1. health and fitness
2. disease

“What is ADAMTS13 Anyway?”
Maxwell Smith, MD
August 19th, 2005
Objectives
• Review the pathology, epidemiology, differential
diagnosis, and treatment of Thrombotic
Thrombocytopenic Purpura (TTP) using a case
presentation
• Describe the discovery of ADAMTS13 (von
Willebrand Factor Cleaving Protease) and its role
in TTP
• Introduce selected testing methods for vWF-CP
and evaluate their use in the diagnosis and
management of TTP
35 year-old Hispanic Female
• Presents with easy bruising and abdominal
pain
• Multiple bruises had developed “all over”
without traumatic history
• Multiple small red spots as well
• No neurologic complaints
• 1 week prior had URI symptoms and
diarrhea
Physical Exam
• Vitals: Temp 38.5, HR 94, BP 121/75
• Active gingival and mucosal bleeding
• Innumerable ecchymoses over all
extremities and trunk
• No neurologic abnormalities
• Scattered petechiae on BUE and BLE
• Cervical lymphadenopathy
Lab Studies
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CBC: WBC 12.8, HCT 30, PLTC 12
BUN/Cr: 14 / 0.9
CoAg: PT 13.2, PTT 40.9, d-dimer 3.91
T-bili: 2.5 (2.4 unconjugated)
LDH 1522
Peripheral smear
– Thrombocytopenia
– 1+ schistocytes
• Direct Coombs neg.
• Drug screen neg.
Brief Differential Diagnosis
• TTP vs. Hemolytic uremic syndrome vs. ITP
• TTP
– Classic pentad
– Patients often have only 3/5
• HUS
– Lack of neurologic defects
– “Prominence” of renal compromise
• ITP
– Isolated thrombocytopenia with no other clinical
findings - A diagnosis of exclusion
– No anemia, fever, neuro, or renal signs/symptoms
TTP - Classic pentad
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Fever
Thrombocytopenia
Transient neurologic dysfunction
Microangiopathic hemolytic anemia
Renal failure
TTP Pathology
• Unique composition of microvascular thrombi
– Platelet rich
– vWF rich
– Fibrin poor (lack of involvement of the traditional
clotting cascade)
• Sheer forces lead to fragmentation of the RBC hemolytic anemia with schistocytes
• Vascular compromise leads to end organ
dysfunction (kidneys, brain, etc.)
TTP - Epidemiology
• Classic history is sudden onset of symptoms
in an otherwise health adult
• Propensity for females of child bearing age
TTP - Treatment
• Plasma exchange
– Started in 1970’s
– Changed prognosis from >90% mortality to
<25% mortality
– Effectiveness related to the proposed etiology
of TTP
“What is ADAMTS13 Anyway?”
• Moake et al., 1982
– In 4 patients with chronic TTP, large multimers
of vWF were identified in their serum
– Multimers were similar to those found in media
surrounding in vitro endothelial cells suggesting
vascular cells as a possible source
– Possible failure of cleavage of the vWF
multimer was causing TTP
ADAMTS13 discovery
• 1996 - 2 groups identified a 300 kD
metalloprotease which cleaved the vWF
multimers at a Tyr-Met bond
– Required divalent cations (inhibited by calcium
chelating agents)
– Kinetics were slow in undisturbed plasma
– Mild denaturation of the vWF protein or fluid
shear stress accelerated the reaction
ADAMTS13 discovery cont.
• Levy et al., 2001
• Mapped the gene for the metalloprotease to
chromosome 9q34 with linkage analysis
• Identified a new member of the ADAMTS
family of zinc metalloproteinases,
ADAMTS13
• Identified 12 mutations in patients with
hereditary TTP clinical picture
Von Willebrand Factor Cleaving Protease, vWF, and Platelets Under
Normal Conditions
vWF-cleaving protease (ADAMTS13)
Tyr-Met AA bond in vWF
Receptor for GP Ib on the platelets
Platelet
vWF and Platelets When Von Willebrand Factor Cleaving Protease
is Absent or Deficient
vWF and Platelets When Von Willebrand Factor Cleaving
Protease is Absent or Deficient, Cont.
