Measurement Of ADAMTS13 Activity Research Articles

Thrombotic thrombocytopenic purpura related to severe ADAMTS13 deficiency in children

Thrombotic thrombocytopenic purpura (TTP) related to a severely deficient activity of the von Willebrand factor cleaving protease, ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats) 13, is a life-threatening event, the onset of which may occur as early as childhood. TTP is either inherited (Upshaw-Schulman syndrome) via ADAMTS13 gene mutations (neonatal onset) or acquired via anti-ADAMTS13 autoantibodies (childhood onset). TTP is due to platelet- and von-Willebrand-factor-rich thrombi of the microvasculature, inducing mechanical hemolytic anemia, consumption thrombocytopenia, and multivisceral ischemia. Clinical course consists of relapsing acute events triggered mostly by infections, associated icterus and hyperbilirubinemia, severe hemolytic anemia with schistocytosis and a negative Coombs test, severe thrombocytopenia, and sometimes symptoms related to visceral ischemia (renal failure, central nervous system vascular events, other organ failure). The recently available ADAMTS13 laboratory investigation combining measurement of ADAMTS13 activity in plasma, search for an ADAMTS13 circulating inhibitor, and anti-ADAMTS13 IgG and ADAMTS13 gene sequencing is a crucial addition to TTP diagnosis. Plasma exchanges are first-line treatment of acquired TTP, combined with steroids and immunosuppressive drugs. Curative treatment of acute events in Upshaw-Schulman syndrome relies on plasma infusions (provider of active ADAMTS13). Guidelines for preventive treatment of relapses are not clearly established but should associate plasmatherapy and caution to triggers of relapses. Therapeutic perspectives are focused on the development of concentrated plasma-derived ADAMTS13 or recombinant ADAMTS13.

Levels of von Willebrand factor antigen and von Willebrand factor cleaving protease (ADAMTS13) activity predict clinical events in chronic heart failure

Decreased activity of ADAMTS13, the von Willebrand factor (VWF) cleaving protease, was recently reported in cardiovascular diseases and in hepatic failure. Chronic heart failure (CHF) is characterised by abnormalities of left ventricular function accompanied by the failure of the liver and dysregulation of endothelial activation. Therefore, the aim of our study was to measure ADAMTS13 activity in CHF, and determine the prognostic value of VWF and ADAMTS13 on major clinical events in CHF. ADAMTS13 activity (measured by FRETS-VWF73 substrate) was decreased in CHF (n = 152, left ventricular ejection fraction <45%), and it correlated negatively with B-type natriuretic peptide (BNP) NYHA (New York Heart Association) classes, markers of synthetic capacity of the liver and endothelial dysfunction (all p < 0.005). Both, high VWF:Ag levels (hazard ratio [HR] 1.52, 95% confidence interval [CI] 1.189-1.943), and low ADAMTS13/VWF:Ag ratios (HR 0.70, 95% CI 0.58-0.84) independently and significantly predicted short-term (1 year follow-up) clinical adverse events in heart failure (HF). Decreased activity of ADAMTS13 with concomitant high VWF:Ag levels is a significant independent predictor of clinical events in CHF. The levels of the two molecules may integrate the impaired synthetic capacity of the liver and the disturbed endothelial regulation and can therefore be a useful tool to predict clinical events in CHF.

Lower ADAMTS13 Activity and Higher Bethesda Units of Antibody Inhibitor in Early Remission Are Associated with a Higher Probability of TTP Exacerbation.

An exacerbation of TTP is defined as a recurrence of TTP requiring therapy within 30 days of the last plasma exchange (PE). In contrast, the term relapse of TTP refers to a de novo event of TTP that by definition occurrs later than 30 days after the last PE. Many researchers believe that an exacerbation probably represents the continuation of disease from an unresolved episode of TTP, thus making it a distinct clinical scenario worthy of investigation. 20–40% of patients with idiopathic TTP experience an exacerbation, resulting in the potential for considerable mortality and morbidity. Biomarkers that can predict a patient's risk of exacerbation may help clinicians make correct decisions regarding the discontinuation of PE, immunotherapy, and the need to maintain or remove the central venous catheters required for PE. In this study, we used samples from our cohort of 36 patients with idiopathic TTP who were followed through their disease onset, treatment, remission, and recurrences. First, we selected the pretreatment samples from a total of 61 episodes of TTP in the 36 patients, including both the initial and subsequent recurrences. In eight of these 61 events, TTP exacerbation occurred after initial response to PE treatment. Second, we selected the early remission samples (the first week of remission after the discontinuation of PE therapy) from a total of 59 events (including both initial onset and recurrences). In nine of these 59 events, TTP exacerbation occurred after the initial response to PE treatment. Next, we measured ADAMTS13 activity and Bethesda Units (BU) of ADAMTS13 inhibitors in both pretreatment and early remission samples. Finally, we used logistic regression analysis to evaluate the performance of these biomarkers with respect to the prediction of exacerbations of TTP. For pre-treatment samples, neither ADAMTS13 activity nor BU predicted the risk of exacerbation when comparing the exacerbation group (N=8) to the non-exacerbation group (N=53). In the early remission samples however, both ADAMTS13 activity and BU of antibody inhibitor demonstrated statistically significant differences between the exacerbation (N=9) and non-exacerbation (N=50) groups. The results are not influenced by the covariates of age, gender, and ethnicity. In conclusion, severely deficient ADAMTS13 activity and elevated BU of ADAMTS13 inhibitors in early remission are highly associated with the probability of TTP exacerbation, providing the rationale for clinicians to either perform additional PE, add additional therapies, or delay the decision to remove the central venous catheter and monitor patients more closely for the need to re-initiate PE.Logistic Regression Analysis of ADAMTS13 Activity and BU of ADAMTS13 inhibitor in Prediction of TTP Exacerbation. Mean and p values are given in the table.Comparison groupsSamplesBiomarkersExacerbatonNon- Exacerbationp valuesPre-tretmentADAMTS13 activity%1.8%3.5%0.3191%BU58.70.5418Early remissionADAMTS13 activity%3.5%32.7%0.0086BU22.62.40.0083

