THROMBOSIS AND HEMOSTASIS
Gain-of-function ADAMTS13 variants that are resistant to autoantibodies against
ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura
Cui Jian,1 Juan Xiao,1 Lingjie Gong,1 Christopher G. Skipwith,1,2 Sheng-Yu Jin,1 Hau C. Kwaan,3 and X. Long Zheng1,2
1Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA; 2University of Pennsylvania Perelman School of
Medicine, Philadelphia, PA; and 3Department of Internal Medicine, Northwestern University, Chicago, IL
hancing specific activity. Site-directed mutagenesis was used to generate a series
of ADAMTS13 variants, and their functional properties were assessed. Of 24
novel ADAMTS13 variants, 2 (ie, M4,
R660K/F592Y/R568K/Y661F and M5,
R660K/F592Y/R568K/Y661F/Y665F) exhibited increased specific activity approximately 4- to 5-fold and approximately 10to 12-fold cleaving a peptide VWF73 substrate and multimeric VWF, respectively.
More interestingly, the gain-of-function
ADAMTS13 variants were more resistant
to inhibition by anti-ADAMTS13 autoanti-
bodies from patients with acquired idiopathic TTP because of reduced binding
by anti-ADAMTS13 IgGs. These results
shed more light on the critical role of the
exosite in the spacer domain in substrate
recognition. Our findings also help understand the pathogenesis of acquired autoimmune TTP. The autoantibody-resistant
ADAMTS13 variants may be further developed as a novel therapeutic for acquired
TTP with inhibitors. (Blood. 2012;119(16):
3836-3843)
Introduction
ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13) cleaves ultra large (UL) von Willebrand
factor (VWF) on endothelial cells,1 soluble VWF in the flowing
blood,2,3 and VWF adhering to sites of injury where VWF-rich
platelet thrombi are formed.4-6 This cleavage by ADAMTS13 is
highly specific, occurring at the Tyr1605-Met1606 bond in the
A2 domain.7 In vivo, fluid shear stress accelerates the cleavage of
cell bound ULVWF1,8 and soluble VWF multimers in circulation.2,3
In vitro, addition of a denaturant, such as urea9 or guanidine,7 also
markedly accelerates the proteolytic cleavage of soluble VWF by
ADAMTS13. These findings greatly facilitate the development of
various biochemical assays for assessing ADAMTS13 activity.
The importance of VWF proteolysis by ADAMTS13 is highlighted by the development of a potentially fatal syndrome,
thrombotic thrombocytopenic purpura (TTP), when plasma
ADAMTS13 activity is severely deficient. This can result from
either hereditary mutations of ADAMTS13 gene10 or acquired
formation of autoantibodies that inhibit plasma ADAMTS13
activity.11-13 Nearly all adult idiopathic TTP patients with severely
deficient plasma ADAMTS13 activity harbor polyclonal immunoglobulin Gs (IgGs) that bind the Cys-rich and spacer domains,
particularly the spacer domain of ADAMTS13.13-17 Recent studies
have shown that exosite 3 (ie, Y659-Y665) and several other
adjacent amino acid residues (ie, R568 and F592) in the spacer
domain compose a major antigenic epitope for IgG autoantibodies
in idiopathic TTP.18,19 This region is also found to play an essential
role in proteolytic cleavage of VWF under various conditions6,20-24
and modulation of arterial thrombus formation in vivo.6
In the present study, we test a hypothesis that a modification of
the exosite 3 in the spacer domain might create an ADAMTS13
variant with reduced binding and inhibition by autoantibodies from
patients with acquired idiopathic TTP while preserving or enhancing its specific activity. To this aim, a series of recombinant
ADAMTS13 variants were engineered by replacing several surface
charged/hydrophobic residues in the exosite 3 with those having
similar chemical structures. Proteolytic activity and sensitivity of
the novel variants to patient anti-ADAMTS13 autoantibodies
were assessed. Of 24 novel ADAMTS13 variants, 2 exhibit
dramatically enhanced specific activity but are more resistant to
inhibition by a panel of autoantibodies from acquired idiopathic
TTP patients. These results indicate that the novel gain-of-function
and autoantibody-resistant ADAMTS13 variants may be further developed for therapy of acquired idiopathic TTP patients with inhibitors.
