12. Variant forms of VWF were recognized in the
1970s, and we now recognize that these variations are the result of synthesis
of an abnormal protein. Gene sequencing identified many of these persons as
having a VWF gene mutation. The genetic causes of milder forms of low VWF are
still under investigation, and these forms may not always be caused by an
abnormal VWF gene. In addition, there are acquired disorders that may result in
reduced or dysfunctional VWF (see section on "Acquired von
Willebrand Syndrome" [AVWS]). Table 2 contains a
synopsis of VWF designations, functions, and assays. Table
3 contains abbreviations used throughout this document.
Table 2. Synopsis of VWF Designations, Properties,
and Assays
Designation |
Property |
Assay |
von Willebrand factor (VWF) |
Multimeric glycoprotein that promotes platelet
adhesion and aggregation and is a carrier for FVIII in plasma |
See specific VWF assays below |
von Willebrand factor ristocetin
cofactor activity (VWF:RCo) |
Binding activity of VWF that causes binding of
VWF to platelets in the presence of ristocetin with consequent
agglutination |
Ristocetin cofactor activity: quantitates
platelet agglutination after addition of ristocetin and VWF |
von Willebrand factor antigen
(VWF:Ag) |
VWF protein as measured by protein assays; does
not imply functional ability |
Immunologic assays such as ELISA*, LIA*, RIA*,
Laurell electroimmunoassay |
von Willebrand factor
collagen-binding activity (VWF:CB) |
Ability of VWF to bind to collagen |
Collagen-binding activity: quantitates binding of
VWF to collagen-coated ELISA* plates |
von Willebrand factor multimers |
Size distribution of VWF multimers as assessed by
agarose gel electrophoresis |
VWF multimer assay: electrophoresis in agarose
gel and visualization by monospecific antibody to VWF |
Factor VIII (FVIII) |
Circulating coagulation protein that is protected
from clearance by VWF and is important in thrombin generation |
FVIII activity: plasma clotting test based on
PTT* assay using FVIII-deficient substrate; quantitates activity |
Ristocetin-induced Platelet
Aggregation (RIPA) |
Test that measures the ability of a person's VWF
to bind to platelets in the presence of various concentrations of
ristocetin |
RIPA: aggregation of a person's PRP* to various
concentrations of ristocetin |
*See Table 3. Nomenclature
and Abbreviations.
The VWF Protein and Its
Functions In Vivo
VWF is synthesized in two cell types. In the vascular
endothelium, VWF is synthesized and subsequently stored in secretory granules
(Weibel-Palade bodies) from which it can be released by stress or drugs such as
desmopressin (DDAVP, 1-desamino-8-D-arginine vasopressin), a synthetic analog
of vasopressin. VWF is also synthesized in bone marrow megakaryocytes where it
is stored in platelet alpha-granules from which it is released following
platelet activation. DDAVP does not release platelet VWF.
VWF is a protein that is assembled from identical
subunits into linear strings of varying size referred to as multimers. These
multimers can be >20 million daltons in mass and >2 micrometers in
length. The complex cellular processing consists of dimerization in the
endoplasmic reticulum (ER), glycosylation in the ER and Golgi, multimerization
in the Golgi, and packaging into storage granules. The latter two processes are
under the control of the VWF propeptide (VWFpp), which is cleaved from VWF at
the time of storage. VWF that is released acutely into the circulation is
accompanied by a parallel rise in FVIII, but it is still not entirely clear
whether this proteinprotein association first occurs within the
endothelial cell.11,12
In plasma, the FVIIIVWF complex circulates as a
loosely coiled protein complex that does not interact strongly with platelets
or endothelial cells under basal conditions. When vascular injury occurs, VWF
becomes tethered to the exposed subendothelium (collagen, etc.). The high fluid
shear rates that occur in the microcirculation appear to induce a
conformational change in multimeric VWF that causes platelets to adhere, become
activated, and then aggregate so as to present an activated platelet
phospholipid surface. This facilitates clotting that is, in part, regulated by
FVIII. Because of the specific characteristics of hemostasis and fibrinolysis
on mucosal surfaces, symptoms in VWD are often greater in these tissues.
Plasma VWF is primarily derived from endothelial
synthesis. Platelet and endothelial cell VWF are released locally following
cellular activation where this VWF participates in the developing hemostatic
plug or thrombus (see Figure 1).
Plasma VWF has a half-life of approximately 12 hours
(range 915 hours). VWF is present as very large multimers that are
subjected to physiologic degradation by the metalloprotease ADAMTS13 (A
Disintegrin-like And Metalloprotease domain [reprolysin type] with
Thrombospondin type I motifs). Deficiency of ADAMTS13 is associated with the
pathologic microangiopathy of thrombotic thrombocytopenic purpura (TTP). The
most common variant forms of type 2A VWD are characterized by increased VWF
susceptibility to ADAMTS13.
