JOURNAL OF COAGULATION DISORDERS

REVEW ARTICLE

von Willebrand Factor Proteolysis by ADAMTS13

Derrick J Bowen
Affiliation: Department of Haematology, School of Medicine, Cardiff University, Cardiff, UK
Submission date: 27th July 2009, Revision date: 1st September 2009, Acceptance date: 5th September 2009

A B S T R A C T
The plasma glycoprotein von Willebrand factor (VWF) is essential for effective hemostasis. It mediates platelet adhesion
and aggregation at a site of vascular damage. Additionally, it transports coagulation factor VIII (an important protein of the
clotting cascade) in the blood, shielding the coagulation factor from inactivation. VWF is found in plasma, platelets,
vascular subendothelium and endothelial cells; its presence throughout the vasculature reflects its fundamental role in
blood clot formation. Plasma VWF circulates as homopolymers (multimers) of different lengths, with the size differences
arising principally through cleavage by the metalloprotease ADAMTS13 (a disintegrin and metalloprotease with
thrombospondin repeats). ADAMTS13-mediated VWF proteolysis contributes to the regulation of VWF bioactivity: newly
synthesized ultralarge multimers are highly thrombogenic in comparison to smaller proteolysed forms. The regulation of VWF
multimer size by ADAMTS13 is essential for normal hemostatic function, as evidenced by the pathological states that occur
when proteolysis is deficient or excessive: ADAMTS13 deficiency is the basis for the life-threatening disorder thrombotic
thrombocytopenic purpura (TTP), in which ultralarge VWF multimers in the circulation predispose to the spontaneous
formation of platelet aggregates, with potentially fatal consequences; conversely, too much proteolysis of VWF underlies
one form of the hemorrhagic disorder von Willebrand disease (VWD): hemostasis is severely compromised by the absence of
large (and sometimes intermediate) multimers in the circulation due to their rapid breakdown by ADAMTS13. This review
aims to provide the clinician or scientist new to this subject area with an overview of ADAMTS13-mediated VWF proteolysis
and the known key factors that can affect this important biochemical process in health and disease.
Keywords: von Willebrand factor, von Willebrand disease, ADAMTS13, proteolysis, thrombotic thrombocytopenic purpura
(TTP)
Correspondence: Derrick J Bowen, Department of Haematology, School of Medicine, Cardiff University, Heath Park,
Cardiff CF14 4XN, UK. E-mail: bowendj1@cf.ac.uk

The formation of a blood clot at a site of vascular

injury is a complex physiological process in which the
glycoprotein von Willebrand factor (VWF) plays a
fundamental role. Clot formation initiates when blood
contacts subendothelial structures, and circulating
platelets adhere, triggering further platelet aggregation to produce the primary hemostatic plug or soft
clot. During this process, platelet activation provides a
procoagulant surface that supports the activity of key
protein complexes in the coagulation cascade. The
latter produces fibrin polymers that become covalently
cross-linked into the soft clot, thereby reinforcing and
stabilizing it. Formation of the platelet-rich primary
hemostatic plug (primary hemostasis) and clot stabilization (secondary hemostasis) are tightly regulated
processes, defects in which give rise to hemorrhagic or
thrombotic disorders.

including those involving VWF. However, at high

shear rates, there appears to be an absolute dependency on VWF. VWF interacts with platelets via two
platelet receptors: glycoprotein Iba (GPIba) [1] and
integrin aIIbb3 (previously referred to as platelet
glycoprotein IIb/IIIa or GPIIb/IIIa) [2]. GPIba is
unique among platelet receptors in that it does not
require prior platelet activation in order to bind its
ligand (VWF); in contrast, aIIbb3 does. VWF interaction with GPIba is rapid and reversible, whereas
interaction with aIIbb3 is slow and irreversible [3, 4].
At high shear rates, the VWFGPIba interaction
tethers platelets long enough for activation and
subsequent irreversible binding via aIIbb3. Although
GPIba is competent to bind VWF without platelet
activation, there is apparently no significant interaction between the two proteins in circulating blood.
This is explained, at least in part, by the requirement
for a critical level of shear stress [4].