The Effect of Plasma Exchange on Rituximab Levels and Response to Rituximab in Patients with Acute Idiopathic Thrombotic Thrombocytopenic Pupura (TTP).

Most cases of acute idiopathic TTP have ADAMTS13 activity <5% and detectable anti-ADAMTS13 IgG. Rituximab, a monoclonal anti-CD20 antibody, is known to be an effective adjunct to eradicate the antibody. The initial doses of Rituximab are often given in the period that the patient is receiving plasma exchange (PEX). In order to ascertain whether Rituximab is removed by PEX and whether this affects response to treatment, we serially measured ADAMTS13 activity, anti-ADAMTS13 IgG level and serum Rituximab concentrations in patients who were treated with Rituximab within 3 days of admission for acute idiopathic TTP. Rituximab levels were measured before and after PEX, after each dose and then in remission. In addition we measured the above parameters in two patients who received Rituximab electively (not concurrently with plasma exchange). The ADAMTS13 activity (normal value >66%) was measured using a collagen binding assay while the anti-ADAMTS13 IgG (normal range <4%) and Rituximab levels (lower limit of detection 10mcg/ml) were measured using ELISA techniques.16 patients (11 female, 5 male; median age 39y (range 13y – 75y)) with acute idiopathic TTP were treated with Rituximab. All patients had PEX from admission to remission, received Rituximab 375mg/m2 once weekly for 4 weeks and pulsed methylprednisolone 1g daily for three days. At presentation, all patients had ADAMTS13 activity <5% and anti-ADAMTS13 IgG (median 39.5%; range 12–85%). The median follow up was 6 months (m) (range 1–12 m). 14 patients received 4 doses of Rituximab, 2 received 6 doses. One patient died of TTP related complications before the third dose. The median number of PEX after the first dose of Rituximab was 15 (range 5–21).The median serum Rituximab concentration following each dose was: 1st dose 154 mcg/ml (range 108–467mcg/ml); 2nd dose 175 mcg/ml (range 116–436mcg/ml); 3rd dose 187.5 mcg/ml (157–301mcg/ml); 4th dose 216 mcg/ml (155–243mcg/ml); 5th 155 mcg/ml; 6th 211 mcg/ml. All PEX patients had Rituximab levels <10 mcg/ml immediately prior to the 2nd dose. Of those still being exchanged, 5/7 had levels <10 mcg/ml prior to 3rd dose and 5/6 patients had levels <10mcg/ml prior to the 4th dose. In contrast all the patients who had finished PEX had detectable Rituximab prior to their next dose. The median reduction in Rituximab per PEX expressed as a percentage of the pre-PEX level was 65 % (range 45–72%). The reduction was similar for 1.0 volume (65%) and 1.5 volume exchanges (70%). Rituximab was detected in the PEX fluid. Following completion of Rituximab therapy, the median concentration was 32mcg/ml (range 11–55mcg/ml) at 1m, 20 mcg/ml (range <10 – 42mcg/ml) at 2m and 16 mcg/ml (range <10– 20mcg/ml) at 3m. Seven patients reached 4 month follow up and had levels <10mcg/ml. Patients who had fewer than 10 PEX had higher median Rituximab levels (20mcg/ml) at 3m than those who had more than 10 PEX (Rituximab <10mcg/ml). There was no significant difference in ADAMTS13 activity or anti-ADAMTS IgG levels between these groups at 3m (patients receiving <10 PEX: mean activity 38% and IgG 6%; Patients receiving >10 PEX: mean activity 36% and IgG 15%). In 2 patients receiving elective Rituximab, the median concentrations before and after each dose were: 235mcg/ml after the 1st dose, 95mcg/ml before and 233mcg/ml after 2nd dose, 119mcg/ml before and 289mcg/ml after the 3rd dose, 167mcg/ml before and 373mcg/ml after 4th dose. Follow up is too short to correlate levels and ADAMTS13 activity/anti-ADAMTS13 IgG levels. There have been no relapses in any of the patients.In conclusion, Rituximab is removed by plasma exchange. The peak doses achieved are higher in those not receiving concurrent PEX. Higher Rituximab levels are associated with fewer PEX. Rituximab was still detectable at a low level in a third of patients 3 months after treatment but not at 4 months. Measurement of Rituximab levels will help to modify Rituximab doses and modification of the dosing schedule using higher initial doses or more frequent dosing may achieve higher levels earlier in treatment and a shorter time to remission. Longer term follow will also help to determine whether the peak Rituximab concentration achieved correlates with duration of remission.