Table 3. Nomenclature and Abbreviations
Designation |
Definition |
ADAMTS13 |
A Disintegrin-like And Metalloprotease domain
(reprolysin type) with ThromboSpondin type 1 motifs, a plasma metalloprotease
that cleaves multimeric VWF |
ASH |
American Society of Hematology |
AVWS |
acquired von Willebrand syndrome |
BT |
bleeding time |
CAP |
College of American Pathologists |
CBC |
complete blood count |
CDC |
Centers for Disease Control and Prevention |
CFC |
clotting factor concentrate |
CI |
confidence interval |
C.I. |
continuous infusion |
CLSI |
Clinical Laboratory Standards Institute (formerly
National Committee for Clinical Laboratory Standards: NCCLS) |
CNS |
central nervous system |
CV |
coefficient of variation |
Cyclic AMP |
adenosine 3′5′cyclic phosphate |
CK |
cystine knot |
D & C |
dilation and curettage |
DARD |
Division for the Application of Research
Discoveries |
DDAVP |
1-desamino-8-D-arginine vasopressin
(desmopressin, a synthetic analog of vasopressin) |
DIC |
disseminated intravascular coagulation |
DNA |
deoxyribonucleic acid |
DVT |
deep vein thrombosis |
ELISA |
enzyme-linked immunosorbent assay |
ER |
endoplasmic reticulum |
FDA |
Food and Drug Administration |
FVIII* |
[blood clotting] factor VIII |
FVIIIR:Ag* |
factor VIII-related antigen (see VWF:Ag) |
FVIII:C* |
factor VIII coagulant activity |
FVIII gene |
factor VIII gene |
GI |
gastrointestinal |
GPIb |
glycoprotein Ib (platelet) |
GPIIb/IIIa |
glycoprotein IIb/IIIa complex (platelet) |
HRT |
hormone replacement therapy |
IgG |
immunoglobulin G |
IGIV |
immune globulin intravenous (also known as
IVIG) |
ISTH |
International Society on Thrombosis and
Haemostasis |
IU/dL |
international units per deciliter |
LIA |
latex immunoassay (automated) |
MAB |
monoclonal antibody |
MeSH |
medical subject headings (in MEDLINE) |
MGUS |
monoclonal gammopathy of uncertain
significance |
NCCLS |
National Committee for Clinical Laboratory
Standards |
NHF, MASAC |
National Hemophilia Foundation, Medical and
Scientific Advisory Committee |
NHLBI |
National Heart, Lung, and Blood Institute |
NIH |
National Institutes of Health |
N.R. |
not reported |
NSAIDs |
nonsteroidal anti-inflammatory drugs |
OCP |
oral contraceptive pill |
PAI-1 |
plasminogen activator inhibitor type 1 |
PCR |
polymerase chain reaction |
PFA-100® |
platelet function analyzer |
PLT-VWD |
platelet-type von Willebrand disease |
PRP |
platelet-rich plasma |
PT |
prothrombin time |
PTT |
partial thromboplastin time (activated partial
thromboplastin time) |
RIA |
radioimmunoassay |
RIPA |
ristocetin-induced platelet aggregation |
SDS |
sodium dodecyl sulfate |
TTP |
thrombotic thrombocytopenic purpura |
tPA |
tissue plasminogen activator |
TT |
thrombin time |
Tx |
Treatment |
VWD |
von Willebrand disease |
VWF* |
von Willebrand factor (FVIII carrier
protein) |
VWF:Ac |
von Willebrand factor activity |
VWF:Ag* |
von Willebrand factor antigen |
VWF:CB* |
von Willebrand factor collagen-binding
activity |
VWF:FVIIIB* |
von Willebrand factor: factor VIII binding
assay |
VWF gene |
von Willebrand factor gene |
VWF:PB assay |
von Willebrand factor platelet-binding assay |
VWFpp |
von Willebrand factor propeptide |
VWF:RCo* |
von Willebrand factor ristocetin cofactor
activity |
WHO |
World Health Organization |
*These abbreviations (for FVIII and VWF and all
their properties) are defined in Marder VJ, Mannucci PM, Firkin BG, Hoyer LW,
Meyer D. Standard nomenclature for factor VIII and von Willebrand factor: a
recommendation by the International Committee on Thrombosis and Haemostasis.
Thromb Haemost 1985 Dec;54(4):871872; Mazurier C, Rodeghiero F.
Recommended abbreviations for von Willebrand Factor and its activities.
Thromb Haemost 2001 Aug;86(2):712.
Factors that affect levels of plasma VWF
include age, race, ABO and Lewis blood groups, epinephrine, inflammatory
mediators, and endocrine hormones (particularly those associated with the
menstrual cycle and pregnancy). VWF is increased during pregnancy (a three- to
fivefold elevation over the woman's baseline by the third trimester), with
aging, and with acute stress or inflammation. Africans and African Americans
have higher average levels of VWF than the Caucasian population.13,14 VWF is reduced by hypothyroidism and
rarely by autoantibodies to VWF. The rate of VWF synthesis probably is not
affected by blood group; however, the survival of VWF appears to be reduced in
individuals who have type O blood. In fact, ABO blood group substance has been
identified on VWF.
The Genetics of VWDM
Since the 1980s, molecular and cellular studies have
defined hemophilia A and VWD more precisely. Persons who have severe VWD have a
normal FVIII gene on the X chromosome, and some are found to have an abnormal
VWF gene on chromosome 12. The VWF gene is located near the tip of the short
arm of chromosome 12, at 12p13.3.15 It spans approximately 178 kb of DNA
and contains 52 exons.16
Intronexon boundaries tend to delimit structural domains in the protein,
and introns often occur at similar positions within the gene segments that
encode homologous domains. Thus, the structure of the VWF gene reflects the
mosaic nature of the protein (Figure 2).