Platelet arrest, adhesion, and aggregation are central to primary hemostasis. At low shear rates, multiple ligandreceptor interactions may participate,

Following platelet arrest and adhesion, aggregation

of further platelets involves a number of ligand
receptor interactions, among which VWFaIIbb3 and

INTRODUCTION

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Journal of Coagulation Disorders

fibrinogenaIIbb3 are important. VWF and fibrinogen

act in concert, serving distinct but synergistic roles in
promoting platelet aggregation [5]. At high shear
rates, surface-bound VWF appears to be capable of
reversible association with circulating VWF, and the
resulting homotypic multimer assemblies may provide
a further contribution to the arrest of passing platelets
[6]. At pathophysiological shear rates, transient VWFmediated platelet aggregation can occur that is
dependent upon GPIba but independent of aIIbb3 and
fibrinogen [7]. The latter may be highly relevant as a
pathological mechanism leading to vessel occlusion at
a site of stenosis, where vessel narrowing may vastly
increase shear stress. However, it may also be relevant
in a normal setting: as the soft clot is laid down, the
lesion aperture narrows while blood pressure remains
unchanged. Exiting blood may therefore be exposed to
ever-increasing shear stress, possibly permitting activation-independent, VWFGPIba-mediated platelet
aggregation, and this may facilitate lesion closure.

of dimers [14], is removed leaving multimers composed

entirely of mature VWF subunits (Figure 1). Posttranslational processing additionally includes extensive glycosylation involving the addition of 12 Nlinked and 10 O-linked carbohydrate moieties
(Figure 1) [15] and sulfation of specific N-linked
carbohydrates [16]. Internal homologies within the
mature subunit give rise to repeated structural
domains [17] (Figure 1). The A1 domain contains the
binding site for GPIba (and also for heparin and
sulfated glycolipids); the C1 domain contains the
ligand motif (Arg-Gly-Asp, RGD) for integrin aIIbb3;
the A3 domain (and possibly also the A1 domain) is
considered to be the binding site for collagen; and the
D9D3 domains bind FVIII (Figure 1).
VWF produced in endothelial cells can be stored in
WeibelPalade bodies [18] or secreted. Secretion can
occur by a constitutive pathway that does not require
stimulation by secretagogues, or by a regulated pathway that does [19]. The former releases multimers
directly upon synthesis; the latter releases the Weibel
Palade stores. Endothelial VWF can be secreted
apically into the bloodstream or basolaterally into
the subendothelial matrix. Only arteries, arterioles,
and large veins have subendothelial deposits of VWF
[20]; in capillaries, VWF may be deposited basolaterally in response to endothelial cell activation [21].
Plasma VWF appears to be derived principally from
endothelial cells [22]. Platelet VWF is stored in agranules from which it is released upon platelet
activation [23]. As much as 15% of the total VWF in
blood is contained within the platelet compartment
[24]. VWF is therefore found in four physiological
compartmentsplasma, platelets, endothelial cells,
and the subendotheliumand this no doubt reflects
its important role in primary hemostasis.

Collagen contributes to the overall process in at least

two ways: first, it may potentiate the ability of VWF
that is bound to it to arrest platelets; second, it can
interact directly with any of several receptors on the
platelet surface, thereby adding to plateletsubendothelium interactions.
While the intricacies and timeline of events in
primary hemostasis may not yet be fully delineated,
the detailed studies to date have identified key
features of VWF involvement and emphasize the
central role that this protein plays at high shear
stress. VWF has evidently evolved to catch platelets
from the passing blood under the adverse circumstance
of high shear.
In addition to its role in primary hemostasis, VWF
contributes towards secondary hemostasis: it transports
and protects coagulation factor VIII (FVIII) in the
circulation [8]. FVIII is an essential protein in the
intrinsic coagulation cascade and is necessary for
efficient fibrin production. FVIII deficiency is characterized by a breakdown of secondary hemostasis, resulting
in clinically significant bleeding (hemophilia A). Thus,
VWF facilitates secondary hemostasis by contributing to
the maintenance of circulating FVIII levels.

