ADAMTS13 [the 13th member of ADintegrin-like And Metalloprotease with ThromboSpondin type 13 motifs] is a metalloprotease which limits platelet aggregation and microthrombi formation in the microcirculation by cleaving Von Willebrand Factor [VWF] between Tyrosine 1605-Methionine 1606 [Tyr1605-Met1606] to generate a series of small molecular weight multimers.
A deficiency of ADAMTS13 either because of an inherited mutation within the ADAMTS13 gene or the development of an autoantibody leads to the potentially lethal syndrome of Thrombotic Thrombocytopenic Purpura [TTP]. TTP is characterised by a pentad of symptoms:
• Microangiopathic haemolytic anaemia [MAHA]
• Renal impairment
• Neurological problems
The gene for ADAMTS13 maps to the long arm of chromosome 9 at 9q34, spans 34kb of DNA and contains 29 exons. ADAMTS13 is synthesised primarily in the liver but there is some evidence that it can also be synthesised by endothelial cells. Cleavage of VWF by ADAMTS13 does not normally occur because of inaccessibility of the Tyr1605-Met1606 peptide bond, which lies buried within the core β-sheet of the VWF. Shear force, various denaturing reagents or the binding of platelets to VWF exposes the ADAMTS13 cleavage site allowing cleavage to take place. The VWF multimer pattern observed in plasma is due to the continued synthesis of VWF and its cleavage by ADAMTS13.
A large number of mutations within the ADAMTS13 gene have been reported in families with congenital TTP. In addition there are a number of polymorphisms within the ADAMTS13 gene that may potentially affect its secretion and therefore plasma levels.
Principles & Methods
A number of different methods and approaches have evolved to measure ADAMTS13 activity and to detect the present of any inhibitors.
|ADAMTS13 Antigen||Several immunoassays for ADAMTS13 antigen exist and but they vary in their ability to detect full-length, mutant and truncated forms of ADAMTS13.
ADAMTS13 antigen may be normal in TTP due to the presence of immune complexes or reduced due to accelerated clearance.
Assays of ADAMTS13 antigen are generally unhelpful in the absence of a functional ADAMTS13 assay.
|Direct ADAMTS13 Activity Assays||Direct assays involve the detection of cleavage of products either of a full-length VWF molecule or a VWF fragment that encompasses the ADAMTS13 cleavage site.
1. SDS Agarose Gel electrophoresis and Western Blotting. In this assay purified VWF is incubated with plasma for 24 hours. Cleavage of the VWF by ADAMTS13 takes place leading to a reduction in multimer sizes. This reduction is visualised by agarose gel electrophoresis followed by Western blotting with a peroxidase-conjugated anti-VWF antibody. The concentration of ADAMTS13 activity in the test sample can be established by reference to a series of diluted normal plasma samples.
2. SDS-PAGE and Western Blotting. This assay is similar to the above assay but involves the visualisation of dimeric VWF fragments following SDS PAGE and Western Blotting. The assay is technically easier that SDS agarose gel electrophoresis and appears a very sensitive method for measuring ADAMTS13 activity levels.
3. Fluorescence Resonance Energy Transfer [FRETS] Assays. FRETS assays require two interacting partners of which one is labelled with a donor fluorophore and the other is labelled with an acceptor fluorophore. FRETS assays for ADAMTS13 involve a chemically modified fragment of the A2 domain of VWF which spans the ADAMTS13 cleavage site. This is readily cleaved by normal plasma but not by ADAMTS13 deficient plasma.
This cleavage is blocked by EDTA and so samples for this assay must be collected into tubes that contain citrate as an anticoagulant and not EDTA.
|Indirect ADAMTS13 Activity Assays||Indirect assays involve the detection of cleavage of products either of a full-length VWF molecule or a VWF fragment that encompasses the ADAMTS13 cleavage site in the A2 domain of VWF.
1. Collagen Binding Assays. Normal plasma or purified VWF is incubated with the test plasma sample in the presence of BaCl2 and 1.5M urea which denatures the VWF. VWF is cleaved by ADAMTS13 and residual VWF is measured by its binding to collagen Type III. The bound VWF is quantitated using an ELISA assay with a conjugated anti-VWF antibody.
2. Ristocetin-Induced Aggregation. This is similar to the collagen-binding assay above but residual VWF is measured by ristocetin-induced platelet aggregation using a platelet aggregometer.
3. Functional ELISA assays. In this assay, a recombinant VWF fragment is immobilised onto an ELISA plate using an antibody to a tag on the VWF. The VWF fragment encodes the A2 domain and the ADAMTS13 cleavage site at Tyr1605-Met1606 and is tagged with S-transferase [GST]-histidine [GST-VWF73-His].
Plasma is added to the immobilised GST-VWF73-His fragment and cleavage of the immobilised fragment occurs at the ADAMTS13 cleavage site. The residual, cleaved VWF fragment is measured by using a second monoclonal antibody that recognises only the cleaved VWF fragment and NOT the interact fragment. ADAMTS13 activity is, therefore, inversely proportional to the residual substrate concentration.
This method forms the basis for the TECHNOZYM® ADAMTS-13 Activity ELISA.
|Anti-ADAMTS13 Autoantibodies||Two types of anti-ADAMTS13 antibodies have been reported in patients with acquired TTP:
1. Neutralising antibodies [~2/3 of cases] that inhibit the action of ADAMTS13. These are classically detected by performing a 1:1 mix of test:normal plasma and then measuring ADAMTS13 activity.
2. Non-neutralising antibodies [~1/3 of cases] that bind to and accelerate the clearance of ADAMTS13 from the plasma. These can be detected by Western blotting but more conveniently using an ELISA assay.
There are a number of causes of a low ADAMTS13 activity measured by the above techniques. In classical TTP due to the presence of an autoantibody that either accelerates clearance of ADAMTS13 or inhibits its activity, ADAMTS13 during the acute phase is low or undetectable. In contrast, levels of ADAMTS13 may be normal or near normal in patients with TTP secondary to bone marrow transplantation, pregnancy, HIV and malignancy.
Measurement of ADAMTS13 activity in patients with a history of classical TTP is important because low levels have been shown to be predictive of relapse. It is unclear at present (and the data is conflicting) as to whether the titre of an inhibitory antibody to TTP is significant i.e. are those individuals with a high titre anti-ADAMTS13 antibody more likely to relapse than those with a low titre.
The normal range for ADAMTS13 ranges widely depending upon the method by which the assay is performed. In general the normal range lies between 50-160% derived from healthy individuals. However, at present no international standard, quality control material or officially recognised units for ADAMTS13 exist and this compounds the problem of establishing a reference range.
What Test Next
Assays of ADAMTS13 are usually performed to establish or to exclude a diagnosis of TTP. If a low ADAMTS13 activity is found, studies should be undertaken to establish if an inhibitor is present and if not one should consider the possibility of congenital TTP [Upshaw-Schülman syndrome]. Not all cases of congenital TTP present in childhood.
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