A partial, unprocessed VWF pseudogene is located at
chromosome 22q11.2.17 This
pseudogene spans approximately 25 kb of DNA and corresponds to exons 2334
and part of the adjacent introns of the VWF gene.18 This segment of the gene encodes
domains A1A2A3, which contain binding sites for platelet glycoprotein Ib (GPIb)
and collagen, as well as the site cleaved by ADAMTS13. The VWF pseudogene and
gene have diverged 3.1 percent in DNA sequence, consistent with a relatively
recent origin of the pseudogene by partial gene duplication.18 This pseudogene is found in humans and
great apes (bonobo, chimpanzee, gorilla, orangutan) but not in more distantly
related primates.19 The VWF
pseudogene complicates the detection of VWF gene mutations because polymerase
chain reactions (PCRs) can inadvertently amplify segments from either or both
loci, but this difficulty can be overcome by careful design of gene-specific
PCR primers.18
Figure 1. VWF and Normal
Hemostasis
![Figure 1. VWF and Normal Hemostasis. This figure is explained in detail in the legend below.](images/Figure1.jpg)
A cross-sectioned blood vessel shows
stages of hemostasis. Top, VWF is the carrier protein for blood clotting factor
VIII (FVIII). Under normal conditions VWF does not interact with platelets or
the blood vessel wall that is covered with endothelial cells. Middle left,
following vascular injury, VWF adheres to the exposed subendothelial matrix.
Middle right, after VWF is uncoiled by local shear forces, platelets adhere to
the altered VWF and these platelets undergo activation and recruit other
platelets to this injury site. Bottom left, the activated and aggregated
platelets alter their membrane phospholipids exposing phosphatidylserine, and
this activated platelet surface binds clotting factors from circulating blood
and initiates blood clotting on this surface where fibrin is locally deposited.
Bottom right, the combination of clotting and platelet aggregation and adhesion
forms a platelet-fibrin plug, which results in the cessation of bleeding. The
extent of the clotting is carefully regulated by natural anticoagulants.
Subsequently, thrombolysis initiates tissue repair and ultimately the vessel
may be re-endothelialized and blood flow maintained. Note: Used by
permission of R.R. Montgomery.
The VWF pseudogene may occasionally serve as a
reservoir of mutations that can be introduced into the VWF locus. For example,
some silent and some potentially pathogenic mutations have been identified in
exons 27 and 28 of the VWF gene of persons who have VWD. These same sequence
variations occur consecutively in the VWF pseudogene and might have been
transferred to the VWF by gene conversion.20-22 The segments involved in the
potential gene conversion events are relatively short, from a minimum of 7
nucleotides20 to a maximum of 385
nucleotides.22 The frequency of
these potential interchromosomal exchanges is unknown.
The spectrum of VWF gene mutations that cause VWD is
similar to that of many other human genetic diseases and includes large
deletions, frameshifts from small insertions or deletions, splice-site
mutations, nonsense mutations causing premature termination of translation, and
missense mutations affecting single amino acid residues. A database of VWF
mutations and polymorphisms has been compiled for the International Society on
Thrombosis and Haemostasis (ISTH)23,24 and is maintained for online access
at the University of Sheffield (http://www.shef.ac.uk/vwf/index.html).
Mutations causing VWD have been identified throughout the VWF gene. In contrast
to hemophilia A, in which a single major gene rearrangement causes a large
fraction of severe disease, no such recurring mutation is common in VWD. There
is a good correlation between the location of mutations in the VWF gene and the
subtype of VWD, as discussed in more detail in "Classification of VWD
Subtypes." In selected families, this information can facilitate the search for
VWF mutations by DNA sequencing.
Figure 2. Structure and Domains
of VWF
![Figure 2. Structure and Domains of VWF. This figure is explained in detail in the legend below.](images/Figure2.gif)
The von Willebrand factor (VWF) protein
sequence (amino acid 12813) is aligned with the cDNA sequence (nucleic
acid 18439). The VWF signal peptide is the first 22 aa, the propeptide
(VWFpp) aa 23763, and mature VWF aa 7642800. Type 2 mutations are
primarily located in specific domains (regions) along the VWF protein. Types
2A, 2B, and 2M VWF mutations are primarily located within exon 28 that encodes
for the A1 and A2 domains of VWF. The two different types of 2A are those that
have increased proteolysis (2A2) and those with abnormal multimer
synthesis (2A1). Type 2N mutations are located within the D′
and D3 domains. Ligands that bind to certain VWF domains are identified,
including FVIII, heparin, GPIb (platelet glycoprotein Ib complex), collagen,
and GPIIb/IIIa (platelet glycoprotein IIb/IIIa complex that binds to the RGD
[arginine-glycine-aspartate] amino acid sequence in VWF). Note: Used by
permission of R.R. Montgomery.