Methods
Constructs
QuickChange site-directed mutagenesis regents from Stratagene were used
to replace one or a clustered of surface charged amino acid residues (R660,
F592, R568, Y661, and Y665) in the 9-10 variable region of the spacer
domain. A pcDNA3.1 vector containing wild-type (WT) ADAMTS13V5-His, as described previously,23 was used as a template. The resulting
Submitted December 18, 2011; accepted January 22, 2012. Prepublished
online as Blood First Edition paper, January 30, 2012; DOI 10.1182/blood2011-12-399501.
The online version of this article contains a data supplement.
There is an Inside Blood commentary on this article in this issue.
© 2012 by The American Society of Hematology
3836
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
BLOOD, 19 APRIL 2012 䡠 VOLUME 119, NUMBER 16
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Thrombotic thrombocytopenic purpura
(TTP) is primarily caused by immunoglobulin G (IgG) autoantibodies against
A Disintegrin And Metalloprotease with
ThromboSpondin type 1 repeats, 13
(ADAMTS13). Nearly all adult idiopathic
TTP patients harbor IgGs, which bind the
spacer domain of ADAMTS13, a region
critical for recognition and proteolysis
of von Willebrand factor (VWF). We hypothesize that a modification of an exosite in the spacer domain may generate
ADAMTS13 variants with reduced autoantibody binding while preserving or en-
BLOOD, 19 APRIL 2012 䡠 VOLUME 119, NUMBER 16
POTENTIAL NOVEL THERAPEUTICS FOR ACQUIRED TTP
3837
Table 1. Clinical characteristics, plasma ADAMTS13 activity, inhibitors of TTP patients, and sensitivity of WT ADAMTS13 and novel variants
to the inhibition by patient plasma
Patient
no.
Platelet
CNS,
ADAMTS13
count, Hemato- LDH, Creatinine, signs and
activity by
ⴛ 109/L
crit, %
U/L
mg/dL
symptoms rF-vWF73, %
Plasma
antiADAMTS13
inhibitors
Plasma
antiADAMTS13IgG,
U/mL
Sensitivity to patient plasma
inhibition
Age,
y
Sex
WT
M1
M2
M3
M4
1
44
Female
15
23
695
1.1
Yes
⬍5
⫹
84.2
⫹⫹
⫹⫹
⫹⫹
⫹
⫺
⫺
2
56
Female
19
29
654
1.1
Yes
⬍5
⫹
35.4
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
M5
3
45
Female
88
28
995
0.7
Yes
⬍5
⫹
81.9
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
4
52
Female
52
20
866
1.1
Yes
⬍5
⫹
132.0
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
5
79
Female
7
27
1660
1.4
Yes
⬍5
⫹
118.0
⫹⫹
⫹⫹
⫹⫹
⫹
⫺
⫺
6
34
Male
15
23
2703
0.9
Yes
⬍5
⫹
127.5
⫹⫹
⫹⫹
⫹⫹
⫹
⫺
⫺
7
34
Male
0
33
859
1.9
No
⬍5
⫹
162.0
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
8
23
Female
11
18
3412
0.8
Yes
⬍5
⫹
132.0
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
9
42
Female
23
17
3259
0.8
Yes
⬍5
⫹
168.0
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
21
Female
9
13
1489
1.5
Yes
⬍5
⫹
72.0
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
61
Female
33
28
511
0.8
Yes
⬍5
⫹
221.0
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
12
42
Male
16
24
6517
1.2
No
⬍5
⫹
147.2
⫹⫹
⫹⫹
⫹⫹
⫹
⫺
⫺
N ⫽ 12
43*
15.5*
23.5*
1242*
1.1*
(83.3)
(100)
(100)
(100)
(100)
(100)
(100)
(50)
(17)
(17)
Values in parentheses indicate positive rates in percentages.
LDH indicates lactate dehydrogenase; ⫺, negative inhibition (⬍ 10% reduction in activity); ⫹, mild inhibition (10%-30% reduction in activity); ⫹⫹, moderate inhibition
(30%-50% reduction in activity); and ⫹⫹⫹, strong inhibition (⬎ 50% reduction in activity) after 50:50 mixing with patient plasma.
*Median values.
variants with a desired mutation or mutations were sequenced to confirm
the accuracy at the Nucleic Acid Core Facility, Children’s Hospital of
Philadelphia.
