adamts13, ttp, and beyond...outline • discovery of adamts13 • pathogenesis and potential...
TRANSCRIPT
ADAMTS13, TTP, and Beyond
X. Long Zheng, M.D., Ph.D.
The University of Alabama at Birmingham, Birmingham, AL
ASFA (April 21, 2017)
Disclosure of Conflict of Interests
• Consultant: Ablynx and BioMedica
Diagnostics
• Speaker’s Bureau: Alexion
• Research collaboration: Lee’s
Pharmaceuticals, Alexion and
Synapse
Outline
• Discovery of ADAMTS13
• Pathogenesis and potential triggers of
TTP
• Mechanism of autoantibody-mediated
inhibition of ADAMTS13 activity
• Diagnostic dilemma and potential novel
therapeutics
• Prospective
• 42 Y AAF presented with lethargy, blurred vision, and
disorientation.
• Thrombotic thrombocytopenic purpura
(TTP)
• Blood smear: >10 schistocytes
• Platelet 24,000 and Hb 9.6
• LDH 1,157, haptoglobin 0.09, creatinine: 1.6, PT and
PTT normal, and DAT (-)
• CT scan: multiple cerebral infarcts.
My first case
1924
A16-year-old adolescent girl
Abrupt onset of petechiae
Hemolytic anemia, followed by paralysis, coma, and
death
Autopsy showed widespread hyaline thrombi in the
terminal arterioles and capillaries of various organs.
Eli Moschcowitz (1924)
Moschcowitz’s disease, now
known as TTP
Moake et al. NEJM, 1982:307. 23:1432-35
Ultra large VWF multimers in patient
plasma with chronic relapsing TTP
Endo
thelia
l
TT
P a
ctive
TT
P R
em
Norm
al
UL
VWF
-500 KDa
-2000 KDa Dr. Joe Moake
- 1982 N
orm
al
TT
P
Lack of a VWF-depolymerase may be the cause of TTP.
Hypothesis:
Deficiency of a plasma VWF–cleaving protease
in patients with chronic relapsing TTP
Dr. Bernhard Lämmle
-1997
Furlan M et al. Blood 1997; 89:3097-3103
Asymptomatic
family members
Chronic
Relapsing TTP
Sample 1 2 1 2 1 2 1 2 1 2
NHP
Urea or
guanidine
Shear
VWF-cleaving
protease
IgG autoantibodies against VWF-cleaving
protease are responsible for acquired TTP
Tsai and Lian. NEJM, 1998
Pro
tease a
ctivity (
%)
Resid
ual a
ctivity (
%)
Acute Rem Acute Rem
Dr. Han-Mou Tsai
- 1998
IgG fraction isolated from acquired TTP patients inhibited
VWF-cleaving protease activity.
200-
150-
100-
50-
0-
200-
150-
100-
50-
0-
Plasma VWF-cleaving protease is essential for
maintaining normal hemostasis
VWF-cleaving
protease
LM Morning Report
Washington University, 1999
Deficiency of VWF-cleaving activity results in
disseminated microvascular thrombosis
Hereditary
(<5%)
Blood Flow
LM morning report
Washington University, 1999
Autoantibodies
(>95%)
Microvascular
thrombosis
Exposure of
subendothelial matrix
What is VWF-cleaving protease?
Structure of von Willebrand Factor-cleaving
Protease (ADAMTS13), a Metalloprotease Involved
in Thrombotic Thrombocytopenic Purpura*
Xinglong Zheng‡§, Dominic Chung§¶, Thomas K. Takayama‖, Elaine M.
Majerus**, J. Evan Sadler**‡§§ and Kazuo Fujikawa¶
Departments of ‡Pathology, **Medicine, and §§Biochemistry and Molecular
Biophysics and Howard Hughes Medical Institute, Washington University
School of Medicine, St. Louis, Missouri 63110 and the Departments of ¶Biochemistry and ‖IUrology, University of Washington, Seattle, Washington
98195
J Biol Chem. 2001 Nov 2;276(44):41059-63. Epub 2001 Sep 13.