Classification of VWD
Subtypes
VWD is classified on the basis of criteria developed
by the VWF Subcommittee of the ISTH, first published in 1994 and revised in
2006 (Table 4).25,26
The classification was intended to be clinically
relevant to the treatment of VWD. Diagnostic categories were defined that
encompassed distinct pathophysiologic mechanisms and correlated with the
response to treatment with DDAVP or blood products. The classification was
designed to be conceptually independent of specific laboratory testing
procedures, although most of the VWD subtypes could be assigned by using tests
that were widely available. The 1994 classification reserved the designation of
VWD for disorders caused by mutations within the VWF gene,25 but this criterion has been dropped
from the 2006 classification26
because in practice it is verifiable for only a small fraction of patients.
VWD is classified into three major categories: partial
quantitative deficiency (type 1), qualitative deficiency (type 2), and total
deficiency (type 3). Type 2 VWD is divided further into four variants (2A, 2B,
2M, 2N) on the basis of details of the phenotype. Before the publication of the
1994 revised classification of VWD,25 VWD subtypes were classified using
Roman numerals (types I, II, and III), generally corresponding to types 1, 2,
and 3 in the 1994 classification, and within type II several subtypes existed
(designated by adding sequential letters of the alphabet; i.e., II-A through
II-I). Most of the latter VWD variants were amalgamated as type 2A in the 1994
classification, with the exception of type 2B (formerly II-B) for which a
separate new classification was created. In addition, a new subtype (2M) was
created to include variants with decreased platelet dependent function
(VWF:RCo) but no significant decrease of higher molecular weight VWF multimers
(which may or may not have other aberrant structure), with "M" representing
"multimer." Subtype 2N VWD was defined, with "N" representing "Normandy" where
the first individuals were identified, with decreased FVIII due to VWF defects
of FVIII binding.
Type 1 VWD affects approximately 75 percent of
symptomatic persons who have VWD (see Castaman et al., 2003 for a
review).27 Almost all of the
remaining persons are divided among the four type 2 variants, and the
partitioning among them varies considerably among centers. In France, for
example, patients’ distribution was reported to be 30 percent type 2A, 28
percent type 2B, 8 percent type 2M (or unclassified), and 34 percent type
2N.28 In Bonn, Germany, the
distribution was reported to be 74 percent type 2A, 10 percent type 2B, 13
percent type 2M, and 3.5 percent type 2N.29 Table 5
summarizes information about inheritance, prevalence, and bleeding propensity
in persons who have different types of VWD.
Table 4. Classification of VWD
Type |
Description |
1 |
Partial quantitative deficiency of VWF |
2 |
Qualitative VWF defect |
2A |
Decreased VWF-dependent platelet adhesion with
selective deficiency of high-molecular-weight multimers |
2B |
Increased affinity for platelet GPIb |
2M |
Decreased VWF-dependent platelet adhesion without
selective deficiency of high-molecular-weight multimers |
2N |
Markedly decreased binding affinity for
FVIII |
3 |
Virtually complete deficiency of VWF |
Note: VWD types are defined as described in
Sadler JE, Budde U, Eikenboom JC, Favaloro EJ, Hill FG, Holmberg L, Ingerslev
J, Lee CA, Lillicrap D, Mannucci PM, et al. Update on the pathophysiology and
classification of von Willebrand disease: a report of the Subcommittee on von
Willebrand Factor. J Thromb Haemost 2006 Oct;4(10):21032114.
Table 5. Inheritance, Prevalence, and Bleeding
Propensity in Patients Who Have VWD
Type |
Inheritance |
Prevalence |
Bleeding Propensity |
Type 1 |
Autosomal dominant |
Up to 1% |
Mild to moderate |
Type 2A |
Autosomal dominant (or recessive) |
Uncommon |
Variableusually moderate |
Type 2B |
Autosomal dominant |
Uncommon |
Variableusually moderate |
Type 2M |
Autosomal dominant (or recessive) |
Uncommon |
Variableusually moderate |
Type 2N |
Autosomal recessive |
Uncommon |
Variableusually moderate |
Type 3 (Severe) |
Autosomal recessive |
Rare (1:250,000 to 1:1,000,000) |
High (severe bleeding) |
The prevalence of type 3 VWD in the population is not
known precisely but has been estimated (per million population) as: 0.55 for
Italy,30 1.38 for North
America,31 3.12 for Sweden,30 and 3.2 for Israel.32 The prevalence may be as high as 6 per
million where consanguinity is common.1
Type 1 VWD
Type 1 VWD is found in persons who have partial
quantitative deficiency of VWF. The level of VWF in plasma is low, and the
remaining VWF mediates platelet adhesion normally and binds FVIII normally.
Laboratory evaluation shows concordant decreases in VWF protein concentration
(VWF:Ag) and assays of VWF function (VWF:RCo). Levels of blood clotting FVIII
usually parallel VWF and may be reduced secondary to reduced VWF. Usually, in
type 1 VWD, the FVIII/VWF:Ag ratio is 1.52.0. In most persons who have
type 1 VWD, this results in FVIII being normal, or mildly decreased, and not
reduced as much as the VWF. VWF multimer gels show no significant decrease in
large VWF multimers.25 The
laboratory evaluation of VWD is discussed in the
"Diagnosis and Evaluation"
section.