Preparations of recombinant WT ADAMTS13 and variants
COS-7 cells were transfected with plasmid and polyethylenimine (PEI)
according to the manufacturer’s instruction (Advanced Cell System).
Serum-free conditioned medium was collected 4 days after transfection and
concentrated 50 to 100 times using a filtration column (Millipore) in the
presence of 1% protease inhibitor cocktail (Sigma-Aldrich).
ELISA
The concentrations of WT ADAMTS13 and variants in the concentrated
conditioned medium were determined by an in-house ELISA. Briefly, a
high-binding microtiter plate was coated with 100 L of monoclonal
anti-disintegrin IgG (40 g/mL; custom-made in Green Mountain Antibody) overnight. The remaining binding sites were blocked for 30 minutes
with 150 L/well of 2.5% BSA in PBS. WT and variants diluted with PBS
were added and incubated for 2 hours. After being washed with PBS,
monoclonal anti–V5-HRP IgG (Invitrogen; 1:1000) was added for detection. Purified ADAMTS13 was used as a calibration. Each quantitation was
repeated 3 times for consistency.
Western blot
The integrity of WT and variants in the concentrated conditioned medium
were assessed by Western blotting after fractionation on 8% SDSpolyacrylamide gel under reduced conditions as described previously.23
After being transferred to a nitrocellulose membrane, recombinant WT and
variants were blotted by anti-V5 IgG (Invitrogen; 1:5000) and IRdye800CWlabeled goat anti–mouse IgG (1:20 000; LI-COR) in 20mM Tris-HCl,
150mM NaCl containing 0.05% Tween-20 and 1% casein (TBSTc). The
fluorescent signals obtained with an Odyssey imaging system (LI-COR)
were converted to gray images.
Proteolytic cleavage of a VWF73 peptide
A 5⬘-maleimide fluorescein-labeled recombinant VWF73 peptide (rFVWF73; 2M) previously described25,26 was incubated with WT and
variants (0.2nM) in 5mM Bis-Tris, pH 6.0, containing 25mM CaCl2 and
0.005% Tween-20 in a 96-well white plate (Corning). The rate of
fluorescence generation was monitored at 37°C with a SpectroMax
fluorescent microtiter plate reader (Molecular Devices; Ex/Em
485/535 nm) every minute for 30 minutes. Normal human plasma from
George Kings Biotech was used as a reference.
Proteolytic cleavage of multimeric VWF under denaturing
conditions
Purified plasma VWF (37.5 g/mL or 150nM) was incubated with WT and
variants in the conditioned medium (0.2nM and 0.04nM) at 37°C for
4 hours on a dialysis membrane (0.25 m, pore size) floating over 50 mL
dialysis buffer (10mM Tris-HCl, pH 8.0, containing 1.5M urea in a conical
tube). The digested materials were withdrawn and denatured with a
multimer sample buffer (70mM Tris-HCl, pH 6.5, 2.4% SDS, 0.67M urea,
and 4mM EDTA) at 60°C for 20 minutes. The denatured VWF was
fractionated with 1% (weight/volume) SeaKem HGT agarose gel (Cambrex). The proteins were then transferred onto a nitrocellulose membrane,
blocked with TBSTc, and incubated with anti-VWF IgG (Dako North
America; 1:5000) in TBSTc. After being washed with TBST 3 times, the
bound primary antibody was detected by an IRDye 800CW-labeled goat
anti–rabbit IgG (LI-COR; 1:10 000) in TBSTc, followed by Odyssey
imaging system (LI-COR) as described previously.6,27
TTP patient plasma
This study was approved by the Internal Review Boards at the Children’s
Hospital of Philadelphia and the University of Pennsylvania, as well as
Northwestern University. Informed consent was obtained from each
participant before blood was drawn in accordance with the Declaration of
Helsinki. Blood (5 mL) was collected into a tube containing 3.8% sodium
citrate and centrifuged at 1500g for 15 minutes at 25°C. Plasma was
collected and stored at ⫺80°C. All patients (n ⫽ 12) included in the study
had acquired idiopathic TTP. Patients did not have other concurrent
conditions, such as hematopoietic progenitor cell transplantation, cancer,
drugs, and pregnancy, which were known to cause secondary TTP. The
median age of TTP patients was 43 years, with a female-to-male ratio of
3:1. The median platelet count, hematocrit, and serum levels of lactate
dehydrogenase were 15.5 ⫻ 109/L, 23.5%, and 1242 units/L, respectively.