Back to Our Patient
• Heme/Onc diagnosed TTP clinically
• Sample drawn for vWF-CP testing and sent
to the Blood Center of Wisconsin
• 7 rounds of plasma exchange over 4-5 days
• Patient discharged home with normal
platelet count
• Lab test pending
Laboratory Testing for vWF-CP
• Genomic Studies
– Limited use unless documented family history of TTP
like illness (FISH analysis for multiple known genetic
mutations in the ADAMTS13 gene)
• Activity & Inhibitor Studies
– Wide variety of methods currently in use
– Initial methods required laboratory and personnel
expertise
– Subsequent methods have decreased turnaround time
and complexity
Flaws in current vWF-CP Testing
• The test result is invariably compared to the
current gold standard for TTP diagnosis clinical (universally accepted to be difficult
and often incorrect) - misclassification bias
• Most measure enzyme activity indirectly
• Most carry out enzymatic reaction in non
physiologic conditions
• No standardization of methods
Questions to be Answered from
the Literature
• 1. What test is our send out lab using and
does it work?
• 2. How well do the various testing methods
compare with each other?
• 3. How has this specific test (or similar
tests) been evaluated and how well has it
performed?
• 1. What test is our send out lab using and
does it work?
Gerritsen et al., 1999
• Assay of von Willebrand Factor (vWF)cleaving Protease Based on Decreased
Collagen Binding Affinity of Degraded
vWF
• Developed a simple activity and inhibitor
assay for vWF-CP
• Evaluated the test in 40 patients
Gerritsen et al., 1999
• Activity Assay
– Deactivate donor plasma vWF-CP with EDTA
– Mix donor and patient plasma
– If vWF-CP is present and functional in the patient
sample, it will cleave vWF-multimers in the donor
plasma (more vWF-CP, more vWF monomers, less
vWF multimers)
– Add solution to plates coated with human type III
collagen (preferentially binds vWF multimers)
– Quantify the collagen bound vWF multimers with a
anti-vWF multimer peroxidase labeled antibody
– Measure absorbance at 492nm
– Calibration curve done with serially diluted donor
plasma samples
Gerritsen et al., 1999
• Inhibitor assay
– Add non-deactivated donor plasma with patient
sample in 1:1 ratio
• If antibodies to vWF-CP are present in the patient
sample, they will bind and deactivate vWF-CP from
the donor plasma
– Perform same test as previously described
Gerritsen et al., 1999
• Plasma sample selection
– Based on clinical findings (classic pentad) and
a history of relatives with a similar condition
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10 “normal” control patients
10 with familial TTP
11 with acquired TTP
9 with HUS
Gerritsen et al., 1999
N 10
10
11
9
10
11
Gerritsen et al., 1999
• Conclusions
– vWF-CP activity can be used to distinguish
TTP from HUS
– The presence of a vWF-CP inhibitor can further
differentiate acquired from hereditary TTP
– The collagen binding assay is sensitive and
specific
Gerritsen et al., 1999
• Study deficiencies
– Low sample number (40)
– Misclassification bias
• Diagnosis of TTP
• Definition of familial
• 2. How well do the various testing methods
compare with each other?
Studt et al., 2003
• Measurement of von Willebrand factorcleaving protease (ADAMTS-13) activity in
plasma: a multicenter comparison of
different assay methods
Studt et al., 2003
• Methods
– Identical aliquots from 30 different patients with
acquired TTP, hereditary TTP, and “other” conditions
were sent to 5 different laboratories
– Each lab used its standard testing method for activity
and presence of inhibitor
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(1) Immunoblot assay
(2) Residual collagen binding assays
(1) Residual ristocetin cofactor activity assay
(1) Immunoradiometric assay
Studt et al., 2003
• Results
Studt et al., 2003
• Results Cont.