The spectrum of mutations occurring in VWD type 1 has
been described extensively in two major studies.33,34 Particularly severe, highly
penetrant forms of type 1 VWD may be caused by dominant VWF mutations that
interfere with the intracellular transport of dimeric proVWF35-39 or that promote the rapid clearance
of VWF from the circulation.38,40,41 Persons who have such mutations
usually have VWF levels <20 IU/dL.33,34 Most of the mutations characterized
to date cause single amino acid substitutions in domain D3.35-37,39,42 One mutation associated with rapid
clearance has been reported in domain D4.38
Increased clearance of VWF from the circulation in
type 1 VWD may account for the exaggerated but unexpectedly brief responses to
DDAVP observed in some patients. Consequently, better data on the prevalence of
increased clearance could affect the approach to diagnosing type 1 VWD and the
choice of treatment for bleeding.
A diagnosis of type 1 VWD is harder to establish when
the VWF level is not markedly low but instead is near the lower end of the
normal range. Type 1 VWD lacks a qualitative criterion by which it can be
recognized and instead relies only on quantitative decrements of protein
concentration and function. VWF levels in the healthy population span a wide
range of values. The mean level of plasma VWF is 100 IU/dL, and approximately
95 percent of plasma VWF levels lie between 50 and 200 IU/dL.43,44 Because mild bleeding symptoms are
very common in the healthy population, the association of bleeding symptoms
with a moderately low VWF level may be coincidental.45 The conceptual and practical issues
associated with the evaluation of moderately low VWF levels are discussed more
completely later in this section. (See "Type 1 VWD Versus
Low VWF: VWF Level as a Risk Factor for Bleeding.")
Type 2 VWD
The clinical features of several type 2 VWD variants
are distinct from those of type 1 VWD, and they can have strikingly distinct
and specific therapeutic needs. As a consequence, the medical care of patients
who have type 2 VWD benefits from the participation of a hematologist who has
expertise in hemostasis. Bleeding symptoms in type 2 VWD are often thought to
be more severe than in type 1 VWD, although this impression needs to be
evaluated in suitable clinical studies.
Type 2A VWD refers to qualitative variants in
which VWF-dependent platelet adhesion is decreased because the proportion of
large VWF multimers is decreased. Levels of VWF:Ag and FVIII may be normal or
modestly decreased, but VWF function is abnormal as shown by markedly decreased
VWF:RCo.46 Type 2A VWD may be
caused by mutations that interfere with the assembly or secretion of large
multimers or by mutations that increase the susceptibility of VWF multimers to
proteolytic degradation in the circulation.47-49 The deficit of large multimers
predisposes persons to bleed.
The location of type 2A VWD mutations sometimes can be
inferred from high-resolution VWF multimer gels. For example, mutations that
primarily reduce multimer assembly lead to the secretion of multimers that are
too small to engage platelets effectively and therefore are relatively
resistant to proteolysis by ADAMTS13. Homozygous mutations in the propeptide
impair multimer assembly in the Golgi and give rise to a characteristic "clean"
pattern of small multimers that lack the satellite bands usually associated
with proteolysis (see "Diagnosis and
Evaluation"); this pattern was initially described as "type IIC"
VWD.50-52 Heterozygous mutations
in the cystine knot (CK) domain can impair dimerization of proVWF in the ER and
cause a recognizable multimer pattern originally referred to as "type
IID."53,54 A mixture of monomers
and dimers arrives in the Golgi, where the incorporation of monomers at the end
of a multimer prevents further elongation. As a result, the secreted small
multimers contain minor species with an odd number of subunits that appear as
faint bands between the usual species that contain an even number of subunits.
Heterozygous mutations in cysteine residues of the D3 domain also can impair
multimer assembly, but these mutations often also produce an indistinct or
"smeary" multimer pattern referred to as "type IIE."55,56
In contrast to mutations that primarily affect
multimer assembly, mutations within or near the A2 domain of VWF cause type 2A
VWD that is associated with markedly increased proteolysis of the VWF
subunits56 (see Figure 2). These mutations apparently interfere with the
folding of the A2 domain and make the Tyr1605Met1606 bond accessible to
ADAMTS13 even in the absence of increased fluid shear stress. Two subgroups of
this pattern have been distinguished: group I mutations enhance proteolysis by
ADAMTS13 and also impair multimer assembly, whereas group II mutations enhance
proteolysis without decreasing the assembly of large VWF multimers.49
Computer modeling of domain A2 suggests that group I mutations affect both
assembly and proteolysis, because group I mutations have a more disruptive
effect on the folding of domain A2 than do group II mutations.57
Type 2B VWD is caused by mutations that
pathologically increase plateletVWF binding, which leads to the
proteolytic degradation and depletion of large, functional VWF
multimers.56,58 Circulating platelets also are coated
with mutant VWF, which may prevent the platelets from adhering at sites of
injury.59
Although laboratory results for type 2B VWD may be
similar to those in type 2A or type 2M VWD, patients who have type 2B VWD
typically have thrombocytopenia that is exacerbated by surgery, pregnancy, or
other stress.60-62 The
thrombocytopenia probably is caused by reversible sequestration of
VWFplatelet aggregates in the microcirculation. These aggregates are
dissolved by the action of ADAMTS13 on VWF, causing the characteristic decrease
of large VWF multimers and the prominent satellite banding pattern that
indicates increased proteolytic degradation.63,64 The diagnosis of type 2B VWD depends
on finding abnormally increased ristocetin induced platelet aggregation (RIPA)
at low concentrations of ristocetin.