A year of breakthrough in
TTP research
Gerritsen HE et al. Partial amino acid sequence of purified
von Willebrand factor-cleaving protease. Blood. 2001; Sep
15.
Soejima K, et al. A novel human metalloprotease
synthesized in the liver and secreted into the blood: possibly,
the von Willebrand factor-cleaving protease? J Biochem.
2001, Oct 1.
Levy GG, et al. Mutations in a member of the ADAMTS gene
family cause thrombotic thrombocytopenic purpura. Nature.
2001; Oct 4.
1 3 6 7 9 12 15 17 20 22 24 28 29
5kb
A Disintegrin And Metalloprotease with ThromboSpondin
type 1 repeats (ADAMTS), 13
Zheng et al. J. Biol. Chem. Sept. 13, 2001
ADAMTS13
CUB1 S P Metallo- protease Dis 1 Cys Spacer 2 3 4 5 6 7 8 CUB2
RQRR ★★ ★★★★ ★ ★ ★ ★
O-glycosylation ★ N-glycosylation
1,427 amino acids 145 -190 kDa
Summary of the discovery of ADAMTS13
-19
24
1st case
description
Moschcowitz
-20
01
Identification
& cloning of
ADAMTS13
Zheng
Gerritsen
Fujikawa
Soejima
Levy
TTP
-19
82
Ultra large
VWF
Moake
-19
78
-19
60
Plasma
factor
deficiency
Schulman
Upshaw -1
99
7
-19
98
VWF-cp &
autoantibody
Furlan
Lammle
Tsai
Pathogenesis of TTP
Hereditary TTP: - ADAMTS13 mutations (>200)
Acquired TTP: - Autoantibodies against ADAMTS13
• 59% missense • 13% nonsense • 13% deletions • 6% insertions • 9% alternative splicing
Age of onset for the 1st
thrombocytopenia in patients
with hereditary deficiency of ADAMTS13 varies greatly
Fujimura et al. ADAMTS13 Biology and Disease
ed. By G.M. Rodgers, Springer 2015
Early onset (75%)
Late onset (25%)
Other genetic or environmental factors may contribute to the
pathogenesis of TTP
All patients except for one have ADAMTS13 activity < 0.5%
Anti-ADAMTS13 IgG is present in all
patients with acquired TTP at UAB
Cao W, Pham HP, Williams III LA, McDaniel JK, Siniard RC, Lorenz RG,
Marques MB, and Zheng XL. Haematologica. 2016;101:1319-1326
In 52 patients with ADAMTS13<5%, 46 patients had inhibitor >0.4 U/ml (positive), but 6 patients <0.4 U/ml (negative).
However, inhibitors are not always demonstrated with functional assays.
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
0.0 1.0 2.0 3.0 4.0 5.0 6.0
An
ti-A
DA
MT
S13 I
gG
(U
/ml)
<0.4 0.4-1.2 1.3-2.4 2.5—5.0 >5.0
Titer (U/ml)
Anti-ADAMTS13 IgG was detected in TTP
patients with inhibitor<0.4 U/ml
Cao et al. Haematologica, 2016
Normal
How do IgG autoantibodies inhibit
ADAMTS13 activity?
Casina et al, PNAS, 2015, Aug 4;112:9620-5
Five small flexible loops in the spacer domain are
specifically targeted by autoantibodies from TTP patients.
Anti-A13
v v v v v v v CUB TSP1 2-8
Veronica Casina Ph,D
Potential triggers
• Infection or system inflammation
• Complement amplification conditions
• Pregnancy
• Certain medications
• Additional genetic factors
These are the same triggers for hemolytic
uremic syndrome (HUS)
I have low ADAMTS13
“Second Hit”
Human neutrophil peptides (HNPs) are
dramatically elevated in acquired TTP
HNPs
MPO
Histone-DNA
Cao W, Pham HP, Williams III LA, McDaniel JK, Siniard RC, Lorenz RG,
Marques MB, and Zheng XL. Haematologica. 2016;101:1319-1326
Fuchs et al. Blood 2012:120:1157-64
Plasma nucleosomes and DNA are
markers of acute TMAs
Plasma levels of complement
factor Bb increase in acquired TTP
C3
C5
FH FI MCP
FB FD
C5b6789
Cao W, Pham HP, Williams III LA, McDaniel JK, Siniard RC,
Lorenz RG, Marques MB, and Zheng XL. Haematologica.