Stimulated release of VWF from its storage organelles places ultralarge multimers, which are highly
thrombogenic, directly at the site of need (Figure 3).
In the case of endothelial cells, these acutely released
ultralarge multimers rapidly form strings and networks that are anchored on to the cell surface via
integrin aVb3 [25]. Such immobilization would expose
them to shear stress in the passing blood flow. This
may potentiate platelet binding; however, it would
also promote VWF proteolysis by the metalloprotease
ADAMTS13 (a disintegrin and metalloprotease with
thrombospondin repeats) [26].

von WILLEBRAND FACTOR

VWF is synthesized by endothelial cells [9] and
megakaryocytes [10]. It is produced as a pre-proprotein comprising 2813 amino acids. As part of posttranslational processing, the pre- and pro-sequences
(22 and 741 amino acids respectively) are removed,
leaving the mature VWF subunit (2050 amino acids)
(Figure 1) [11, 12]. Prior to removal of the prosequence, pro-VWF forms dimers via C-terminal disulfide bonds, and these dimeric units subsequently join
via their N-termini to give polymers (multimers) [13].
The pro-peptide, which is needed for multimerization
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ADAMTS13
ADAMTS13 is synthesized predominantly in the liver
[27]; however, other tissues and cells (including
endothelial cells and platelets) also produce the
enzyme [28, 29]. The primary translation product is
1427 amino acids long and comprises a signal peptide
(33 amino acids), a propeptide (41 amino acids), and a
multidomain mature protein (1353 amino acids) [30]
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Figure 1. von Willebrand factor structure. (A) Relative extent of pre-, pro-, and mature VWF. (B) Pre-pro-VWF domain structure and
position of carbohydrate moieties in the mature subunit. Internal homologies within the protein give rise to repeated domains (A1D4); D9
is a partial homolog of the D domains; CK is a cysteine-rich region (cysteine-knot); H and D represent N-linked and O-linked glycosylation
sites respectively. The A2 domain contains the ADAMTS13 cleavage site at Tyr1605Met1606, the Tyr1584Cys polymorphic residue (*) that
affects proteolysis, two N-linked carbohydrates (Asn1515 and Asn1574, of which Asn1574 may influence proteolysis), and five O-linked
carbohydrates. The numeric scale indicates amino acid residue number. (C) Location of VWF functional domains involved in ligand
binding and multimer formation (RGD represents the arg-gly-asp motif, which is the ligand for integrin aIIbb3). (D) Formation of VWF
multimers. Mature VWF dimerizes via C-termini and dimers form multimers via N-termini. ADAMTS13 proteolysis within any of the constituent
A2 domains yields multimeric forms found in plasma

(Figure 2). The latter includes a metalloprotease

domain, disintegrin domain, cysteine-rich and spacer
region, and several thrombospondin type 1 motifs, all
of which characterize the enzyme as a member of the
ADAMTS family [30]. The metalloprotease domain has
the consensus sequence HEXXHXXGXXHD characteristic of certain zinc-dependent metalloendopeptidases

[31], a predicted calcium ion binding site (Glu83,

Asp173, Cys281), and a met turn in which Met249
supports the active site zinc ion. These features are
characteristic of the metzincin family of metalloendopeptidases [31], which achieve optimal activity with
both zinc and calcium ions. Cooperative activation of
ADAMTS13 has been demonstrated by zinc and

Figure 2. Structure of ADAMTS13. Pre- and pro-sequences are followed by a metalloprotease domain containing the active site, a
disintegrin domain, a TSP-1 (thrombospondin-1) motif, a Cys-rich region, a spacer domain, seven additional TSP-1 motifs, and two CUB
domains

