Plasma ADAMTS13 Activity Research Articles

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw–Schulman syndrome)

ADAMTS13, the specific von Willebrand factor (VWF)-cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw-Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations. Six probands with congenital TTP and their family were tested for ADAMTS13 activity in plasma using a two-site immunoradiometric assay and for ADAMTS13 gene mutations using polymerase chain reaction and sequencing. ADAMTS13 activity was severely deficient (< 5%) in the six probands and one mildly symptomatic sibling but normal (> 50%) in all the parents and the asymptomatic siblings. Ten novel candidate ADAMTS13 mutations were identified in all families, showing either a compound heterozygous or a homozygous status in all probands plus the previous sibling and a heterozygous status in the parents. The mutations were spread all over the gene, involving the metalloprotease domain (I79M, S203P, R268P), the disintegrin domain (29 bp deletion in intron/exon 8), the cystein-rich domain (acceptor splice exon 12, R507Q), the spacer domain (A596V), the 3rd TSP1 repeat (C758R), the 5th TSP1 repeat (C908S) and the 8th TSP1 repeat (R1096stop). This study emphasizes the role of ADAMTS13 mutations in the pathogenesis of congenital TTP and suggests that several structural domains of this metalloprotease are involved in both its biogenesis and its substrate recognition process.

Assays of ADAMTS-13 activity

Various assays for determination of ADAMTS-13 activity in plasma have been developed, all comprising two steps. The first step consists of proteolyzing a substrate by ADAMTS-13. Substrates were either exogenous von Willebrand factor (VWF) (purified from human plasma concentrates or recombinant VWF [rVWF]), purified VWF fragments, or endogenous VWF (from the tested plasma sample). All assays required a step in which substrate unfolding is performed using either urea or guanidine. Test plasmas were used at various dilutions and ADAMTS-13 was activated in most cases by divalent cations. The second step consists of quantifying the digestion products or the residual VWF remaining after proteolysis. Cleavage of VWF was thus estimated using electrophoresis (generation of proteolytic fragments or decrease of the size of multimers), functional methods (decrease of the collagen binding activity or of ristocetin cofactor activity), or immunological methods (enzyme-linked immunosorbent assay [ELISA] or immunoradiometric assay (IRMA) using selected monoclonal antibodies to VWF). Since 1998, all of these assays have been used to demonstrate the relevance of a deficient ADAMTS-13 activity in thrombotic thrombocytopenic purpura (TTP). However, improvements are required, as these methods remain cumbersome, time-consuming, and too remote from physiology to be routinely helpful for rapid laboratory diagnosis.

Von Willebrand factor-cleaving protease (ADAMTS-13) activity determination in the diagnosis of thrombotic microangiopathies: the Swiss experience

Severe deficiency of von Willebrand factor (VWF)-cleaving protease (ADAMTS-13) activity (<5% of normal) is a specific finding for acute idiopathic thrombotic thrombocytopenic purpura (TTP), a disorder that presents as thrombocytopenia, microangiopathic hemolytic anemia, and often organ dysfunction such as neurological disturbances or renal failure, and fever. Between January 2001 and July 2003, ADAMTS-13 activity was determined in plasma samples of 396 consecutive patients referred to our laboratory for diagnostic purposes. Plasma samples with ADAMTS-13 activity less than 5% were in addition tested for the presence of inhibitory antibodies. Patients were assigned to 10 predefined clinical categories according to information provided by the referring clinician: thrombotic microangiopathy (TMA) not further specified; neoplasia- or chemotherapy-associated TMA; TMA following hematopoietic stem cell transplantation; TMA with additional/alternative disorder; idiopathic TTP; hemolytic-uremic syndrome (HUS) not specified; HUS with diarrhea prodrome (D+HUS); atypical HUS; other hematological disorder; and no clinical information available. Severe ADAMTS-13 deficiency was found in 69 (17%) patients, including 42 with acquired idiopathic TTP, either at initial presentation or at relapse, 14 with confirmed or suspected hereditary TTP, 10 with TMA not further specified, two with neoplasia- or chemotherapy-associated TMA, and one in continued clinical remission 3.4 years after splenectomy for plasma-refractory TTP. Forty-three (62%) patients with ADAMTS-13 activity less than 5% displayed inhibitory antibodies. Severe ADAMTS-13 deficiency was found in 60% of patients diagnosed with acute idiopathic TTP, but in none of 130 patients diagnosed with HUS or in any of the 14 patients with hematopoietic stem cell transplantation-associated TMA. Thus, plasma ADAMTS-13 activity less than 5% does not identify all patients clinically diagnosed with TTP, and severe ADAMTS-13 deficiency is not invariably associated with clinical manifestations of microvascular platelet clumping.

Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods

A severely deficient ADAMTS-13 activity (<5%) is a key laboratory finding confirming the diagnosis of thrombotic thrombocytopenic purpura (TTP), whereas a mildly or moderately decreased activity is found in various other conditions. Laboratory tests for ADAMTS-13 activity must reliably identify a severe deficiency and detect inhibitory antibodies against ADAMTS-13. We carried out a multicenter comparison of different assays for ADAMTS-13 activity in plasma, including the quantitative immunoblotting of degraded von Willebrand factor (VWF) substrate, the residual collagen binding activity and ristocetin cofactor activity of degraded VWF, and an immunoradiometric assay. The main goal was to investigate whether all assays concordantly identified severe ADAMTS-13 deficiency and detected inhibitory antibodies. ADAMTS-13 activity was determined by five laboratories in 30 plasma samples of patients with hereditary and acquired TTP and other conditions. ADAMTS-13 activity values of the samples ranged from <3% to > 100%. Concerning the identification of a severe ADAMTS-13 deficiency, good interassay and interlaboratory agreement was observed with only one false-negative and two false-positive results by two laboratories using a collagen binding assay. For samples with normal or mildly to moderately reduced ADAMTS-13 activity, results were less concordant. There was good agreement for the detection of strong inhibitors. We conclude that all assays investigated are useful as a screening test in suspected TTP. Further assay improvement is needed, however.

Von Willebrand factor-cleaving protease (ADAMTS-13) activity in thrombotic microangiopathies: diagnostic experience 2001/2002 of a single research laboratory.

Severe deficiency of von Willebrand factor-cleaving protease (ADAMTS-13) activity (<5% of normal) is specific for classical thrombotic thrombocytopenic purpura (TTP), a disorder presenting with thrombocytopenia, microangiopathic haemolytic anaemia and often with organ dysfunction such as neurological symptoms, renal failure, and fever. A certain, though according to several case series, variable percentage of patients with clinically diagnosed TTP and most patients with other forms of thrombotic icroangiopathies (TMA) do not show severe ADAMTS-13 deficiency. We determined ADAMTS-13 activity in 508 plasma samples of 309 patients referred to our laboratory in 2001 and 2002. Plasma samples with ADAMTS-13 activity <5% were additionally tested for the presence of inhibitory antibodies. Patients were assigned to ten predefined clinical categories according to information provided in the referral letter (TMA not specified; TMA associated with neoplasia or chemotherapy; TMA following haematopoietic stem cell transplantation; TMA with additional disorder; idiopathic TTP; haemolytic-uraemic syndrome (HUS) not specified; HUS with diarrhoea prodrome; atypical HUS; other haematological disorder; no clinical information available). We detected 50 (16%) patients with severe ADAMTS-13 deficiency. Forty-four (88%) of these patients had been classified as idiopathic TTP, 2 as neoplasia- or chemotherapy-associated, and 4 as non-specified TMA. Among the patients labelled as acute idiopathic TTP, the prevalence of severe ADAMTS-13 deficiency was 63% (44/70). Inhibitory antibodies were found in 31 (62%) patients with ADAMTS-13 activity <5%. Of the 44 patients with acute idiopathic TTP, at initial presentation or at relapse, with ADAMTS-13 activity <5%, 11 were identified to have (probable) constitutional severe ADAMTS-13 deficiency. Severe ADAMTS-13 deficiency is found in about 60% of patients diagnosed with idiopathic TTP but in none of 111 diagnosed with HUS. Plasma ADAMTS-13 activity <5%, however, does not identify all patients clinically diagnosed with TTP. Detection of inhibitory antibodies against ADAMTS-13 helps to differentiate between acquired and constitutional forms of TTP, which may be important for treatment strategies.