2016;101:1319-1326
How to distinguish TTP from aHUS
clinically?
• Platelets < 30,000/μl
• Normal or near normal
renal function
• Response to plasma
exchange
• ADAMTS13 activity
(<10%)*
• Less severe
thrombocytopenia
• Significant renal
dysfunction
• Lack of complete response
to plasma exchange
• Normal ADAMTS13
(>10%)
*ADAMTS13 activity is the single most important test to
distinguish TTP from aHUS at the present time.
Bentley. ADAMTS13 Biology and Disease
ed. By G.M. Rodgers, Springer 2015
How is ADAMTS13 test performed?
Tyr-Met
Met
Plasma (or ADAMTS13)
Normal
TTP Patient
Time (min)
Rat
e o
f fl
uo
resc
ence
ge
ner
atio
n (
AU
/se
c)
FRETS-VWF73
Kokame K et al. Br J Haematol. 2005, 129:93-100.
ADAMTS13 activity measured by an ELISA
10
63
10
Current & future therapies
• Hereditary TTP
Plasma infusion vs.
Recombinant ADAMTS13 or
AAV gene therapy.
• Acquired TTP
Plasma exchange & immunotherapy
Other potential novel therapies
Dr. Paul Coppo
Plasma infusion resulted in an intermittent
recovery of platelet counts in hereditary TTP
Furlan & Lammle. Best Pract. Res. Clin. Haematol. 2001, 14, 437-454
AAV8-mediated liver expression of rADAMTS13
results in cure of hereditary TTP in mice
B
Jin et al. Blood. 2013,121(19):3825-9
0 14
Shigatoxin
i.v.
CBC
(Days) Adamts13-/-
ADAMTS13
activity AAV8-
mMDTCS
i.v.
7 21
Sheng-yu, MD, PhD
Kaplan-Meir Survival Curve
Jin et al. Blood. 2013,121(19):3825-9
AAV8-hAAT-LacZ 2.6x1011 vg/kg
Surv
ival
rat
e (%
)
AAV8-hAAT-mMDTCS
2.6x1011 vg/kg
Stx-2
Antibody-resistant rADAMTS13
T633
R636
P590
L591
T669 Y661F R660K
Y665F
β1
β10
β3
β8 β7 β6
β7-β8
loop
β9-β10
loop R568K
F592Y
Motif A
Molecular Surgery to ADAMTS13
Cleavage of FRETS-VWF73 by WT and rADAMTS13 variants
rADAMTS13: M4 and M5 exhibit increased activity by 4-5 fold
Jian et al, Blood, 2012; 119:3836-43.
Cui Jian
rADAMTS13-M4 and M5 were more resistant than WT to
autoantibody from acquired TTP
rADAMTS13
+ normal or patient
plasma
37C, 60 min
Residual activity N: normal human plasma
P: TTP patient plasma
ADAMTS13 variants are resistant to ≈85% TTP patient plasma IgGs.
Jian et al, Blood, 2012; 119:3836-43.
The autoantibody-resistant (ar)
ADAMTS13 may be used to treat acquired
TTP without plasma exchange.
arADAMTS13
VS.
Apheresis
Anti-ADAMTS13
IgG
Thrombin
Quiescent
Platelets
VWF
multimers
ADAMTS13
Cleaved VWF
& platelets
ADP
Collagen
Hypothesis:
Shear
Activated
Platelets
Delivery of rADAMTS13 via platelets
Can human platelets uptake
rADAMTS13 in vitro?