Central nervous system symptoms were present in 10 of 12 patients. Renal
insufficiency (creatinine ⬎ 1.4 mg/dL) was only present in 3 of 12 patients.
All patients exhibited severely deficient plasma ADAMTS13 activity
(⬍ 5% of normal), and positive inhibitors detected by a 50:50 mixing study
with peptide-based and multimer assays. An anti-ADAMTS13 IgG ELISA
from American Diagnostica was used to confirm the presence of IgG
autoantibodies in all patients (Table 1).
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10
11
3838
JIAN et al
Inhibition of cleavage of VWF by autoantibodies from TTP
patients
Recombinant WT and variants (0.2nM) were incubated at 25°C for
30 minutes with 35M of human monoclonal antibody against the spacer
domain of ADAMTS13 previously isolated from a patient with acquired
idiopathic TTP (mAb II-1)6,14-16 (kindly provided by Dr Jan Voorberg at
Sanquin-AMC Landsteiner Laboratory, Amsterdam, The Netherlands) or
with heat-inactivated (56°C for 60 minutes) normal human plasma or
patient plasma (2.5-10 L) in PBS for 30 minutes. The residual proteolytic
activity was determined by the cleavage of VWF multimers using agarose
gel electrophoresis and Western blotting as described previously.6,27
The percentage of inhibition was determined by comparing the residual
activity in WT and variants after incubation with control plasma versus
patient plasma.
A modified immunoprecipitation protocol plus Western blotting was used to
detect the antigen-antibody interaction in solution as described previously.13,19 WT and variants (50 ng) were incubated 5 to 10 L of normal
human plasma or patient plasma and 30 L of protein A/G-Sepharose 4B
(Invitrogen) in 50mM Tris-HCl, pH 7.6, containing 0.15M NaCl, 1% BSA,
1% Triton X-100, and 0.1% Tween-20 at 4°C, overnight. After being
washed with same buffer, bound recombinant WT and variants were eluted
from the beads and determined by Western blotting with anti-V5 IgG
(Invitrogen; 1:5000) in TBSTc, followed by IRDye 800CW-labeled goat
anti–mouse IgG (1:20 000; LI-COR) as previously described.13
Model of ADAMTS13 and VWF interaction
The interaction between ADAMTS13-spacer domain and VWF-A2 was
modeled using the HHPred server plugin in PyMol software (http://
www.pymol.org). The supplemental Videos (available on the Blood Web
site; see the Supplemental Materials link at the top of the online article)
were made with Adobe Photoshop CS 5 software.
Results
Identification of the optimal residue at position of 660 for
ADAMTS13 activity
We28 and others18 have previously shown that R660 in the spacer
domain of ADAMTS13 plays an essential role for substrate
recognition. A substitution of arginine at the position of 660 with
alanine (R660A) nearly abolished proteolytic activity toward
various substrates.18,28 To determine the optimal residue at this
position, we prepared a series of ADAMTS13 variants by replacing
the R with 18 other amino acid residues. The resulting constructs
were transiently expressed in COS-7 cells, which ran at approximately 195 kDa with little degradation on an SDS-polyacrylamide
gel under reduced condition (Figure 1A). The specific activity was
assessed by the cleavage of rF-VWF73 and multimeric VWF. A
replacement of R660 with any other residues except for K (M1,
R660K) resulted in dramatically reduced cleavage of rF-VWF73
(Figure 1B) and multimeric VWF (Figure 1C). These results
suggest that a positively charged residue, such as arginine or lysine
at position 660 in the spacer domain, is required for ADAMTS13
activity.
Identification of gain-of-function ADAMTS13 variants
Based on our preliminary results described in the previous section,
we performed additional site-directed mutagenesis experiments by
sequentially replacing several other charged/hydrophobic residues
on the surface loop in the spacer domain (ie, R660, F592, R568,
Y661, and Y665) with K, Y, K, F, and F, respectively (Figure 2A).
The resulting ADAMTS13 variants (ie, M2, M3, M4, and M5)
were expressed in COS-7 cells. All ran at approximately 195 kDa
on SDS-polyacrylamide gel under reduced conditions (Figure 2B).
