Title: Update on ADAMTS13 October 1, 2004
1Update on ADAMTS13October 1, 2004
- VWF proteolysis and bleeding
- VWF proteolysis and TTP
- Recognition of VWF by ADAMTS13
2The Von Willebrand Factor Precursor
3von Willebrand Factor Multimers
120 nm
Adapted from Fowler et al, J Clin Invest
761491-1500, 1985
4Mutations in VWD Type 2
U. of Sheffield (Ian Peake) http//www.shef.ac.
uk/vwf/mutations.html
Type 2A
5VWF Multimers
NP
1
2B
3
2A
2A
Plt
Proteolysis
6Thrombotic Thrombocytopenic PurpuraA Disorder of
VWF Proteolysis?
Inreasing incidence? Often strikes young adults,
mainly females
Untreated, mortality gt90 Treated with
plasmapheresis, mortality lt20
7VWF Multimers in TTP
Endothelial Cell
Normal Plasma
Active
Remission
Unusually large Multimers
Moake et al, N Engl J Med 3071432-5, 1982
8VWF Cleaving Protease in Plasma
- Discovered in 1996 by Tsai and by Furlan
- Requires Ca2 and Zn2 ions
- Cleaves VWF between Tyr1605 - Met1606
- Activated by shear stress, mild denaturants
- Absent in children with congenital TTP
- Absent in most adults with idiopathic TTP
- (acquired IgG autoantibody inhibitor)
9VWF Cleaving Protease(ADAMTS13)
Metalloprotease
Thrombospondin 1
Disintegrin
A Disintegrin-like And Metalloprotease with
ThromboSpondin-1 repeats
Zheng, Chung et al, J Biol Chem 27641059-63,
2001 Levy et al, Nature 413488-494, 2001
10VWF and Platelet Adhesion
11Shear and VWF Proteolysis
- Proteolysis increased by
- Shear stress (aortic stenosis)
- VWD type 2A mutations
- Denaturants (urea, guanidine)
12Clinical Correlations
Association
Odds Ratio
- ADAMTS13 lt5 and
- Survival b,c 5.4
- Idiopathic TTP a,b,c 8
- Female sex b,c 3.6
- African ancestry b,c 7.5
- ADAMTS13 Inhibitor and
- Relapse a,b,c 13.5
aVeyradier et al, Blood 981765-72, 2001 bVesely
et al, Blood 10260-8, 2003 cZheng et al, Blood
1034043-9, 2004
13ADAMTS Structures
Domains
ADAMTS-
4
5/11,6,8
1,15
16
9,20
14,18
2,3,10,17,19
7,12
13
14VWF Cleaving Protease(ADAMTS13)
Ligand binding?
15ADAMTS13 in COS-7 Cells
FL
MD1CS8
MD1CS
MD1C
MD1
MD
M
Zheng et al, J Biol Chem 27830136-41, 2003
16ADAMTS13 in COS-7 Cells
MD1CS8
MD1CS
control
MD1C
MD1
MD
FL
M
(kDa)
207-
118-
81-
53-
36-
Western blot anti V5
17Recombinant ADAMTS13 ActivityAgainst Plasma VWF
FL EDTA
MD1CS8
MD1CS
FL Ab
Plasma
MD1C
MD1
MD
FL
M
(kDa)
300-
176-
140-
The spacer may recognize VWF
18Recombinant ADAMTS13 ActivityAgainst Plasma VWF
The spacer may recognize VWF
19Functions of Metzincin Propeptides
- Latency
- Pro-enzymes are zymogens
- Cysteine-switch (PRCGvPDv) coordinates Zn
- Activated by proteolysis
- Folding
- Act as endogenous chaperones
- Deletion causes misfolding
20ADAMTS13 Propeptide Reagents
Anti-Propeptide Antibody
SPGAPLKGRPPSPGFQRQR
-GPSHFQQSCRQRQRQRQRRAAGG-
WT
-GPSHFQQSCRQRQRQKQDRAAGG-
R71K/R73D
-GPSHFAAGG-
delPro
Majerus et al, J Biol Chem 27844643-48, 2003
21ADAMTS13 delPro is Secreted and ActiveExpression
in HeLa Cells
Vec
WT
delPro
Vec
WT
delPro
C
M
C
M
C
M
EDTA
210-
210-
131-
VWF Cleavage
Anti-V5
22ProADAMTS13 is Secreted and Active
WT
R71K/R73D
Furin
Anti-Pro
ADAMTS13 Secretion
Anti-V5
VWF Cleavage
VWF (176)2
LoVo
HeLa
23The ADAMTS13 Propeptide
- Does not mediate latency
- No cysteine-switch
- Secreted proADAMTS13 is active
- Intracellular proADAMTS13 is active
- Dispensable for folding
- ADAMTS13 delPro is secreted and active
24ADAMTS13 Substrates
A1
A2
A3
Nishio et al, Proc Natl Acad Sci USA
10110578-83, 2004
25ADAMTS13 Substrates
26Time Course of ADAMTS13 Cleavage
27Time Course of ADAMTS13 Cleavage
A2A3
A2
Fraction Cleaved
A1A2A3
A1A2
Time (hours)
28Activation of ADAMTS13Dependence on VWF Domain A1
GPIba
Heparin
29Heparin Activates ADAMTS13 Promotes Cleavage of
Plasma VWF
(µg/ml)
(mg/ml)
30VWF and GPIba
Interacting surfaces
A1
GPIb
31VWF and GPIba
Interacting surfaces
90
90
GPIb
A1
32VWF and GPIba
Positive
Negative
A1
GPIb
33VWF A Domains
Positive
Negative
A1
A2
34VWF Cleaving Complex
A1
A2
35VWF Cleaving Complex
A2
A1
GPIb
Platelet
36VWF Cleaving Complex
Stretch
A2
A1
?