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Figure 3. A model of events in primary hemostasis, illustrated by a single site of vascular damage at which blood exits the vessel. (A) In an
intact blood vessel, VWF is compartmentalized: ultralarge, highly thrombogenic multimers are localized to the a-granules of platelets and
WeibelPalade bodies of endothelial cells; plasma VWF (which comprises smaller, less thrombogenic forms) is prevented from
immobilization by the endothelial layer, which also masks matrix-bound subendothelial VWF (if present). (B) Vessel damage allows exiting
blood to contact the subendothelial matrix (containing collagen to which plasma VWF can bind) and matrix-bound VWF (if present). (C)
In the earliest stages, platelets arrest and adhere to immobilized VWF. (D) Platelet adhesion is followed by activation and release of agranule contents including ultralarge VWF multimers. Local agonists may stimulate endothelial cells to release ultralarge VWF multimers
from WeibelPalade bodies. (E) VWF-mediated platelet recruitment and ADAMTS13-mediated VWF proteolysis may take place
concurrently during subsequent stages. The former would contribute to clot growth; the latter would limit it. Both are favored by high
shear, which may itself be facilitated by the narrowing of the lesion aperture as the soft clot evolves. Factors that enhance proteolysis
(such as the cysteine 1584 variant of VWF or, to a lesser extent, blood group O) or inhibit proteolysis (such as the serine 475 variant of
ADAMTS13) may imbalance VWF-mediated platelet recruitment and predispose to a hemorrhagic or thrombotic tendency respectively.
For clarity, peripheral constituents of the blood have been omitted from (D) and (E)

calcium together [32]. The C-terminus of ADAMTS13

contains two CUB domains that are common to many
proteins involved in developmental processes, but
whose function in ADAMTS13 is uncertain (CUB
abbreviates: complement component Clr/Cls, Uegf
and bone morphogenic protein 1 domain). Circulating
ADAMTS13 is highly glycosylated [33, 34] and is
primarily full length (pre-pro-sequences attached) [35].

phenomena could contribute to the control of clot

growth (Figure 3), particularly when ADAMTS13 is
available from the various local sources (plasma,
endothelial cells, and activated platelets). Evidence that
excess cleavage has a negative impact on VWF hemostatic function is provided by certain forms of the
bleeding disorder von Willebrand disease (VWD) that
result from a mutant VWF that is highly susceptible to
ADAMTS13 proteolysis [36]. Conversely, a deficiency or
dysfunction of ADAMTS13 leads to the survival of
uncleaved, or partially cleaved, ultralarge VWF in
plasma, which predisposes to the spontaneous formation
of intravascular platelet aggregates and is the biochemical basis of the life-threatening disorder thrombotic
thrombocytopenic purpura (TTP) [37, 38, 41].

ADAMTS13 cleaves VWF at the peptide bond between

Tyr1605 and Met1606, located in the VWF A2 domain
(Figure 1) [3638]. This proteolysis gives rise to multimers of different lengths (Figure 1) and underlies the
characteristic triplet structure of plasma VWF [39]. The
proteolysis is important because it appears to be the
predominant physiological mechanism through which
multimer size is regulated. Although ADAMTS13 and
VWF both circulate in plasma, their interaction leading
to proteolysis is limited by a requirement for VWF to
undergo shear stress, the basis for which may be
stretching of the protein and exposure of the otherwise
buried A2 domain [26, 40].