The specific activity was assessed by the cleavage of both
rF-VWF73 and multimeric VWF. The variants M1, M2, and
M3 exhibited similar activity to WT in cleaving VWF73 peptide (Figure 2C) and multimeric VWF (Figure 3). However, the
variants M4 and M5 exhibited increased specific activity by
approximately 4- to 5-fold (P ⬍ .001) and approximately 10- to
12-fold (P ⬍ .001) in cleaving rF-VWF73 peptide (Figure 2C) and
multimeric VWF (Figure 3), respectively. These results demonstrate, for the first time, that gain-of-function ADAMTS13 variants
can be engineered and identified through a modification of exosite
3 in the spacer domain.
Identification of ADAMTS13 variants resistant to
autoantibodies in TTP patients
Previous studies have shown that exosite 3 along with several other
adjacent residues in the spacer domain contains major binding sites
for anti-ADAMTS13 autoantibodies in patients with acquired
idiopathic TTP.18,19 We therefore hypothesized that a modification
in this region may alter the binding and inhibition of ADAMTS13
variants by patients’ anti-ADAMTS13 autoantibodies. First, we
assessed the inhibitory effect of mAb II-1 that specifically recognizes the exosite 3 in the spacer domain15,16 on proteolytic cleavage
of multimeric VWF by WT and variants. We showed that mAb II-1
dramatically inhibited proteolytic activity of WT and M2, but not
M1, M3, M4, and M5 (Figure 4A), suggesting that R660 is critical
for autoantibody inhibition. When plasma from TTP patients was
used as the source of autoantibodies against ADAMTS13, proteolytic activity of WT, M1, and M2 was almost completely inhibited
after 30 minutes of incubation (Figure 4B-C; Table 1), whereas the
variant M3 was only variably inhibited, but the variants M4 and M5
were not inhibited by the same amount of patient plasma under the
same conditions (Figure 4B-C; Table 1). These results indicate that
the novel gain-of-function ADAMTS13 variants, especially M4
and M5, are more resistant to inhibition by both monoclonal and
polyclonal anti-ADAMTS13 autoantibodies in patients with acquired idiopathic TTP.
Binding of patient anti-ADAMTS13 IgGs to recombinant WT and
variants
To determine whether the resistance of ADAMTS13 variants to
autoantibodies was the result of impaired binding interactions
between the variants and autoantibodies, we performed an immunoprecipitation experiment plus Western blotting as described previously.13,19 As shown, mAb II-1 bound to WT and M2 consistently,
but not much to the variants M1, M3, M4, and M5 (Figure 5). These
results are in agreement with the data from the functional study in
Figure 4A, suggesting that the reduction in inhibitory activity is
caused by impaired binding of monoclonal antibody to the variants.
Moreover, plasma anti-ADAMTS13 IgGs from all 12 patients
with acquired idiopathic TTP also consistently bound to WT, M1,
and M2 but only variably to M3 and rarely to M4 and M5 (Figure
5). Control IgGs from healthy donors did not detectably bind
WT and variants, whereas monoclonal anti-V5 IgG bound to all
constructs (Figure 5). These results indicate that the inhibition by
polyclonal anti-ADAMTS13 IgGs from acquired TTP patients is
also primarily mediated by the direct binding of anti-ADAMTS13
IgGs to the exosite 3 in the spacer domain.