Type 2B VWD mutations occur within or adjacent to VWF
domain A1,23,55,65-68
which changes conformation when it binds to platelet GPIb.69 The mutations appear to enhance
platelet binding by stabilizing the bound conformation of domain A1.
Type 2M VWD includes variants with decreased
VWF-dependent platelet adhesion that is not caused by the absence of
high-molecular-weight VWF multimers. Instead, type 2M VWD mutations reduce the
interaction of VWF with platelet GPIb or with connective tissue and do not
substantially impair multimer assembly. Screening laboratory results in type 2M
VWD and type 2A VWD are similar, and the distinction between them depends on
multimer gel electrophoresis.67
Mutations in type 2M VWD have been identified in
domain A1 (see Figure 2), where they interfere with
binding to platelet GPIb.23,55,67,70-72 One family has been reported in
which a mutation in VWF domain A3 reduces VWF binding to collagen, thereby
reducing platelet adhesion and possibly causing type 2M VWD.73
Type 2N VWD is caused by VWF mutations that
impair binding to FVIII, lowering FVIII levels so that type 2N VWD masquerades
as an autosomal recessive form of hemophilia A.74-76 In typical cases, the FVIII level is
less than 10 percent, with a normal VWF:Ag and VWF:RCo. Discrimination from
hemophilia A may require assays of FVIIIVWF binding.77,78
Most mutations that cause type 2N VWD occur within the
FVIII binding site of VWF (see Figure 2), which lies
between residues Ser764 and Arg1035 and spans domain D′ and part of
domain D3.23,79,80 The most common mutation, Arg854Gln,
has a relatively mild effect on FVIII binding and tends to cause a less severe
type 2N VWD phenotype.77 Some
mutations in the D3 domain C-terminal of Arg1035 can reduce FVIII
binding,81-83 presumably through
an indirect effect on the structure or accessibility of the binding site.
Type 3 VWD
Type 3 VWD is characterized by undetectable VWF
protein and activity, and FVIII levels usually are very low (19
IU/dL).84-86 Nonsense and
frameshift mutations commonly cause type 3 VWD, although large deletions,
splice-site mutations, and missense mutations also can do so. Mutations are
distributed throughout the VWF gene, and most are unique to the family in which
they were first identified.23,87,88
A small fraction of patients who have type 3 VWD
develop alloantibodies to VWF in response to the transfusion of plasma
products. These antibodies have been reported in 2.69.5 percent of
patients who have type 3 VWD, as determined by physician surveys or
screening.85,89 The true incidence is uncertain,
however, because of unavoidable selection bias in these studies. Anti-VWF
alloantibodies can inhibit the hemostatic effect of blood-product therapy and
also may cause life-threatening allergic reactions.85,90
Large deletions in the VWF gene may predispose patients to this
complication.89
VWD Classification, General Issues
The principal difficulties in using the current VWD
classification concern how to define the boundaries between the various
subtypes through laboratory testing. In addition, some mutations have
pleiotropic effects on VWF structure and function, and some persons are
compound heterozygous for mutations that cause VWD by different mechanisms.
This heterogeneity can produce complex phenotypes that are difficult to
categorize. Clinical studies of the relationship between VWD genotype and
clinical phenotype would be helpful to improve the management of patients with
the different subtypes of VWD.
The distinction between quantitative (type 1) and
qualitative (type 2) defects depends on the ability to recognize discrepancies
among VWF assay results,80,91 as discussed in "Diagnosis and
Evaluation." Similarly, distinguishing between type 2A and type 2M VWD requires
multimer gel analysis. Standards need to be established for using laboratory
tests to make these important distinctions.
The example of Vicenza VWD illustrates some of these
problems. Vicenza VWD was first described as a variant of VWD in which the
level of plasma VWF is usually <15 IU/dL and the VWF multimers are even
larger than normal, like the ultralarge multimers characteristic of platelet
VWF.92 The low level of VWF in
plasma in Vicenza VWD appears to be explained by the effect of a specific
mutation, Arg1205His, that promotes clearance of VWF from the circulation about
fivefold more rapidly than normal.41 Because the newly synthesized multimers
have less opportunity to be cleaved by ADAMTS13 before they are cleared,
accelerated clearance alone may account for the increased multimer size in
Vicenza VWD.93 Whether Vicenza VWD
is classified under type 1 VWD or type 2M VWD depends on the interpretation of
laboratory test results. The abnormally large multimers and very low RIPA
values have led some investigators to prefer the designation of type 2M
VWD.94 However, the VWF:RCo/VWF:
Ag ratio typically is normal, and large VWF multimers are not decreased
relative to smaller multimers, so that other investigators have classified
Vicenza VWD under type 1 VWD.41
Regardless of how this variant is classified, the markedly shortened half-life
of plasma VWF in Vicenza VWD is a key fact that, depending on the clinical
circumstance, may dictate whether the patient should receive treatment with
DDAVP or FVIII/VWF concentrates.
Type 1 VWD Versus Low VWF: VWF
Level as a Risk Factor for Bleeding
Persons who have very low VWF levels, <20 IU/dL,
are likely to have VWF gene mutations, significant bleeding symptoms, and a
strongly positive family history.33,34,37,95-99 Diagnosing such persons as having
type 1 VWD seems appropriate because they may benefit from changes in lifestyle
and from specific treatments to prevent or control bleeding. Identification of
affected family members also may be useful, and genetic counseling is
simplified when the pattern of inheritance is straightforward.