It is presently unclear which sites on ADAMTS13

and VWF contribute towards the interaction of the
two proteins under physiologic conditions. Data to
date support the involvement of several sites of
interaction on both proteins, at least under static/
denaturing conditions. The isolated metalloprotease
domain of ADAMTS13 is ineffective in cleaving VWF
[42, 43]; efficient proteolysis requires the presence of
the non-catalytic domains. The disintegrin-like domain

Therefore, shear stress, on the one hand, promotes

VWF interaction with platelets, while on the other, it
enhances cleavage by ADAMTS13. These two opposing
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recruit platelets and, as noted earlier, this may provide a

mechanism contributing to the control of clot growth.
ADAMTS13 can limit the activation-independent, VWF
GPIba-mediated aggregation of platelets that takes place
at pathologically elevated shear stress [7] and may
thereby provide a control mechanism against thrombogenesis at sites experiencing such shear.

appears to interact with VWF via Arg349 and Leu350

in ADAMTS13 and Asp1614 and Ala1612 in VWF [44].
The ADAMTS13 TSP-1 (first motif) and Cys-rich
domains, respectively, appear to bind VWF regions
Gln1624Val1630 and Ile1642Gln1652 [43, 45]. The
spacer domain binds a C-terminal portion of the VWF
A2 domain (residues 16531668) [46]. The carboxyterminal TSP-1 repeats and CUB domains may modulate ADAMTS13VWF interaction [46, 47]. Data from
mutagenesis experiments suggest that ADAMTS13 is
relatively flexible and does not require a fixed spacing
between its catalytic site and the distal VWF binding
sites in the Cys-rich and spacer domains [45]. This
elasticity may facilitate interaction between the two
proteins in flow conditions.

VWF Glycosylation
Studies prior to the discovery of ADAMTS13 provided evidence that the carbohydrate component of
VWF may protect the protein from degradation [58].
Subsequent studies have indicated that VWF glycosylation can affect ADAMTS13-mediated proteolysis.
ABO blood group sugarsA, B, and Hare attached
to the N-linked carbohydrates of VWF [59, 60]
(Figure 1) and appear to alter the rate of
ADAMTS13 cleavage to a small extent: proteolysis
was faster for VWF of blood group O compared with
non-O (O > B . A > AB); however, the differences
between blood groups were not considerable [61]. VWF
from the plasma of individuals with the Bombay
phenotype (characterized by a failure to attach A, B,
and H sugars to carbohydrate structures) was proteolysed more rapidly than that of both blood group O and
blood group AB (Bombay . O . AB) [62].

A model has been proposed in which ADAMTS13

VWF interaction comprises two components acting in
concert: tight binding is provided by the Cys-rich and
spacer domain interactions, whereas the weaker
interactions (in particular of the disintegrin domain)
may assist in positioning the VWF scissile bond into
the active site cleft of the metalloprotease domain [44].

FACTORS INFLUENCING VWF PROTEOLYSIS

BY ADAMTS13
Various factors can affect the efficiency of VWF
cleavage by ADAMTS13. Those that contribute
towards the normal spectrum of proteolysis include
shear stress, VWF glycosylation, amino acid variants
in both proteins, and ligandVWF interaction; these
are discussed in detail below. Additionally, there are
factors that underlie the extremes of proteolysis found
in the pathologies of TTP and VWD. These are
summarized briefly below, but are reviewed in detail
elsewhere [4852].

The physiological significance of these findings is

uncertain. Plasma VWF level correlates with ABO
blood group (O , A , B , AB) and is approximately
25% less in blood group O than in non-O [63, 64]. The
rank order and magnitude of proteolysis in the ABO
blood groups do not correlate with those of VWF level;
therefore, proteolysis is unlikely to explain the
differences in the amount of VWF between these blood
groups. The increased VWF proteolysis associated
with blood group O is small; however, it could have a
subtle deleterious effect on primary hemostasis. In
conjunction with a low plasma VWF level and/or other
deleterious factors, this may compromise clot formation (Figure 3) [48].

Shear Stress
The velocity of blood flow is dependent upon several
parameters including blood pressure, vessel diameter,
and fluid viscosity. Within a given vessel, flow differs
according to the axial location within the lumen: it is
zero at the vessel wall and increases towards the lumen
centre [53]. This gives rise to shear stress, produced by
the faster movement of one layer over an adjacent layer.
In the circulatory system, shear stress is highest in the
microvasculature [54, 55]. In vessels occluded by an
atherosclerotic plaque, shear stress at the site of stenosis
can greatly exceed even the highest normal levels [56].