Studt et al., 2003
• Conclusion
– In general, correlation fairly good [Spearman
rank order correlation coefficient = 0.89 - 0.97
(p<0.001)]
– Deviations were more common in the collagen
binding assay suggesting it is more delicate
Studt et al., 2003
• Problems
– Dose not address the accuracy or
reproducibility of the labs (each sample was
only tested once)
– Poor correlation of data at the higher activity
level compensated for correlation at the lower
activity levels
Tripodi et al., 2004
• Measurement of von Willebrand factor
cleaving protease (ADAMTS13): results of
an international collaborative study
involving 11 methods testing the same set
of coded plasmas
Tripodi et al., 2004
• Method
– Normal plasma (100% activity) and plasma
from a patient with familial TTP (0% activity)
were mixed to have ADAMTS activity, by
volume, of 0%, 10%, 20%, 40%, 80%, and
100%
Tripodi et al., 2004
• Results
– Linearity (expected vs. observed) = from 0.98
to 0.39 (1 = perfect linearity)
– Reproducibility = from <10% to 83%
(coefficient of variation)
– Better correlation between the very low and
high ADAMTS13 levels
Tripodi et al., 2004
• Conclusion
– Best methods included measuring vWFCP by
ristocetin cofactor, residual collagen binding,
and immunoblotting
– Varied inter-laboratory agreement
• 3. How has this specific test (or similar
tests) been evaluated and how well has it
performed?
Furlan et al., 1998
• Von Willebrand Factor-Cleaving Protease in
Thrombotic Thrombocytopenic Purpura and
the Hemolytic -Uremic Syndrome
Furlan et al., 1998
• Methods
– Plasma from patients with a clinical diagnosis of TTP
or HUS along with a worksheet containing clinical and
laboratory data was sent for study (selection bias)
– Patients were classified as TTP, acute or in remission
and as HUS, acute or in remission by the PI without
knowledge of the vWFCP testing (based on the worksheet)
– vWFCP testing using an immunoabsorbent assay
• Results
Furlan et al., 1998
• Results Cont.Furlan
et al., 1998
Furlan et al., 1998
• Conclusion
– Nearly absent levels of vWFCP is a sensitive
test for acute TTP (low false negative)
– Plasma from patients with non-familial TTP
tends to have a vWFCP inhibitor while plasma
from those with familial TTP does not
Furlan et al., 1998
• Problems
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Test bias
Selection bias
No “normal” patients included
No referred patients were excluded
Gold standard (PI interpretation of work sheet)
- misclassification bias
Bianchi et al., 2002
• Von Willebrand factor-cleaving protease
(ADAMTS13) in thrombocytopenic
disorders: a severely deficient activity is
specific for thrombotic thrombocytopenic
purpura
Bianchi et al., 2002
• Methods
– 68 patients with thrombocytopenia (<140 K) recruited
for study with the following clinical diagnoses
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Sepsis (17)
HIT (16)
Osteomyelofibrosis (3)
Myelodysplastic syndrome (4)
ITP(10)
Acute leukemia (6)
Severe aplastic anemia (2)
Miscellaneous (10)
– ADAMTS13 activity and inhibitor measurement with
immunoblotting method
• Results
Bianchi et al., 2002
Only 18% had levels <30%
10% is lowest level
Bianchi et al., 2002
• Conclusion
– These results along with prior publications
indicate that very low levels (<5% activity) of
ADAMT13 activity is very specific for TTP
Bianchi et al., 2002
• Problems
– Test bias
– Misclassification bias
– Did not include normal patients or patients with
TTP in current study
Peyvandi et al., 2004
• Von Willebrand factor cleaving protease
(ADAMTS13) and ADAMTS13
neutralizing antibodies in 100 patients with
TTP