On the other hand, VWF levels of 3050 IU/dL,
just below the usual normal range (50200 IU/dL), pose problems for
diagnosis and treatment. Among the total U.S. population of approximately 300
million, VWF levels <50 IU/dL are expected in about 7.5 million persons, who
therefore would be at risk for a diagnosis of type 1 VWD. Because of the strong
influence of ABO blood group on VWF level,43 about 80 percent of U.S. residents who
have low VWF also have blood type O. Furthermore, moderately low VWF levels and
bleeding symptoms generally are not coinherited within families and are not
strongly associated with intragenic VWF mutations.100-102 In a recent Canadian study of 155
families who had type 1 VWD, the proportion showing linkage to the VWF locus
was just 41 percent.98 In a
similar European study, linkage to the VWF locus depended on the severity of
the phenotype. If plasma levels of VWF were <30 IU/dL, linkage was
consistently observed, but if levels of VWF were >30 IU/dL, the proportion
of linkage was only 51 percent.97
Furthermore, bleeding symptoms were not significantly linked to the VWF gene in
these families.97
Family studies suggest that 2532 percent of the
variance in plasma VWF is heritable.103,104 Twin studies have reported
greater heritability of 6675 percent,105,106 although these values may be
overestimates because of shared environmental factors.104,107 Therefore, it appears that, at least
in the healthy population, a substantial fraction of the variation in VWF level
is not heritable.
Few genes have been identified that contribute to the
limited heritability of VWF level. The major genetic influence on VWF level is
ABO blood group, which is thought to account for 2030 percent of its
heritable variance.13,106,108 The mean VWF level for blood type O
is 75 U/dL, which is 2535 U/dL lower than other ABO types, and 95 percent
of VWF levels for type O blood donors are between 36 and 157 U/dL.43 The Secretor locus has a smaller
effect. Secretor-null persons have VWF levels slightly lower than
Secretors.109
Table 6. Bleeding and VWF Level in Type 3 VWD
Heterozygotes
Reference (First author, year) |
Setting |
Population |
Results |
Castaman et al. 2002a111 |
1 family with type 3 proband |
11 heterozygous |
None with bleeding; 6 who had VWF <50
IU/dL |
Eikenboom et al. 199821 |
8 families with type 3 probands |
22 heterozygous |
2 who had mild bleeding among 9 who had VWF
<50 IU/dL |
Zhang et al. 1995112 |
13 families with type 3 probands |
55 heterozygous |
22 who had mild bleeding among 38 who had VWF
<50 IU/dL; 9 who had mild bleeding among 17 who had VWF >50 IU/dL |
Schneppenheim et al. 1994113 |
22 families with type 3 probands |
44 heterozygous |
5 who had epistaxis, bruising, or menorrhagia
among 24 who had VWF <50 IU/dL; 1 who had postoperative bleeding among 20
who had VWF >50 IU/dL |
Eikenboom 1993114 |
1 family with type 3 probands |
4 heterozygous |
2 who had mild bleeding among 4 who had VWF
<50 IU/dL |
Inbal et al. 1992115 |
4 families with type 3 probands |
20 heterozygous |
None who had bleeding; 15 who had VWF <50
IU/dL |
Nichols et al. 1991116 |
1 family with type 3 proband |
6 heterozygous |
None who had bleeding; 2 who had VWF <50 IU/dL
|
Mannucci et al. 198944 |
15 families with type 3 probands |
28 heterozygous |
None who had bleeding; 19 who had VWF <50
IU/dL |
An effect of the VWF locus has been difficult to
discern by linkage analysis. One study suggested that 20 percent of the
variance in VWF levels is attributable to the VWF gene,108 whereas another study could not
demonstrate such a relationship.110
In sum, known genetic factors account for a minority
of the heritable variation in VWF level, and moderately low VWF levels
(3050 IU/dL) do not show consistent linkage to the VWF locus.97,98,100,101 The diagnosis and management of
VWD would be facilitated by better knowledge of how inherited and environmental
factors influence the plasma concentration of VWF.
The attribution of bleeding to a low VWF level can be
difficult because mild bleeding symptoms are very common, as discussed in the
section on "Diagnosis and
Evaluation" and the risk of bleeding is only modestly increased for persons
who have moderately decreased VWF levels.45 For example, in the course of
investigating patients who have type 3 VWD, approximately 190 obligate
heterozygous relatives have had bleeding histories obtained and VWF levels
measured (see Table 6). The geometric mean VWF level was
47 IU/dL,45 with a range
(±2 SD) of 16140 IU/dL. Among 117 persons who had VWF <50
IU/dL, 31 (26 percent) had bleeding symptoms. Among 74 persons who had VWF
>50 IU/dL, 10 (14 percent) had bleeding symptoms. Therefore, the relative
risk of bleeding was 1.9 (P = 0.046, Fisher's exact test) for persons who had
low VWF. There was a trend for an increased frequency of bleeding symptoms at
the lowest VWF levels: among 31 persons who had VWF levels <30 IU/dL, 12 (39
percent) had symptoms. Bleeding was mild and consisted of epistaxis, bruising,
menorrhagia, and bleeding after tooth extraction. The one person who
experienced postoperative bleeding had a VWF level >50 IU/dL.113
The management of bleeding associated with VWF
deficiency would be facilitated by better understanding of the heritability of
low VWF levels (in the range of 2050 IU/dL), their association with
intragenic VWF mutations, and their interactions with other modifiers of
bleeding risk. Such data could provide a foundation for treating VWF level as a
biomarker for a moderate risk of bleeding, much as high blood pressure and high
cholesterol are treated as biomarkers for cardiovascular disease (CVD)
risk.