ADAMTS13 is a Risk Factor for MI, Stroke and Vascular Death in the Oxford Vascular Study.

Normal haemostasis requires a balance between ultra-high MW VWF synthesis/release and its degradation by ADAMTS13. Although a severe acquired/inherited deficiency of ADAMTS13 occurs in TTP, as VWF is an established risk factor for arterial thrombotic disease it has been recently hypothesized that intermediate to low levels of ADAMTS13 could also be an important risk indicator. A new rapid ADAMTS13 Technozym® ELISA activity assay (Technoclone, Vienna, Austria) was therefore used to measure ADAMTS13 activity within plasma samples from a cohort of 521 patients within the Oxford Vascular Study (OXVASC). OXVASC is a large population based study of all patients with acute vascular events (TIA, stroke, acute coronary syndromes and acute peripheral vascular events) and includes a substudy of various risk factors (including platelet function, aspirin responsiveness and classical coagulation markers) for thrombosis. The ADAMTS13 normal range was 68.96 – 144.36% (n = 20, mean = 106.7%, 95% CI = 95.8–115.5%). Within 521 OXVASC samples, the mean ADAMTS13 level was found to be significantly lower (p < 0.0001, Mann-Whitney test) at 72.34% (95% CI = 70.33–74.35%). The minimum and maximum values obtained were 29.17% and 201.86% respectively. Although plasma VWF levels were significantly elevated, they were not inversely proportional to ADAMTS13 activity (r = 0.093). Furthermore low ADAMTS13 levels were not significantly associated with aspirin non-responsiveness (p = 0.0759) as defined by the Collagen/Epinephrine cartridge on the PFA-100, although VWF levels were significantly higher in non-responsive patients (p = <0.0001). Increased VWF levels were predictive of the combined outcome of stroke, MI or vascular death (HR per decile increase 1.12, 95% CI 1.06–1.19, p = 0.0002), whereas lower ADAMTS13 levels were associated with increased risk (HR per decile decrease 0.91 95% CI, 0.85–0.97, p = 0.004). In conclusion, low ADAMTS13 activity was not associated with high VWF levels and there was no association with aspirin non-responsiveness in a high shear dependent assay (PFA-100). However, low levels of ADAMTS13 were predictive of recurrent major vascular events. ADAMTS13 may therefore have some potential as a risk factor in patients with arterial disease.

Thrombotic microangiopathy in a 17-year-old patient: TTP, HUS or a bit of both?

Thrombotic microangiopathies are characterized by the development of hyaline thrombi in small vessels resulting in thrombocytopenia, microangiopathic hemolysis, and organ dysfunction. Thrombotic-thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are two major clinical syndromes of thrombotic microangiopathies. Although differential diagnosis between TTP and HUS is commonly determined in the clinical setting, recent evidence suggests major pathophysiological differences between the two diseases. Autoimmune inhibitors or genetic mutations of a von Willebrand factor (VWF) cleaving metalloprotease (ADAMTS13) leads to the accumulation of unusually large multimeric forms of VWF in TTP, facilitating adherence of platelets and development of microthrombi. In contrast, classic HUS is caused by infection with verocytotoxin-producing bacteria. This toxin induces endothelial injury, apoptosis and inflammation. Endothelial injury results in increased shear-stress, fostering cleavage of VWF, but thrombosis eventually develops. One would assume that measurement of ADAMTS13 activity and/or detection of verocytotoxin could easily contribute to the differential diagnosis of TTP or HUS. We report on a case of a young patient with thrombotic microangiopathy and renal involvement with low ADAMTS13 concentrations which did not respond well to plasmapheresis therapy, but subsequent to the detection of verocytoxin producing E. coli with the serotype O157:H7, reacted well to antibiotic treatment. Sequencing of the ADAMTS13 gene revealed no mutations and no anti-ADAMTS13 antibodies could be detected. This case shows overlapping presentations as well as etiologies for both TTP and HUS, a finding also underscored by a recent animal model in which verocytoxin triggered development of TTP in ADAMTS13-deficient mice.

Low ADAMTS13 Activity in Clinical Remission Predicts the Risk of TTP Relapses.

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a devastating disorder characterized by frequent recurrences. A predictive biomarker that allows for the early identification and intervention prior to the overt relapse of disease is needed. In the present study, the assay for ADAMTS13 activity was modified to adequately detect ADAMTS13 activity as low as 0.5%. Serial samples (n=157) collected from 24 TTP patients during periods of sustained clinical remission were then studied. These patients were followed up quarterly for an average of 23 months, during which time 9 episodes of TTP relapses from 6 patients occurred. We measured ADAMTS13 activity in these samples and then employed logistic regression modeling to evaluate the relationship between ADAMTS13 activity levels and the risk of TTP relapses. Our data demonstrated that lower ADAMTS13 activity and younger age are significantly associated with a higher probability of relapse in the 3 months following the date the specimens were taken. In contrast, ADAMTS13 antibody IgG levels are not predictive of TTP relapses. Identification of low ADAMTS13 activity during remission as a key risk factor for TTP relapses provides a new screening strategy to identify patients who may benefit from prophylactic therapy prior to disease relapses.

