In 1982, Moake discovered that patients with relapsing acquired or congenital TTP had unusually large multimers of von Willebrand Factor (VWF) circulating in their plasma.  He proposed that TTP patients lacked a VWF protease that normally cleaved ultra-large VWF to prevent it from causing intravascular platelet aggregation and thrombosis. In 1996, VWF-cleaving protease was identified in human plasma and the following year VWF-cleaving protease was shown to be missing from the plasma of patients with congenital TTP. Soon after, adults with acquired idiopathic TTP were reported to have severe VWF-cleaving protease deficiency caused by IgG autoantibodies that inhibit the enzyme.  VWF-cleaving protease was named ADAMTS13 because it is the 13th member of the "ADisintegrin-like And Metalloprotease with Thrombo-spondin repeats" family of metalloproteases.  ADAMTS13 regulates the size of VWF multimers by cleaving a specific peptide bond in the A2 domain.

Severe congenital ADAMTS13 deficiency (Upshaw-Schulman syndrome) is an autosomal recessive condition which may present in children or adults as episodes of TTP. More than 70 mutations within the ADAMTS13 gene have been reported in families with congenital TTP. These patients have undetectable ADAMTS13 activity but do not have an inhibitor to the protease. They can usually be managed with plasma infusion alone since there is not an autoantibody to remove. The severity of the patient’s disease determines the frequency of plasma infusions. For patients with more severe frequent episodes, they can be managed with plasma infusions every 3 weeks since the ADAMTS13 half-life is 2 to 3 days. Other patients will only need plasma infusion when they are challenged such as with infection, surgery, or pregnancy.

In acquired TTP, autoantibodies inhibit ADAMTS13 function by binding to functional regions of ADAMTS13 and inhibiting enzyme activity (neutralizing inhibitor) or by causing accelerated ADAMTS13 clearance (non-neutralizing inhibitor­). Approximately 66 to 75% of patients with idiopathic TTP have severe ADAMTS13 deficiency at the time of diagnosis. Severe ADAMTS13 deficiency is defined in the literature as <10% of normal activity and has 90% sensitivity and 90% specificity for diagnosis of TTP.  Severe deficiency predicts good response to therapeutic plasma exchange. Approximately 80-90% of severely deficient patients respond to first-line therapies. Although severe ADAMTS13 deficiency predicts good response to therapeutic plasma exchange, it also indicates a higher risk of relapse. Approximately 30% of patients with severe deficiency relapse compared to 9% of patients without severe deficiency.

Patients with TTP secondary to bone marrow transplantation, HIV, pregnancy or malignancy almost never have severe ADAMTS13 deficiency. In addition, ADAMTS13 deficiency rarely if ever occurs in hemolytic uremic syndrome caused by Shiga toxin–producing Escherichia coli. ADAMTS13 testing is not particularly useful for patients with secondary TTP because this subset can be identified without it.  Patients with secondary TTP do not respond to therapeutic plasma exchange.

Clinicians determine the duration of therapeutic plasma exchange based on platelet count recovery resolution of hemolysis, improvement in clinical symptoms, rather than recovery of ADAMTS13 activity. Measurement of ADAMTS13 activity after plasma exchange is not routinely used to assess recovery, but might help to identify patients with a high risk of imminent relapse. Relapses occur in 60% of patients with persistent severe ADAMTS13 deficiency compared with only 19% of patients without deficiency.

The most common assay for ADAMTS13 deficiency is based on enzyme activity. Assays based on activity can detect deficiency due to both neutralizing and non-neutralizing autoantibodies. The most widely used activity assays are fluorescent resonance energy transfer (FRET) methods utilizing a VWF peptide (VWF73) containing a fluorescent moiety and a fluorescent quencher that flank the ADAMTS13 cleavage site. Cleavage of the VWF peptide by ADAMTS13 results in fluorescent emission that is directly proportional to ADAMTS activity. Some FRET methods may underestimate activity in specimens with high bilirubin concentrations. 

Autoantibodies to ADAMTS13 are present in approximately two-thirds of acquired TTP cases. The presence of detectable antibody at diagnosis correlates with a higher risk of relapsing disease. High-titer antibodies also have been associated with a delayed response to plasma exchange, refractory disease, and early death.

Most cases of acquired TTP with severe ADAMTS13 deficiency have neutralizing antibodies that inhibit ADAMTS13 enzyme activity. Bethesda assays are used to detect antibodies that neutralize ADAMTS13 function. Residual ADAMTS13 activity is measured after incubating patient plasma with an equal volume of normal pooled plasma. Recovery of lower ADAMTS13 activity in the mixture than expected indicates the presence of autoantibody. The amount of inhibitor that decreases residual activity to 50% of expected is defined as 1 Bethesda unit (BU).

From 10 to 15% of patients with TTP and severe ADAMTS13 deficiency have non-neutralizing inhibitors, which are not detected by Bethesda assays. These inhibitors can be detected by enzyme immunoassays using a solid phase coated with recombinant ADAMTS13 protein. Some patients may have both neutralizing and non-neutralizing inhibitors.

Elevated levels of plasma free hemoglobin above 2 g/dL and bilirubin above 15 mg/dL can reduce ADAMTS13 activity. Conversely, recent plasma exchange or transfusion can normalize ADAMTS13 activity and mask the diagnosis of TTP.  ADAMTS13 autoantibodies can be detected in other immune disorders including systemic lupus erythematosus, antiphospholipid syndrome, and hypergammaglobulinemia.

Investigators at the University of Utah and ARUP Laboratories have developed a clinical prediction score based on routine laboratory test results to predict the likelihood of severe ADAMTS13 deficiency and response to therapeutic plasma exchange. The point based prediction score is calculated by assigning points for the following laboratory values:

 Laboratory test Points
Creatinine >2 mg/dL -11.5
Platelets > 35 K/mcL -30
D-dimer >4 mcg/mL -10
Reticulocytes >3% +21
Indirect bilirubin >1.5 mg/dL +20.5

The points are added together to estimate the probability of severe ADAMTS13 deficiency as seen in the following table.

Total score Probability of severe ADAMTS13 deficiency
<20 0%
20-30 40%
>30 100%

Both ADAMTS13 activity and autoantibody should be ordered together. Specimen requirement is two citrated (blue top) tubes of blood. Specimen should be collected prior to plasma transfusion or therapeutic plasma exchange. Specimen should not be collected in lavender top tubes containing EDTA since ADAMTS13 is a metalloproteinase that requires metal ions for its function. Serum is unacceptable because ADAMTS13 is degraded by thrombin during clotting. Plasma that cannot be tested within 4 hours of collection should be stored frozen.


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