Global removal of VWF N-linked carbohydrates has

been shown to increase affinity for, and proteolysis by,
ADAMTS13, and to enable proteolysis in the absence
of denaturant [65]. Mutation of the two asparagine
residues vicinal to the Tyr1605Met1606 cleavage site
demonstrated that loss of the carbohydrate moiety at
Asn1574, but not Asn1515, increased the susceptibility
of VWF to proteolysis and allowed cleavage in the
absence of denaturant [65]. Thus, VWF N-linked
carbohydrates collectively may help to support a
globular conformation, and Asn1574 may additionally
influence accessibility of the scissile Tyr1605Met1606
bond to the ADAMTS13 active site.

ADAMTS13 does not proteolyse VWF significantly

under static conditions, but does cleave it slowly in vitro
in the presence of denaturing agents [37]. However, at
shear stress levels equal to those found in the microvasculature, proteolysis is extremely rapid and does not
require denaturants [26, 38, 57]. In a physiological
setting, ADAMTS13 therefore functions efficiently
under the very conditions in which VWF functions to
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In contrast, N-linked carbohydrates on ADAMTS13

do not appear to modulate its activity: they can be
enzymatically removed without affecting proteolysis.
However, they do appear to be necessary for efficient
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secretion of the metalloprotease and may promote

correct conformational maturation of the enzyme
during its biosynthesis [34].

[71], possibly due to Cys1584 inducing a conformational change in the A2 domain that abolishes the
blood group effect. As discussed above, increased
proteolysis of VWF may be detrimental to hemostatic
function (Figure 3). Cys1584 is enriched in type 1
VWD [72, 75]; however, whether this relates to its
effect on VWF proteolysis is uncertain. There is
adequate evidence showing that, in addition to
increased proteolysis, the variant is associated with
decreased VWF level, increased clearance of the
protein, and decreased VWF functionality [64, 76]. It
is likely that the prevalence of Cys1584 in type 1 VWD
reflects all of these deleterious effects [77].

Amino Acid Variants

Both VWF and ADAMTS13 show amino acid variation in the normal population. Several coding sequence
variants have been described in ADAMTS13, including
Arg7Trp, Gln448Glu, Pro475Ser, Pro618Ala, Arg625His,
Ala732Val, and Ala900Val [41]. The Pro475Ser variant,
which is prevalent in the Japanese population [66] but
absent or rare in other populations (Caucasians, AfroAmericans, Chinese) [67, 68], has a significant effect on
proteolytic activity. The serine allele is associated with
very low ADAMTS13 activity despite normal secretion.
Based upon allele frequencies at the genetic level, as
many as 10% of the Japanese population may be
heterozygous for Ser475 and consequently may have
decreased ADAMTS13 levels [66].

The amino acid variation Val1565Leu located in the

VWF A2 domain has also been shown to affect
proteolysis, however to a much smaller extent than
Tyr1584Cys [71]. The rarer leucine allele correlates
with a minor increase in proteolytic susceptibility;
however, the magnitude of the effect is so small that it
may have no physiological relevance.

The polymorphism Pro618Ala in ADAMTS13 also has

a significant effect on ADAMTS13 level, mediated by a
deleterious effect of the Ala618 allele on secretion and
proteolytic activity [69]. Ala732Val has a small but
demonstrable effect, the valine allele correlating with
impaired catalytic activity [69].

Interestingly, both Tyr1584 and Val1565 are highly

conserved in VWF from different species, they each
occur in regions that are homologous between species,
and they are near to the ADAMTS13 scissile bond [71].
These features provide an argument for a direct effect
of the variant residues Cys1584 and Leu1565 on VWF
proteolysis.

Studies on the interplay between ADAMTS13 amino

acid variants have indicated that the same polymorphisms can be either positive or negative modifiers
depending on the allelic combination present.
Additionally, polymorphisms may interact synergistically and not just additively [69]. These observations
may be relevant to normal variations in hemostatic
function (although this has not been formally investigated), and there is evidence supporting their importance in the phenotypic expression of TTP [69].