Peyvandi et al., 2004
• Methods
– 3 of the following present:
thrombocytopenia,
hemolytic anemia,
increased LDH, and
neurologic symptoms
– Residual collagen binding
assay used for activity and
inhibitor levels
– Low ADAMTS13 activity
was <46%
• Results
Peyvandi et al., 2004
2/15 patients with <20% ADAMTS13 activity and no inhibiting
antibodies had mutations in the ADAMTS13 gene
Peyvandi et al., 2004
• Conclusions
– ADAMTS13 activity deficiency, regardless of
the cutoff is not exclusively diagnostic for TTP
in patients with solid clinical evidence of TTP
– May be other mechanisms involved in the
inhibition of ADAMTS13 activity or other
pathways involved
Peyvandi et al., 2004
• Problems
– Referral bias
– Misclassification bias
Back to Our Patient
• Pre-plasmapheresis sample was sent to the
Blood Center of Wisconsin for vWF-CP
testing using the Gerritsen method
(approximately 4-5 day TAT)
– ADAMTS13 Activity = <4% (RR>60%)
– ADAMTS13 Inhibitor = 2 IU (RR <0.5IU)
• 1 inhibitor unit will decrease the expected enzyme
activity by 50%
Will These Test Results Help Our
Patient?
• TAT requires presumptive diagnosis be made and
treatment began prior to test results
• Supports the clinical impression of acquired TTP
• Will not be helpful in monitoring treatment or
during remission
• Patient was treated appropriately with
plasmapheresis and recovered well, all without
test results
Conclusion
• Review the pathology, epidemiology, differential
diagnosis, and treatment of Thrombotic
Thrombocytopenic Purpura (TTP) using a case
presentation
• Describe the discovery of ADAMTS13 (von
Willebrand Factor Cleaving Protease) and its role
in TTP
• Introduce selected testing methods for vWF-CP
and evaluate their use in the diagnosis and
management of TTP
Closing Thoughts
• Sensitivity and specificity of vWF-CP activity
may be more closely related to the ability of
clinicians to determine between the various causes
of thrombocytopenia, rather than being specific to
TTP
• As with many tests, the initial reports of a
“perfect” test have not been substantiated (often,
with much less fanfare)
• Testing for vWF-CP needs further development in
order to have a positive impact on the
management of thrombocytopenic patients
References
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Bianchi V. et al. Von Willebrand factor cleaving protease (ADAMTS13) in
thrombocytopenic disorders: a severely deficient activity is specific for TTP. Blood
2002; 100; 710-713.
Blood Center of Wisconsin. ADAMTS 13 Activity and Inhibitor, June 2005.
<www.bcw.edu>
Furlan M. et al. Von Willebrand factor cleaving protease in TTP and HUS. NEJM;
1998; 339; 1578-84.
Gerritsen H. et al. Assay of von Willebrand factor cleaving protease based on decreased
collagen binding affinity of degraded vWF. Thrombosis and Haemostasis1999; 82;
1386-9.
Mannucci P. TTP: A simpler diagnosis at last? Thrombosis and Haemostasis 1999; 82;
1380-1.
Peyvandi F. et al. Von Willebrand factor cleaving protease (ADAMTS13) and
ADAMTS13 neutralizing antibodies in 100 patients with TTP. British Journal of
Haematology 2004; 127; 433-439
Sadler J. A new name in thrombosis, ADAMTS13. PNAS 2002; 99; 11552-11554.
Studt j. et al. Measurement of von Willebrand factor cleaving protease (ADAMTS13)
activity in plasma: a multicenter comparison of different methods. Journal of
Thrombosis and Haemostasis 2003; 1; 1882-1887.
Tripodi A. et al. Measurement of von Willebrand factor cleaving protease
(ADAMTS13): results of an international collaborative study involving 11 methods
testing the same set of coded plasmas. Journal of Thrombosis and Haemostasis 2004; 2;
1601.