The ADAMTS13 Gene as the Immunological Culprit in Acute Acquired TTP - First Evidence of Genetic Out-Breeding Depression in Humans.

Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disorder due to autoantibodies against ADAMTS13. The case of identical twins suffering from acute acquired TTP with severe autoantibody-mediated ADAMTS13 deficiency (Studt JD et al., Blood 2004; 103:4195) led us to speculate on unknown genetic determinants of acquired TTP and on the immunological basis of ADAMTS13 autoimmunity. We investigated 37 patients (pts) with acute TTP with acquired severe ADAMTS13 deficiency of <10% of the normal, as well as 60 healthy controls. We measured ADAMTS13 activity, anti-ADAMTS13 IgG by ELISA and searched for a functional ADAMTS13 inhibitor. The ADAMTS13 gene was sequenced and 15 short tandem repeats (STR) at loci not linked to the ADAMTS13 gene were analyzed. Genotypic data of 25 polymorphic markers on the ADAMTS13 gene were used for haplotype inference. Inter-allelic diversity was assessed by summing up the number of individual heterozygous base positions for the polymorphic markers on the ADAMTS13 gene and the STR loci separately. Identified ADAMTS13 mutations were expressed in COS-7 cells and their effect on biosynthesis, secretion and activity was determined. We found that 4 of 37 pts with severe acquired ADAMTS13 deficiency were heterozygous carriers of an ADAMTS13 mutation. The mutation P457L, found in two pts, is a known mutation reported in hereditary TTP. R1096H and A1145T are new mutations. Expression studies revealed that P457L and A1145T mutants were secreted normally, but showed reduced activity of 44% and 11%, respectively. Secretion of the R1096H mutant, however, was reduced. A particular ADAMTS13 haplotype (designated H6) inferred from single nucleotide polymorphism (SNP) data was significantly more frequent in pts than in controls (p<0.0001). Moreover, individual ADAMTS13 inter-allelic diversity was significantly higher in pts than in controls (p=0.012). Primarily, non-synonymous SNPs, which encode amino acid exchanges, contributed to this phenomenon (p=0.0014). The increased inter-allelic diversity in pts was found to be restricted to and specific for the ADAMTS13 gene, as individual inter-allelic diversity at 15 neutral STR markers was similar in pts and controls. In conclusion, heterozygous ADAMTS13 missense mutations are found in a high frequency in pts with acquired TTP. The mutations P457L, R1096H and A1145T, reduce baseline ADAMTS13 activity in vitro by at least 50% and may render heterozygous carriers more susceptible for TTP. In addition, ADAMTS13 mutations, such as R1096H, may promote autoantibody production due to increased intracellular degradation and MHC presentation. The ADAMTS13 haplotype H6 and particularly the increased inter-allelic diversity represent risk factors for autoimmunity towards ADAMTS13 and acute acquired TTP. ADAMTS13 autoimmunity as a result of increased inter-allelic diversity represents the first example of genetic out-breeding depression in humans. As autoantigens in general seem to have increased numbers of SNPs, increased inter-allelic diversity may represent an important pathophysiological feature in other autoimmune disorders as well.

Investigation of von Willebrand Factor Gene Mutations in Korean von Willebrand Disease Patients

We intended to find the mutations of von Willebrand factor (VWF) gene as the most important contributing factor of von Willebrand disease (VWD) in Korean patients. In 40 known vWD patients mutations of vWF gene were sought by direct sequencing of PCR products targeting exons 18, 19, 20, 26, 28 and 52 frequently implicated as the locations of mutation. For factors other than VWF gene contributing to VWD phenotype, we tested ABO blood group and measured ADAMTS13 activity in VWD patients. Twenty-seven cases (67.5%) were type 1 vWD, 3 cases (7.5%) type 3, and 5 cases (12.5%) type 2A. Three cases were type 2A or 2B (7.5%) and 2 cases were suspected to be type 2N (5.0%). Among them six candidate missense mutations were found: V1279I, R1306W, R1308C, and V1316M were previously reported in type 2B and type 1 vWD, and C858W and T1477I were novel findings. All patients were heterozygotes. Blood group O was overly represented in VWD patients, while ADAMTS13 activity of the patients was not significantly different from that of normal control. Mutation of VWF gene detected by genetic studies can significantly improve the diagnostic accuracy, especially in subtype assignment of VWD. Two novel mutations, C858W and T1477I associated with VWD were found and expected to contribute to the elucidation of its pathophysiology.