The possibility that the amino acid polymorphisms

Asp1472His, Gln1571His, Pro1601Thr, and Gly1643Ser
in the VWF A domains influence ADAMTS13-mediated
proteolysis has been investigated using recombinant
expressed proteins. The advantage of this approach is
that potential confounding variables are standardized,
thereby allowing comparison solely of the effect of each
amino acid allele. Using recombinant VWF and
recombinant ADAMTS13, His1472, His1571, and
Thr1601 in VWF all conferred mild resistance to
proteolysis, whereas Ser1643 potentiated proteolysis
[78]. The disadvantage of recombinant studies is that
VWF and ADAMTS13 expressed in vitro may differ
from their in vivo counterparts in a number of ways (for
example in terms of glycosylation) that could influence
the results. In this context, it is interesting to note that,
in contrast to the above result, His1472 appeared to
have no effect on proteolysis when plasma VWF, rather
than the recombinant protein, was used [71].

Amino acid variation in VWF can also affect

ADAMTS13-mediated proteolysis. The Tyr1584Cys
variation in the VWF A2 domain has been extensively
studied. The cysteine allele is associated with mildly
enhanced proteolysis [70, 71], the basis for which may
be the formation of new inter- or intramolecular
disulfide linkages that alter the conformation of the
A2 domain and enhance access to the cleavage site [40,
72, 73]. That Cys1584 is necessary for enhanced
proteolysis and not simply linked to a causative
change elsewhere in VWF has been shown by comparing the coding sequences of the protein in related
individuals whose plasma VWF showed either normal
or increased proteolysis. Two sequence variants were
unique to VWF that showed increased proteolysis:
Arg484 and Cys1584. Arg484, in the absence of
Cys1584, did not influence VWF proteolysis, indicating
that Cys1584 is necessary for the effect [74].

In combination, these detailed studies indicate that

ADAMTS13-mediated VWF proteolysis appears to be
highly dependent upon the amino acid variants present
at certain influential positions in both proteins. One
important inference from this is that the rate of
proteolysis could be quite different between two
individuals who have similar VWF protein levels and
also similar ADAMTS13 protein levels, potentially
leading to differences in hemostatic efficiency despite
the similar phenotypic values. This may contribute to

The mildly enhanced proteolysis associated with

Cys1584 is considerably greater than that of blood
group O. It is not additive with that of blood group O
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normal variation in healthy individuals, and it may be

relevant to the phenotypic expression of TTP and VWD.

characteristic feature of a subset of type 2A VWD

(group 2 type 2A) [86]. In this subset, missense point
mutations in the region of the VWF gene (VWF,
located at 12p1213) encoding the A2 domain bring
about amino acid changes that cause the protein to be
proteolysed without the need for shear stress. These
amino acid substitutions may induce conformational
changes that alter the accessibility of the cleavage site
within the VWF A2 domain, or they may directly alter
ADAMTS13VWF interaction without structural perturbation [40, 87, 88]. In the circulation, the mutant
VWF is spontaneously and rapidly cleaved by
ADAMTS13, resulting in the loss of large (and sometimes intermediate) multimer forms [86, 87, 89]. This,
together with the possibility that ultralarge multimers
secreted at a time of hemostatic challenge may be
cleaved far more rapidly than normal, severely
compromises hemostatic function. Characteristic clinical features include mucocutaneous bleeding, epistaxis, easy bruising, bleeding after dental extraction,
heavy menstrual periods, and excessive blood loss
during childbirth (for a review, see http://www.nhlbi.
nih.gov/guidelines/vwd/index.htm).