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Binding of patient anti-ADAMTS13 IgGs to WT and variants
BLOOD, 19 APRIL 2012 䡠 VOLUME 119, NUMBER 16
BLOOD, 19 APRIL 2012 䡠 VOLUME 119, NUMBER 16
POTENTIAL NOVEL THERAPEUTICS FOR ACQUIRED TTP
3839
Model of interactions between the spacer domain and A2
domain
To gain insight into the molecular mechanisms underlying the
enhanced specific activity of ADAMTS13 variants, we performed
modeling based on the existing crystal structural information of the
proximal C-terminal fragment of ADAMTS13 (ie, DTCS29; Figure
6A) and the A2 domain of VWF.30 As shown, the spacer domain
composes 10 -sheets (ie, 1-10), a pocket formed by various
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Figure 1. Western blot and proteolytic activity of
ADAMTS13 and mutants. (A) Western blotting with
anti-V5 IgG detects WT and single point mutants at the
position 660 in the concentrated condition medium
(⬃ 50nM per lane). Arrowhead indicates the intact fulllength ADAMTS13, ⬃ 195 kDa; and double stars, degradation product. (B) Relative proteolytic activity (%) of WT
and single point variants assessed by the cleavage of
rF-VWF73. Data are mean ⫾ SD (n ⫽ 3). All ADAMTS13
mutants except for R660K had relative activity less than
20% of WT. (C) Proteolytic cleavage of multimeric VWF
by ADAMTS13 and single point mutants under denaturing conditions. Plasma-derived VWF (37.5 g/mL or
150nM) was incubated at 37°C with 0.2nM of recombinant WT-ADAMTS13 and point mutants in the presence
of 1.5M urea for 4 hours. The proteolytic cleavage of
VWF was determined by 1% agarose gel electrophoresis
and Western blotting. ⫹ indicates the presence of 10mM
EDTA in the reaction; ⫺, the absence of EDTA in the
reaction; HMW, high molecular weight multimers; and
P, cleavage product.
-sheets containing a cluster of hydrophobic residues (L591, F592,
L637, F638, L668, and T669), and a ring formed by Y661 and
Y665 lined by basic residues R568, R589, R660, and R636 (Figure
6B). This pocket appears to directly interact with the ␣6-helix
(residues between D1653 and R1668) in the central A2 domain of
VWF (Figure 6D). The hydrophobic residues in the A2 domain
presumably face exosite 3 to make strong hydrophobic contacts in
conjunction with some hydrogen bonding outside of the pocket
3840
JIAN et al
activity. Of 24 novel ADAMTS13 variants prepared, 2 (ie, M4 and
M5) exhibited dramatically enhanced specific activity toward
rF-VWF73 peptide (Figure 2) and multimeric VWF (Figure 3).
More importantly, these 2 gain-of-function variants are more
resistant than WT and other variants to inhibition by either
monoclonal or polyclonal autoantibodies against ADAMTS13 in
patients with acquired idiopathic TTP (Figure 4; Table 1). As
shown, 10 of 12 TTP patient plasmas (83%) do not appear to inhibit
proteolytic activity of the variants M4 and M5, whereas the same
amount of patient plasma completely inhibits the proteolytic
activity of WT, M1, and M2 but only variably inhibits M3 activity
under the same conditions. Plasma from 2 patients weakly inhibits
the M4 and M5 activity (Table 1). These results further confirm,
with a gain-of-function rather than a loss-of-function approach
described in the literature,18,28 the critical role of exosite 3 in the
spacer domain in substrate recognition.
Molecular modeling of the interaction between the spacer
domain and VWF-A2 domain suggests that the ␣5-helix of
VWF-A2 domain appears to directly interact with the residues in
the exosite 3 in the spacer domain (Figure 6; supplemental Video
1), primarily through hydrophobic interactions. A substitution of
the amino acid residues R, F, R, Y, and Y with K, Y, K, F, and F at
the positions of 568, 592, 660, 661, and 665, respectively, appears
to increase hydropathy and hydrophobic interactions between the
exosite 3 in the spacer domain and the ␣5-helix in VWF-A2
domain (Figure 6; supplemental Video 1).
(supplemental Video 1). A substitution of R with K residue or
Y with F residue or vice versa appears to alter hydrophobicity of
the exosite 3 (Figure 6C). The corresponding changes of the
hydropathy index were noted as follows: R3K: ⫺4.5 3 ⫺3.9 and
Y3F: ⫺1.3 3 ⫹2.8, thereby enhancing the interaction between
VWF and ADAMTS13. Furthermore, a substitution of F592 with
Y may open up the pocket even more, thereby better engaging the
substrate. There was also a corresponding backbone shift that
appears to take place in the 2-, 5-, 6-, and 9-sheets to
compensate for the increased hydrophobicity (Figure 6C). These
changes allow greater engagement of the exosite 3 with the
A2 domain, particularly the amino acid residues between residues
D1653 and R1668 (supplemental Video 1). Together, our findings
suggest that the modification of an exosite in the spacer domain is a
viable approach to improve ADAMTS13 function while reducing
autoantibody binding and inhibition.