ADAMTS13 assays and ADAMTS13-deficient mice

Thrombotic thrombocytopenic purpura can be induced by acquired or congenital deficiency of the plasma von Willebrand factor-cleaving protease, ADAMTS13. Measurement of ADAMTS13 activity is important for the diagnosis and treatment of microangiopathies including thrombotic thrombocytopenic purpura. Phenotypic analysis of mice lacking the Adamts13 gene is valuable for understanding the pathogenesis of microangiopathies. The minimum substrate for ADAMTS13 activity was identified as 73 amino acid residues in the A2 domain of von Willebrand factor, called VWF73. Several new assays have been developed using this sequence. The VWF73-based assays are rapid, quantitative, and easy to handle, and are well correlated with the measures from previous assays. Mice lacking the Adamts13 gene were produced. The mice were viable and fertile. They showed a prothrombotic state but no symptoms of spontaneous thrombocytopenia, hemolytic anemia, or microvascular thrombosis were observed. VWF73-based ADAMTS13 assays will significantly facilitate the accurate diagnosis of microangiopathies and contribute to the improved clinical treatment of these diseases. Accumulated clinical information on patients with ADAMTS13 deficiency and mice lacking the Adamts13 gene indicates that additional environmental or genetic susceptibility factors are required to trigger thrombotic thrombocytopenic purpura.

Alterations of ADAMTS-13 Activity as a Common Indicator of the Endothelial Dysfunction Developing in Different Thrombotic Microangiopathies.

Injury to the endothelial cell is a key factor for the initiation and development of thrombotic microangiopathies (TMA). Endothelial dysfunction implies alterations of the von Willebrand factor (vWF) pathway. Severe deficiency of the vWF-cleaving protease activity has been traditionally associated with thrombotic thrombocytopenic purpura (TTP). However, there is ongoing evidence indicating that ADAMTS-13 may be a causal factor in the pathogenesis of other TMA.We evaluated whether ADAMTS-13 deficiency and autoantibodies inactivating the protease were prevalent in TMA with different physiopathological origin. Patients included in the study presented TMA associated with: TTP (n=13), hemolytic uremic syndrome (HUS) (n=4), gastrointestinal angiodysplasia (GA) (n=11), or stem cell transplantation (SCT) (n=3). We measured ADAMTS-13 activity and antigen (ADAMTS-13:Ag), the presence and inhibitory activity of autoantibodies to the protease, vWF:Ag, and vWF:multimeric pattern. ADAMTS-13 activity and inhibitory activity of autoantibodies were analyzed using a modification of the immunoblotting assay described by Furlan et al (1996). ADAMTS-13:Ag and the presence of IgG were measured by ELISA kits.The vWF:multimeric pattern was resolved by electrophoresis in agarose gels.In patients with TTP, undetectable ADAMTS-13 activity was found in 10 out of 13 patients, 9 of which had IgG against the metalloprotease and reduced ADAMTS-13:Ag levels. Plasma exchange normalized the ADAMTS-13:Ag levels and decreased IgG title. Interestingly, one case of TTP with autoantibodies was associated with clopidogrel treatment. Levels of vWF:Ag in these patients varied from 50 to 180%.As for patients with HUS, in 2 out of 4 ADAMTS-13 activity was absent and ADAMTS-13:Ag was reduced, but no IgG to the metalloprotease was detected. It should be pointed out that one of the cases of HUS was subsequent to cocaine abuse. In TMA associated with SCT and GA, ADAMTS-13 activity was around 20% in all the patients studied, although levels of the metalloprotease were within the control range (700–1400ng/ml). vWF:Ag levels were found to be significantly increased (to around 200%) in the former group of patients.In summary, presence of autoantibodies to ADAMTS-13 concurred with decreased antigen levels, reinforcing the hypothesis of an accelerated clearance of the metalloprotease in vivo. Among the patients studied, we found a clopidogrel associated TTP and a cocaine caused HUS. Presence of antibodies to ADAMTS-13 associated with clopidogrel treatment would suggest an immunological mechanism, which cannot be ruled out in the case of cocaine associated HUS. Overall, our results indicate that the absence of ADAMTS-13 activity is not specific for TTP. A reduced activity of the metalloprotease may be an indicator of endothelial dysfuntion as a common feature in different TMA.

A Rapid Sensitive Assay for Measuring ADAMTS13 Activity in Plasma Utilizing a Novel Recombinant Alexa488-vWF86 (Q1599\\P1611C) FRET Substrate.