VWFLigand Binding
X-ray crystallography studies indicate that the VWF
A1 domain undergoes large structural changes on
binding with GPIba [79]. Additionally, proteolysis of a
recombinant peptide containing the VWF A1A2A3
region was increased when a mutant fragment of
GPIba bound [80]. Together, these observations suggest that binding of GPIba to VWF increases the
susceptibility of the latter to proteolysis by
ADAMTS13 via a conformational change. Heparin, a
glycosaminoglycan stored in the secretory granules of
mast cells and released into the blood at a site of
vascular damage, also increases VWF proteolysis [80].
The obsolete antibiotic ristocetin, which is used in the
laboratory to trigger plateletVWF aggregation,
causes increased proteolysis [61]. For both heparin
and ristocetin, the mechanism of the effect may be
through the induction of conformational changes upon
binding to VWF.

CLINICAL SIGNIFICANCE

For both TTP and VWD, the parameters that

influence VWF proteolysis by ADAMTS13 may contribute towards differences in penetrance and severity.
This is likely to be most important in the case of
mutations in either protein that cause a mild deficiency: the additive or synergistic effect of negative
modifiers acting on such mutations could tip the
balance in favor of clinical presentation. This is
certainly believed to be the case for the Tyr1584Cys
polymorphism in VWF: the cysteine allele is found in
association with phenotypes ranging from asymptomatic to overt type 1 VWD.

ADAMTS13-mediated proteolysis is a natural part of

VWF processing and, as is the case for all other
biochemical processes, displays a normal range in the
population [81]. At the extremes of the normal range,
the level of proteolysis may well predispose to
thrombotic (low level) or hemorrhagic (high level)
tendencies. Beyond theses extremes, the overt pathological disorders of TTP and a subset of VWD arise.
TTP is caused by a marked deficiency of ADAMTS13
activity, brought about either by mutation of the
relevant gene (ADAMTS13, located at 9q34) [41] or by
acquired antibodies arising from an autoimmune
response to the protein [81, 82]. In the case of the
former (hereditary TTP), heterogeneous mutations
have been characterized in ADAMTS13, including
missense and nonsense point mutations, splice site
mutations, and deletions [51]. Characterization of
antibodies in acquired TTP has shown that diverse
ADAMTS13 epitopes may be targeted [83]. Irrespective
of the root cause of ADAMTS13 deficiency, the
outcome is the survival of ultralarge VWF multimers
in the circulation, leading to spontaneous platelet
aggregation with potentially disastrous consequences
[84]. These include damage or failure of the major
organs fed by the circulatory system (heart, kidneys,
brain, eyes). Five classical symptoms are indicative of
TTPneurologic disturbance, kidney failure, fever,
thrombocytopenia, and microangiopathic hemolytic
anemia. The last is believed to arise from shearinduced damage of red blood cells passing the platelet
aggregates (for a review, see [85]).

SUMMARY
In an undamaged, healthy vessel, blood is maintained
in a liquid state by several key phenomena: the
endothelial surface lining the lumen is anticoagulant,
endothelial cells themselves secrete anticoagulant
molecules, hemostatic proteins in the blood circulate
in inactive forms, and platelets, although primed ready
for clot formation, do not do so because the appropriate triggers are unavailable to them.
All this changes when vascular damage occurs and
the various signals that activate clot formation come
into play. Amid the complex array of biochemical
activities that occur, VWF capture of platelets at the
site of damage is fundamental. The efficiency of the
entire process of clot formation reflects a considerable
number of variables. VWF proteolysis by ADAMTS13
is just one biochemical process in the complex milieu,
but it is of pivotal importance, as evidenced by the
severe pathologies arising from its perturbation.
Proteolysis is itself influenced by many variables,
including the amount of ADAMTS13 and VWF in the

In contrast to the above, excessive VWF proteolysis

by ADAMTS13 may predispose to bleeding. This is the
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Journal of Coagulation Disorders

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blood, shear stress, amino acid polymorphisms within

each protein, VWFGPIba binding, and ABO blood
group. These provide for a large number of possible
permutations, some of which may be important to the
phenotypic expression of TTP or VWD.
Disclosure: The author has no financial interests to
disclose related to the contents of this article.

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