Von Willebrand factor-cleaving protease (ADAMTS-13) activity in thrombotic microangiopathies: diagnostic experience 2001/2002 of a single research laboratory.

Severe deficiency of von Willebrand factor-cleaving protease (ADAMTS-13) activity (<5% of normal) is specific for classical thrombotic thrombocytopenic purpura (TTP), a disorder presenting with thrombocytopenia, microangiopathic haemolytic anaemia and often with organ dysfunction such as neurological symptoms, renal failure, and fever. A certain, though according to several case series, variable percentage of patients with clinically diagnosed TTP and most patients with other forms of thrombotic icroangiopathies (TMA) do not show severe ADAMTS-13 deficiency. We determined ADAMTS-13 activity in 508 plasma samples of 309 patients referred to our laboratory in 2001 and 2002. Plasma samples with ADAMTS-13 activity <5% were additionally tested for the presence of inhibitory antibodies. Patients were assigned to ten predefined clinical categories according to information provided in the referral letter (TMA not specified; TMA associated with neoplasia or chemotherapy; TMA following haematopoietic stem cell transplantation; TMA with additional disorder; idiopathic TTP; haemolytic-uraemic syndrome (HUS) not specified; HUS with diarrhoea prodrome; atypical HUS; other haematological disorder; no clinical information available). We detected 50 (16%) patients with severe ADAMTS-13 deficiency. Forty-four (88%) of these patients had been classified as idiopathic TTP, 2 as neoplasia- or chemotherapy-associated, and 4 as non-specified TMA. Among the patients labelled as acute idiopathic TTP, the prevalence of severe ADAMTS-13 deficiency was 63% (44/70). Inhibitory antibodies were found in 31 (62%) patients with ADAMTS-13 activity <5%. Of the 44 patients with acute idiopathic TTP, at initial presentation or at relapse, with ADAMTS-13 activity <5%, 11 were identified to have (probable) constitutional severe ADAMTS-13 deficiency. Severe ADAMTS-13 deficiency is found in about 60% of patients diagnosed with idiopathic TTP but in none of 111 diagnosed with HUS. Plasma ADAMTS-13 activity <5%, however, does not identify all patients clinically diagnosed with TTP. Detection of inhibitory antibodies against ADAMTS-13 helps to differentiate between acquired and constitutional forms of TTP, which may be important for treatment strategies.

Is ADAMTS-13 deficiency specific for thrombotic thrombocytopenic purpura? No

Is ADAMTS-13 deficiency specific for thrombotic thrombocytopenic purpura? No

Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes

Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes

Evaluation of ADAMTS-13 activity in plasma using recombinant von Willebrand Factor A2 domain polypeptide as substrate.

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF), and is absent or severely reduced in the plasma of patients with thrombotic thrombocytopenia purpura (TTP). Under physiologic flowing conditions, the enzyme cleaves endothelial cell-derived ultra-large VWF multimers at the Y842/M843 peptide bond located in the A2 domain, where many mutations associated with Type 2A VWD cluster. These VWF mutants are more susceptible for cleavage activity, decreasing the large VWF multimers in the plasma. The susceptibility of a recombinant VWF-A2 domain to ADAMTS-13 and the potential application in detecting enzyme activity were investigated. In vitro, fluid phase cleavage of VWF by ADAMTS-13 requires denaturing conditions and prolonged incubation in order to estimate enzyme activity. We have measured ADAMTS-13 activity based on enzyme cleavage of a recombinant VWF-A2 domain under non-denaturing conditions. In our assay, enzyme activity was absent in plasma from congenital and acquired TTP patient, and blocked by each EDTA, monoclonal antibody VP-1 (peptide-specific antibody against residues 828-842 of VWF), and an ADAMTS-13 antibody purified from plasma of an acquired TTP patient. This novel recombinant VWF-A2 protein has potential utility as matrix for a rapid clinical measurement of plasma ADAMTS-13 activity.