Discussion
In the present study, we have demonstrated that a positively
charged residue (ie, arginine or lysine at position 660) is critical for
ADAMTS13 enzymatic function (Figure 1). This observation
prompts us to test a hypothesis that replacement of the critical
residues in the exosite in the spacer domain may generate
ADAMTS13 variants that are resistant to binding and inhibition by
anti-ADAMTS13 autoantibodies from patients with acquired idiopathic TTP while preserving or enhancing specific proteolytic
Figure 3. Proteolytic cleavage of multimeric VWF by ADAMTS13 and variants
under denaturing conditions. (A) Human plasma-derived VWF (37.5 g/mL or
150nM) predenatured with 1.5M urea was mixed with WT, M1, M2, M3, M4, and
M5 (0.04nM and 0.2nM) in the absence or in the presence of EDTA (10mM; last lane)
and dialyzed against 10mM Tris-HCl, pH 8.0, containing 1.5M urea at 37° for 4 hours.
The cleavage of VWF was determined by agarose (1%) gel electrophoresis and
Western blotting with rabbit anti-VWF IgG (1:5000), followed by IRDye 800CWlabeled goat anti–rabbit IgG (1:10 000). (B) The relative activity was determined by
ImageJ quantitation of the ratio of cleavage product (P, arrowhead) to high molecular
weight (HMW) VWF multimers in each sample. The specific activity was normalized
to that of WT (1 arbitrary unit). Data are mean ⫾ SEM from 3 independent
experiments (n ⫽ 3).
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Figure 2. Characterization of single and compound ADAMTS13 variants.
(A) Schematic domain organization of full-length ADAMTS13 showing a signal
peptide (S), metalloprotease domain (M), disintegrin domain (D), 8 TSP1 repeats
(1-8), Cys-rich domain (C) and spacer domain (Spa), as well as 2 CUB domains
(C1 and C2; top), surface representation of exosite 3 and adjacent residues in the
spacer domain of ADAMTS13 (left), and names of various ADAMTS13 variants with
amino acid substitution (right). (B) Western blotting with anti-V5 detects recombinant
WT and variants in the conditioned medium (50 ng per lane). Arrowhead indicates
full-length protein of ADAMTS13 WT and variants (⬃ 195 kDa) with little degradation.
(C) Relative specific activity of ADAMTS13 variants compared with WT. Data are
mean ⫾ SD (n ⫽ 3). **P ⬍ .001, statistically highly significant.
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POTENTIAL NOVEL THERAPEUTICS FOR ACQUIRED TTP
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Figure 4. Inhibition of proteolytic activity of ADAMTS13 and variants by
autoantibodies. Recombinant WT or variants M1 to M5 (final concentration of
0.2nM) was incubated without (⫺) or with (⫹) 35M of mAb II-1 (A) or 5 to 10 L of
heat-inactivated normal human plasma (N) or plasma from TTP patient 1 (P; B) for
30 minutes. The residual activity was determined by the cleavage of multimeric VWF.
EDTA (10mM) was included in the last lane as a negative control. The relative
residual activity was determined by the ratio of product (P) to high molecular weight
VWF (HMW) multimer using ImageJ Version 1.45m software and normalized to the
activity in the presence of normal human plasma. (C) The percentage of inhibition
(mean ⫾ SD) by a panel of 12 TTP patient plasmas. **P ⬍ .001, statistically highly
significant difference between WT and 3 variants (ie, M3, M4, and M5).
Our findings also provide novel insight into the mechanism
underlying pathogenesis of acquired idiopathic TTP that is primarily caused by anti-ADAMTS13 autoantibodies. Despite the polyclonal nature of autoantibodies against ADAMTS13 in these
patients,13,31 the inhibitory activity of anti-ADAMTS13 autoantibodies appears to be primarily mediated through their binding to the
exosite 3 in the spacer domain, as the alteration in this region
dramatically reduced binding (Figure 5) and inhibition by patient
autoantibodies (Figure 4). These results do not appear to be
contradictory to our previous findings in which multiple domains
of ADAMTS13 are targeted by anti-ADAMTS13 IgGs in patients
with acquired idiopathic TTP.13 The anti-ADAMTS13 IgGs that
bind the C-terminal TSP1 2 to 8 repeats and CUB domains are less
abundant and exhibit lower affinity than those recognizing the
spacer domain.13 In the presence of 1% Triton X-100, the interaction between the middle and distal C-terminal domains and
anti-ADAMTS13 IgGs can be disrupted.19 Indeed, our current
results are highly consistent with those reported by Pos et al,19 in
which a replacement of R568, F592, R660, Y661, and Y665 with A
Figure 5. Binding of anti-ADAMTS13 IgGs from TTP patients to ADAMTS13 and
variants. WT or variants M1 to M5 (50 ng) were incubated with mAb II-1 (35M) or
TTP patient plasmas (5-10 L each, depending on plasma IgG concentrations). The
immune complexes were pulled down with protein A-Sepharose 4B and detected by
Western blotting with anti-V5. Anti-V5 IgG coupled Sepharose 4B beads were used
for a positive control. Normal IgG from healthy persons (nIgG) was used for a
negative control.