ADAMTS13 (von Willebrand Factor cleaving protease) is a zinc metalloprotease that cleaves the Tyr(1605)/Met(1606) bond within the A2 domain of von Willebrand Factor (vWF). Low levels of ADAMTS13 activity (<5%) are strongly associated with the life threatening hemostatic condition thrombotic thrombocytopenic purpura (TTP). A rapid clinical assay for ADAMTS13 is necessary to provide critical information for the early diagnosis of TTP and to guide and monitor the treatment of TTP.Present methods for measuring ADAMTS13 activity (e.g. collagen binding, vWF multimer analysis and ELISA) are cumbersome, take a long time to complete and lack sensitivity. A fluorescence resonance energy transfer (FRET) assay for ADAMTS13 has been described, which uses the costly and difficult to synthesize truncated A2 domain VWF73aa peptide as substrate (1). However, the lower limit of detection of ADAMTS13 assays using VWF73 FRET substrate is only about 5–10% of normal ADAMTS13 activity levels (1,2). Recently, Zhang et al (3) reported a novel recombinant VWF A2 FRET substrate of 86 amino acids in which cysteine residues were substituted for residues Q1599 and P1611, respectively. The cysteine residues were chemically labeled with fluorescein maleimide to produce a FRET substrate whereby the two fluorescein dyes flanking the ADAMTS13 Tyr/Met cleavage site interact sufficiently such that auto-quenching of fluorescence occurs.In this study, we developed a rapid more sensitive fluorescent assay for ADAMTS13 using a novel recombinant VWF86(Q1599C/P1611C) FRET substrate. VWF86(Q1599C/P1611C) peptide was chemically coupled via the cysteine residues to the brighter, more photostable Alexa488 fluorochrome. Assay conditions were optimized to obtain higher analytical sensitivity < 5% normal ADAMTS13 activity (0.5 ng/ml) and shorter assay times (20–30 minutes). ADAMTS13 activity in 50 normal individuals as well as subjects with TTP, thrombosis and lupus anticoagulant will be presented. The rapid FRET assay using the novel recombinant Alexa488-vWF86 (Q1599\\P1611C) substrate should prove clinically useful for quantitation of ADAMTS13 activity and the rapid diagnosis of TTP.

Comparison of FRETS-VWF73 to full-length VWF as a substrate for ADAMTS13 activity measurement in human plasma samples

Comparison of FRETS-VWF73 to full-length VWF as a substrate for ADAMTS13 activity measurement in human plasma samples -

Superoxide Inhibits ADAMTS-13 Activity and Regulates Sickle Cell Adhesion.

The major clinical manifestations of sickle cell anemia are due to a vaso-occlusive process that leads to severe pain crises, tissue infarction, and in some instances, death. The pathophysiology of vaso-occlusion is extremely complex, the proximate cause appearing to be an increase in the adhesive interaction between sickle erythrocytes and vascular endothelium. There is ample evidence that it is the highest molecular weight forms of VWF, the ultra-large multimers (ULVWF), expressed on endothelium support sickle erythrocyte adhesion most potently. We postulate that variation in the activity of the plasma metalloprotease, ADAMTS-13, which processes ULVWF to the VWF form normally found in plasma will be an important determinant of the propensity for individuals with sickle cell anemia to develop vaso-occlusive crises. It has also been postulated that increased production of reactive oxygen species (ROS) also contributes to the pathology of sickle cell disease. ROS have been implicated in diverse cellular processes, acting through oxidation of cysteine residues. ADAMTS-13 is a cysteine-rich protein; we therefore hypothesized that its function could be affected by ROS. We measured ADAMTS-13 activity in the plasmas of sickle cell disease patients, both in the “steady state” and at various times during vaso-occlusive crises. We used three assays: a static assay that measures the ability of patient plasma to reduce the size of endothelial-derived ULVWF, a static assay that assesses the ability of patient plasma to cleave a recombinant VWF A2 fragment, and an assay performed under flow that evaluates the ability of patient plasma to cleave ULVWF attached to the surfaces of histamine-stimulated human umbilical vein endothelial cells (HUVECs). Five of nine patients with sickle cell anemia had significantly lower ADAMTS-13 activity than measured in normal controls during the chronic phase of the illness, some with activity measuring below 50% of normal levels. We also evaluated activity in two patients during acute vaso-occlusive crisis. Both patients had levels on the day of hospitalization that were indistinguishable from the levels found in patients with thrombotic thrombocytopenic purpura. Recovery of activity correlated with clinical recovery, to the point that in both cases activity was normal upon hospital discharge. We then compared superoxide generation facilitated by sickle erythrocytes with that generated by normal erythrocytes and found, as others have a significantly higher generation by sickle erythrocytes. We also found that superoxide (produced using a hypoxanthine-xanthine oxidase system) significantly decreased the activity of both plasma and recombinant ADAMTS-13 in vitro. Superoxide was also able to stimulate release of ULVWF and cell-membrane expression from cultured HUVEC. These results suggest that the enhanced production of superoxide in patients with sickle cell disease plays an important role in promoting erythrocyte adhesion to the endothelium by both stimulating release of ULVWF and inhibiting its cleavage by ADAMTS-13.