25
rADAMTS13 (ng)
0 20 40 80
rA13 (μg/ml)
230-
180-
116-
66-
40-
12-
(kDa) 0 20 40 80
rA13 (μg/ml)
37 °C 25 °C
50 100 12.5 0
Donor platelets + rADAMTS13
A13 VWF
Abdelgawwad, et al. unpublished
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 25 50 75 100 125 150 175
Pla
tele
t C
ove
rag
e
Time (Seconds)
Control
Platelets
0
0.2
0.4
0.6
0.8
1
0 25 50 75 100 125 150 175
Pla
tele
t C
ove
rag
e
Time (Seconds)
Control
Platelets
N=3
N=3
PBS
Plts
w/rA13
50 μg/ml
PBS
Plts
w/rA13
100 μg/ml
Platelet-delivered rADAMTS13 is efficacious in
reducing the rate of thrombus formation under shear
Abdelgawwad, et al. unpublished
Moh
Transgenic mice expressing rADAMTS13
in megakaryocytes and platelets in mice
TGPlt+A13
Adamts13-/-
Pickens et al. Blood, 2015,125(21):3326-34
Brandy Pickens, PhD
Genotype Number Platelet count (×109/L)
TG 20 896 ± 156**
KO 20 631 ± 93
WT 16 708 ± 179
The data presented are the mean ± SD.
TG, Adamts13−/−PltA13; KO, Adamts13−/−; WT, wild-type.
** P < .001 when compared with KO and WT mice, respectively.
Higher baseline platelet counts in
transgenic mice than in Adamts13-/- and
wild- type mice
Pickens et al. Blood, 2015,125(21):3326-34
Platelet-delivered rADAMTS13 reduces the rate
of thrombus formation in mesenteric arteriole
after FeCl3 injury
0
2
4
6
8
10
12
14
01:22.9 06:22.9 11:25.5 16:25.5 21:26.3 26:26.8 31:26.8
Th
rom
bu
s F
orm
ati
on
(C
han
ge i
n f
lou
rescen
ce)
KO
WT
TG
Time (min)
Pickens et al. Blood, 2015,125(21):3326-34
Platelet-delivered rADAMTS13 significantly reduced
thrombocytopenia and mortality rate in the presence
of anti-ADAMTS13 IgG
B 10 μg/gram rVWF
*****
Pickens et al. Blood, 2015,125:3326-34
C
0 14
rVWF (i.v.)
CBC
(Hours)
scFv4-20 (i.p.)
7 24
ADAMTS13 activity
WT vs. TGPtA13
A
Take home…...
• Identification of ADAMTS13 provides a new avenue for studying the mechanism, diagnosis, and treatment of TTP.
• Mapping studies let us identify the critical
region in ADAMTS13 where autoantibody
bind, which shed new light on the
mechanism of autoantibody-mediated
inhibition of ADAMTS13 activity.
• rADAMTS13 and AAV8-based gene therapy
may become the treatment of choice for
hereditary TTP.
• arADAMTS13 variants, platelet-delivery of
rADAMTS13, as well as antagonists for
VWF-platelet interaction may be further
developed as novel therapeutics for
acquired TTP.
• Whether anti-complement therapy is needed
or not for a subset of acquired TTP patients
is yet to be determined.
Prospective
Low ADAMTS13 is associated with many thrombotic
and inflammatory diseases:
Ischemic cerebral infarction
Myocardial infarction
Preeclampsia/eclampsia
Malignant malaria
Septic DIC and ICU patients with renal failure
Trauma-associated mortality.
Acknowledgement
Marisa Marques, M.D.
Lance Williams, M.D.
Huy P. Pham, M.D., M.P.H.
Robin Lorenz, M.D., Ph.D.
Bruce Sachais, MD, PhD.
Don Siegel, M.D., Ph.D.
Douglas Cines, M.D.
Walter Englander, Ph.D.
Adam Miszta, PhD, MSc. Eng.
Bas de Laat, Ph.D.
Current Wendy Cao, M.D., Ph.D.
Vikram Pillai, Ph.D.
Catherine Zander, Ph.D.
Liang Zheng, Ph.D.
Jenny McDaniel, M.D.
Jay Li, Ph.D.
Nicole Kocher, B.S.
Moh Abdelgawwad, M.D., M.S.
05.07.2016
Grant supports: NBF, AHA, and NIH-R01s
Collaborators