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abolishes the binding of anti-ADAMTS13 IgGs from most TTP
patients.19 However, alanine substitution in their study results in
generation of loss-of-function ADAMTS13 variants, which have
little value for therapy.
Our findings may change the way we treat acquired TTP with
inhibitors. Plasma exchange remains the main therapy for these
patients.32,33 It alone appears to be inadequate to restore severe
deficiency of plasma ADAMTS13 activity when high-titer antiADAMTS13 IgGs are present.33 Infused ADAMTS13 is rapidly
neutralized by anti-ADAMTS13 IgGs in TTP patient plasma. Low
ADAMTS13 activity and persistence of anti-ADAMTS13 IgGs in
patients correlate with an increased rate of relapse.11,33,34 Other
immunosuppressive therapies, such as cyclosporine,35,36 cyclophosphamide,37,38 and rituximab (anti-CD20 antibody),37,38 may reduce
the antibody formation, but they take weeks to months to have a
clinical effect. Therefore, an autoantibody-resistant ADAMTS13
variant may have a value to instantaneously restore plasma
ADAMTS13 activity in patients with inhibitors. The infused
ADAMTS13 variants are more likely to survive and work better
despite the presence of polyclonal anti-ADAMTS13 IgGs in these
patients. The binding affinity of anti-ADAMTS13 IgGs toward
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JIAN et al
BLOOD, 19 APRIL 2012 䡠 VOLUME 119, NUMBER 16
Figure 6. Modeling of the spacer domain and VWF-A2
interaction. (A) Surface representation of proximal
C-terminal DTCS fragment of ADAMTS13. (B) Close-up
view of the hydrophobic cluster in the exosite 3 in the
spacer domain of ADAMTS13. This pocket contains a
cluster of hydrophobic residues (L591, F592, L637,
P638, L668, T669, and ring of Y661 and Y665), lined
by basic residues (R568, R589, R636, and R660),
supported by 8 -sheets (ie, 1, 2, 3, 6, 7, 8, 9, and 10).
(C) A substitution of these surface residues with those in
yellow appears to increase hydrophobicity of this pocket.
(D) VWF-A2 (1653-1668) forms an amphipathic helix
(␣6). Hydrophobic residues facing to the top and charged
residues to the bottom. This amphipathic helix may
govern specificity to the exosite 3 in the spacer domain
by inserting its hydrophobic side into the pocket.
Acknowledgments
The authors thank Dr Jan Voorberg, Sanquin-AMC Landsteiner
Laboratory, Amsterdam, The Netherlands, for providing human
monoclonal antibody against spacer domain of ADAMTS13
(mAb II-1).
This work was supported in part by the American Heart
Association (National Established Investigator Award 0940100N;
X.L.Z.) and the National Institutes of Health (grant P01HL074124,
Project 3; X.L.Z.).
Authorship
Contribution: C.J. and X.L.Z. designed research, performed experiments, and wrote and revised the manuscript; J.X., L.G., C.G.S.,
and S.-Y.J. performed experiments and revised the manuscript; and
H.C.K. provided patient samples and clinical information and
revised the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: X. Long Zheng, Department of Pathology and
Laboratory Medicine, Children’s Hospital of Philadelphia, 34th St
and Civic Center Blvd, 816G ARC, Philadelphia, PA 19104;
e-mail: zheng@email.chop.edu.
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POTENTIAL NOVEL THERAPEUTICS FOR ACQUIRED TTP