Measurement of ADAMTS13 activity and inhibitors

Acquired or congenital deficiency in the plasma von Willebrand factor-cleaving protease ADAMTS13 causes life-threatening thrombotic thrombocytopenic purpura. This condition is characterized primarily by thrombocytopenia and microangiopathic hemolytic anemia, accompanied by variable degrees of neurologic dysfunction, renal failure, and fever. Measurement of ADAMTS13 activity is important in the diagnosis of microangiopathies such as thrombotic thrombocytopenic purpura. This review introduces both established and emerging assays for ADAMTS13 activity, focusing on their impact on clinical practice. Previously established assays are useful screening methods to identify suspected thrombotic thrombocytopenic purpura. Novel assays measuring ADAMTS13 activity using either recombinant peptides or synthetic substrates directly measure the activity quantitatively. These assays can also detect neutralizing autoantibodies in the plasma of patients with acquired ADAMTS13 deficiency. Although ADAMTS13 in control subjects exhibits a broad variation in activity, ranging from 30 to 200%, significant decreases in ADAMTS13 activity have been observed in several physiologic and pathologic conditions. A portion of thrombotic thrombocytopenic purpura patients, however, did not display severe ADAMTS13 deficiency, suggesting that as-yet-unidentified environmental or genetic factors may contribute to the etiology of thrombotic thrombocytopenic purpura. New assays measuring ADAMTS13 activity will contribute significantly to the accurate diagnosis of microangiopathies, ultimately leading to improved clinical treatment of these diseases. These assays may also help to clarify the role of ADAMTS13 activity in additional thrombotic disorders, including disseminated intravascular coagulation, stroke, and myocardial infarction.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

FRETS‐VWF73, a first fluorogenic substrate for ADAMTS13 assay

A plasma metalloprotease, ADAMTS13, cleaves von Willebrand factor (VWF) multimers and downregulates their activity in platelet aggregation. Functional ADAMTS13 deficiency leads to the accumulation of hyperactive large VWF multimers, inducing a life-threatening disease, thrombotic thrombocytopenic purpura (TTP). Although measuring ADAMTS13 activity is important in TTP diagnosis, existing methods require time and skill. Here, we report a fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity. We developed a synthetic 73-amino-acid peptide, FRETS-VWF73. Cleavage of this substrate between two modified residues relieves the fluorescence quenching in the intact peptide. Incubation of FRETS-VWF73 with normal human plasma quantitatively increased fluorescence over time, while ADAMTS13-deficient plasma had no effect. Quantitative analysis could be achieved within a 1-h period using a 96-well format in commercial plate readers with common filters. The FRETS-VWF73 assay will be useful for the characterization of thrombotic microangiopathies like TTP and may clarify the importance of ADAMTS13 activity as a predictive marker for various thrombotic diseases.

Relapse of Thrombotic Thrombocytopenic Purpura: Is It a Continuum of Disease?

The recurrence of thrombotic thrombocytopenic purpura (TTP) in 20 to 50% of patients who survive their initial episode, is not only a challenge to manage clinically, but brings to light our lack of understanding of the underlying pathophysiology. Recurrence may occur close to apparent hematologic remission or remotely, months later. Given that surveillance of TTP relies on surrogate measures such as hematologic recovery, normalization of serum lactate dehydrogenase (LDH) and resolution of clinical signs, it is plausible that some recurrences are actually a continuum of disease. Standardizing definitions for remission, exacerbation, and relapse are important for consistency of patient management and interpreting the medical literature. When TTP recurs, several adjuncts to therapeutic plasma exchange (TPE) have been used, including splenectomy, vincristine, and rituximab. Although it would seem intuitive that more severe disease would predict a greater likelihood of relapse, the degree of thrombocytopenia at presentation, the persistence of schistocytosis at discontinuation of TPE, the elevation of LDH, and the length of time to attain clinical remission are not reliable indicators of the clinical outcome of any individual TTP patient. Measurement of ADAMTS13 activity and its inhibitor offer new promise for prognostic and pathophysiologic significance; however, more extensive scientific investigation is needed.

The Frequency and the Significance of ADAMTS-13 Neutralising Inhibitors in 62 Patients with Non-Familial Thrombotic Thrombocytopenic Purpura.