GPIb
Platelet
37Surface, Cofactor, Enzyme, Substrate
38VWF, ADAMTS13, and TTPFeedback Regulation of
Platelet Adhesion
- VWF Multimer Assembly
- Large multimers are functional
- Assembly defects cause VWD
39VWF, ADAMTS13, and TTPFeedback Regulation of
Platelet Adhesion
VWF Multimer Assembly
- Proteolysis by ADAMTS13
- Cleaves VWF Tyr1605-Met1606
- Increase causes VWD (type 2A)
- Decrease causes (some) TTP
40VWF, ADAMTS13, and TTPFeedback Regulation of
Platelet Adhesion
VWF Multimer Assembly
Proteolysis by ADAMTS13
- ADAMTS13 Biochemistry
- MDTCS cleaves VWF in vitro
- ProADAMTS13 is active
- GPIba or heparin accelerates cleavage
41VWF, ADAMTS13, and TTP
- ADAMTS13, TTP
- Xinglong Zheng
- Elaine Majerus
- Patricia Anderson
- Kenji Nishio
- Agnès Veyradier
- Tim Goodnough
- Morey Blinder
- Seattle
- Dominic Chung
- Tom Takayama
- Kazuo Fujikawa
- Earl Davie
- VWF Assembly
- Angie Purvis
- Hushan Ao
- Akira Katsumi
- Imre Bodó
- Jeroen Eikenboom
- Milan Kapadia
- Lisa Westfield
- Elodee Tuley
42(No Transcript)
43TTP and ADAMTS13 Response to Plasma Exchange
- Prospectively enroll adults with TTP
- Plasma samples on admission and every 2-3 days
during therapy - Assay for VWF cleaving protease and inhibitor
Zheng et al, Blood 1034043-9, 2004
44TTP and ADAMTS13 Response to Plasma Exchange
Clinical Characteristics of 37 Patients
Sex 30 female/7 male Age 47 (16-79) LDH
(IU/dl) 939 (263-28,000) Hematocrit () 28
(16-33) Platelets (K/µl) 18 (2-147) Creatinine
(mg/dl) 2.0 (0.6-6.4)
45TTP and ADAMTS13 Response to Plasma Exchange
ADAMTS13 Deficient Category Number
Deficient Inhibitor Survival Idiopathic 20 16
7 17 BMT 8 0 0 4 Cancer/Chemo 4 0 0 0 Peripart
um 2 0 0 2 Other 3 0 0 1 Total 37 16 7 24
46ADAMTS13 Deficient, No Inhibitor Response to
Plasma Exchange
- 9 patients with
- Low protease
- No inhibitor
- Recurrences in 3
- and 1 death
- Plasma exchange
- Good response
- Increased protease
Why protease deficient? Clearance antibody? Other
factors?
47ADAMTS13 Deficient, With Inhibitor Response to
Plasma Exchange
- 7 patients with
- Low protease
- Inhibitor
- Multiple relapses in 4
- and 2 deaths
- Plasma exchange
- Good response
- No change in protease
- Persistent inhibitor
Why is PE effective?
48TTP and ADAMTS13Why Does PE Work for Inhibitors?
- Stress precipitates TTP in congenital ADAMTS13
deficiency - Childhood triggers vaginal delivery, URI,
pneumonia, otitis media - Adult triggers any infection, pancreatitis,
pregnancy - Resolution of stress may end an attack
49Mortality and Category
Dead
Alive
Totals
Idiopathic
18 (26)
50
68
Non-Idiopathic
45
66 (59)
111
Totals
84 (47)
95
179
OR (survival) 4.1 (2.1-7.8)
Vesely et al, Blood 10260-8, 2003 and our data
P lt 0.00005
50Mortality and ADAMTS13
Dead
Alive
Totals
ADAMTS13 lt5
6 (18)
28
34
ADAMTS13 gt5
55
90 (62)
145
Totals
96 (54)
83
179
OR (survival) 5.4 (2-16)
Vesely et al, Blood 10260-8, 2003 and our data
P lt 0.0005
51Relapses and ADAMTS13 Inhibitors
Relapse
No Relapse
Totals
Inhibitor
15 (43)
20
35
No Inhibitor
18
1 (5)
19
Totals
16 (30)
38
54
OR (relapse) 13.5 (2-86)
Veyradier et al, Blood 981765-72, 2001, Vesely
et al, Blood 10260-8, 2003 and our data P lt
0.01
52Clinical Correlations
Character
Odds Ratio
- ADAMTS13 lt5
- Survival 5
- Idiopathic TTP 8
- Female sex 4
- African ancestry 8
- Obesity (high)
- ADAMTS13 Inhibitor
- Relapse 13
53TTP and ADAMTS13Implications for Future Studies
- PE is relatively ineffective for non-idiopathic
TTP - ADAMTS13 and inhibitor assays may be useful to
guide therapy - Plasma exchange should be reevaluated for TTP not
caused by ADAMTS13 deficiency
54VWF, ADAMTS13, and TTPFeedback Regulation of
Platelet Adhesion
VWF Multimer Assembly
Proteolysis by ADAMTS13
ADAMTS13 Biochemistry
- TTP Diagnosis and Treatment
- Stratify by ADAMTS13 level
- Recombinant ADAMTS13