Acquired von Willebrand
Syndrome
Acquired von Willebrand syndrome (AVWS) refers to
defects in VWF concentration, structure, or function that are not inherited
directly but are consequences of other medical disorders. Laboratory findings
in AVWS are similar to those in VWD and may include decreased values for
VWF:Ag, VWF:RCo, or FVIII. The VWF multimer distribution may be normal, but the
distribution often shows a decrease in large multimers similar to that seen in
type 2A VWD.117,118 AVWS usually
is caused by one of three mechanisms: autoimmune clearance or inhibition of
VWF, increased shear-induced proteolysis of VWF, or increased binding of VWF to
platelets or other cell surfaces. Autoimmune mechanisms may cause AVWS in
association with lymphoproliferative diseases, monoclonal gammopathies,
systemic lupus erythematosis, other autoimmune disorders, and some cancers.
Autoantibodies to VWF have been detected in less than 20 percent of patients in
whom they have been sought, suggesting that the methods for antibody detection
may not be sufficiently sensitive or that AVWS in these settings may not always
have an autoimmune basis.
Pathologic increases in fluid shear stress can occur
with cardiovascular lesions, such as ventricular septal defect and aortic
stenosis, or with primary pulmonary hypertension. The increased shear stress
can increase the proteolysis of VWF by ADAMTS13 enough to deplete large VWF
multimers and thereby produce a bleeding diathesis that resembles type 2A VWD.
The VWF multimer distribution improves if the underlying cardiovascular
condition is treated successfully.117-122
Increased binding to cell surfaces, particularly
platelets, also can consume large VWF multimers. An inverse relationship exists
between the platelet count and VWF multimer size, probably because increased
encounters with platelets promote increased cleavage of VWF by ADAMTS13. This
mechanism probably accounts for AVWS associated with myeloproliferative
disorders; reduction of the platelet count can restore a normal VWF multimer
distribution.123-125 In rare
instances, VWF has been reported to bind GPIb that was expressed ectopically on
tumor cells.118,126
AVWS has been described in hypothyroidism caused by
nonimmune mechanism.127 Several
drugs have been associated with AVWS; those most commonly reported include
valproic acid, ciprofloxacin, griseofulvin, and hydroxyethyl starch.117,118
AVWS occurs in a variety of conditions, but other
clinical features may direct attention away from this potential cause of
bleeding. More studies are needed to determine the incidence of AVWS and to
define its contribution to bleeding in the many diseases and conditions with
which it is associated.
Prothrombotic Clinical Issues and VWF in Persons Who
Do Not Have VWD
Whether elevation of VWF is prothrombotic has been the
subject of several investigations. Both arterial and venous thrombotic
disorders have been studied.
Open-heart surgery. Hemostatic activation
after open-heart surgery has been suggested as a mechanism of increased risk of
postoperative thrombosis in this setting. A randomized trial comparing coronary
artery surgery with or without cardiopulmonary bypass ("off-pump") found a
consistent and equivalent rise in VWF:Ag levels at 14 postoperative days
in the two groups,128 suggesting
that the surgery itself, rather than cardiopulmonary bypass, was responsible
for the rise in VWF. There is no direct evidence that the postoperative rise in
VWF contributes to the risk of thrombosis after cardiac surgery.
Coronary artery disease. Three large
prospective studies of subjects without evidence of ischemic heart disease at
entry have shown, by univariate analysis, a significant association of VWF:Ag
level at entry with subsequent ischemic coronary events.129-131 However, the association remained
significant by multivariate analysis in only one subset of subjects in these
studies,129 a finding that could
have occurred by chance. These findings suggest that the association of VWF
with incidence of coronary ischemic events is relatively weak and may not be
directly causal.
Thrombosis associated with atrial
fibrillation. A prospective study of vascular events in subjects with
atrial fibrillation found, by univariate analysis, a significant association of
VWF:Ag level with subsequent stroke or vascular events. The association with
vascular events remained significant with multivariate analysis.132
Thrombotic thrombocytopenic purpura (TTP).
The hereditary deficiency or acquired inhibition of a VWF-cleaving protease,
ADAMTS13, is associated with the survival in plasma of ultralarge VWF
multimers, which are involved in the propensity to development of platelet-rich
thrombi in the microvasculature of individuals who have TTP.133,134
Deep vein thrombosis (DVT). In a case-control
study of 301 patients, evaluated at least 3 months after cessation of
anticoagulation treatment for a first episode of DVT, plasma levels of VWF:Ag
and FVIII activity were related to risk of DVT, according to univariate
analysis. In multivariate analysis, the relation of VWF level with risk of DVT
was not significant after adjustment for FVIII levels.135
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