Neutralising inhibitors against ADAMTS-13 are detected in 51–67% of patients with Thrombotic Thrombocytopenic Purpura (TTP). These ADAMTS-13 inhibitors have not been very well characterised and the diagnostic or the prognostic value of these inhibitors is not established. In the present study, we measured ADAMTS-13 activity and the corresponding inhibitor titer in 96 samples from 62 patients with TTP at various stages of their disease. All patients presented with non-familial TTP. For patients with severe ADAMTS-13 activity without detectable inhibitor heritable ADAMTS-13 deficiency was excluded by either normalisation of ADAMTS-13 activity in remission or by detecting normal ADAMTS-13 activity in first degree family members. ADAMTS-13 activity was quantified by measuring the residual ristocetin cofactor activity of the substrate. The inhibitor against ADAMTS-13 was detected by mixing patient plasma, either neat or diluted, with normal plasma. The inhibitor concentration neutralising 50% of ADAMTS-13 activity in a 1:1 dilution of patient plasma with normal plasma was defined as 1 U/ml. Inhibitors were considered non-detectable (<0.4 U/ml), if residual ADAMTS-13 activity in the mixture was higher than 75%. Samples with ADAMTS-13 activity >6.25% were heat-inactivated (30 min at 56°C) before testing for inhibitory activity. We found severe ADAMTS-13 deficiency in 89% (24/27) of the samples from patients with acute untreated TTP. 87% (21/24) of these samples were positive for inhibitory activity. The inhibitor titer ranged from 0.4 to 62 U/ml with a median of 1 U/ml. One patient with acute TTP demonstrated ADAMTS-13 activity of 34% despite an inhibitor titer of 0.6 U/ml. The sensitivity of a positive inhibitor test for the diagnosis of TTP was thus 82%. The inhibitor titer before initiation of therapy could not be correlated with the platelet count, the CRP-level or the response to PE-therapy, if patients with an index episode and patients with a relapse were analysed separately. Severe ADAMTS-13 activity was detected in 15/31 samples collected from patients during plasma exchange therapy. 93% (14/15) of these samples were positive for an inhibitor with a titer ranging between 0.6 and 47 U/ml (median: 3 U/ml). 12 of 37 patients tested in remission presented with severe ADAMTS-13 deficiency, 6 of them were positive for inhibitory activity (range: 1–52 U/ml; median: 5 U/ml). The inhibitor titer for patients, which were analysed during acute untreated TTP as well as in remission (n=5), was notable not related to the stage of disease. Five patients with positive inhibitory activity at admission demonstrated mild ADAMTS-13 deficiency in remission without detectable inhibitor. In contrast, we detected inhibitory activity of 0,6–0,8 U/ml in 3 samples from two patients with measurable ADAMTS-13 activity. These low titer inhibitors were only detectable, if samples were heat inactivated before performing the inhibitor assay. Our data demonstrate, that inhibitors against ADAMTS-13 are very heterogeneous. It is highly suspected, that some of these inhibitors can either completely or partly neutralise ADAMTS-13 function in vivo without being detectable in vitro. Inhibitors against ADAMTS-13 might, on the other side, not always completely inhibit ADAMTS-13 function, since they can occur in patients with high residual ADAMTS-13 activity. Inhibitor titers show a wide variation and the clinical significance of the inhibitor titer before, during and after therapy needs to be further investigated.

Long-Term Follow-Up of 21 Patients with Thrombotic Thrombocytopenic Purpura (TTP) and Severe ADAMTS13 Deficiency: Demonstration of Persistent ADAMTS13 Deficiency and Neurocognitive Abnormalities.

Many patients who have recovered from TTP report cognitive abnormalities, often described as difficulty with attention, comprehension, and memory. These abnormalities have never been objectively documented. Also there have been reports of patients with continued abnormal ADAMTS13 activity following complete hematologic recovery; the frequency, duration, and clinical importance of persistent ADAMTS13 deficiency has not been described in a cohort of consecutive patients with long-term follow-up. To evaluate these unresolved issues, we measured neurocognitive function, ADAMTS13 activity, platelet count, and hematocrit (hct) in patients from the Oklahoma TTP-HUS Registry. The Registry has measured ADAMTS13 activity on 172/193 (89%) consecutive patients treated with plasma exchange for their initial episode of clinically diagnosed TTP-HUS from 11/13/1995 to 4/30/2003. 4/30/2003 was selected to assess patients with at least 1 year of follow-up after their initial episode. 29/172 (17%) patients with apparently acquired TTP had ADAMTS13 activity <10% at the onset of their initial episode; 22 patients survived. Although severe ADAMTS13 deficiency characteristic of TTP is usually defined as <5%, our 6 surviving patients with initial ADAMTS13 activity of 5–9% all had ADAMTS13 inhibitors; 2 have relapsed and had ADAMTS13 activity of <5% at the time of their 2nd episode. 21/22 patients, who have had follow-up of 0.25–8.25 years from their most recent episode, were evaluated. 9/21 (43%) patients have had between 1–4 relapses. None of the patients had symptoms or signs of TTP at the time of this assessment. The Rey Auditory-Verbal Learning Test (RAVLT) assesses new learning and recent memory. For new learning 11 (52%) patients and for recent memory 7 (33%) patients were below the 16th percentile for age/gender specific norms (p<0.01 and p=0.03 respectively). The Digit Span (DS) score, which assesses attention and concentration, was below the 25th percentile in 8 (38%) patients (p=0.17). 11/20 (55%) had moderate-severe depression symptoms documented by the Beck Depression Inventory-Second Edition (BDI–II) (8 patients) or by prescribed medication for depression (3 patients). ADAMTS13 activity was abnormal (<50%) in 11/21 patients (52%); 2 patients, who were 4.0 and 8.25 years from their most recent episode, had activity <3% with inhibitors; in the other 9 abnormal patients ADAMTS13 activity was 14–50% (median 35%). 19 patients were not thrombocytopenic (platelet counts 174–659, median 286). 1 patient (ADAMTS13 > 100%) with a platelet count of 105 had developed systemic lupus erythematosus after his initial TTP episode; 1 patient (ADAMTS13=50%) with a platelet count of 134 has had intermittent, mild thrombocytopenia since her initial episode 7 years ago. Five patients had mild anemia (hct 32–40%). Relapses, cognitive function, symptoms of depression, platelet count, and hct were not significantly different between patients with normal or abnormal ADAMTS13 activity. The neurocognitive abnormalities documented here may be related to the initial diffuse thrombotic disease. Depression may contribute to these abnormalities. Persistent abnormal ADAMTS13 activity many years after apparent recovery is common but of uncertain clinical importance since ADAMTS13 levels were not associated with clinical outcomes.