Inhibitory anti-ADAMTS13 Antibodies Research Articles

Abstract 17158: Effects of Human Monoclonal Antibodies on ADAMTS13 in Immune Thrombotic Thrombocytopenic Purpura

Introduction: Immune thrombotic thrombocytopenic purpura (iTTP) is caused by antibody-mediated deficiency of the metalloprotease ADAMTS13, which cleaves VWF. Myocardial infarction and ischemic strokes are common and often fatal sequelae of iTTP. Inhibitory anti-ADAMTS13 antibodies appear to affect catalytic turnover more than apparent substrate binding affinity (PMID 37023370). Exploring how antibodies affect VWF cleavage by ADAMTS13 in physiologic conditions is needed to better understand the pathophysiology of iTTP. Hypothesis: Anti-ADAMTS13 antibodies may influence ADAMTS13 function by affecting catalytic turnover and/or apparent substrate binding affinity differently in physiologic conditions than in standard assay conditions. Aims: Characterize how single chain fragments of the variable loop (scFv’s) previously identified from a phage display library derived from several iTTP patients’ antibodies affect ADAMTS13-mediated VWF cleavage in conditions that more closely represent plasma and see if/how they differ from scFv-mediated effects in standard conditions. Methods: ADAMTS13 in normal human plasma was titrated with increasing concentrations of the substrate FRETS-VWF73 to derive maximum velocity ( V max ) and apparent Michaelis constant ( K M,app ) in standard assay conditions (pH 6.0) and conditions closer to physiologic (pH 7.0). The same parameters were derived in the presence of saturating concentrations of scFv’s. Results: Compared to VWF cleavage by ADAMTS13 in the absence of scFv, the inhibitory scFv4-20 decreased V max at both pH 6.0 and pH 7.0; K M,app decreased at pH 6.0 and increased at pH 7.0. The stimulatory scFv3-3 and scFv4-41 increased V max at each pH, while K M,app in the presence of each scFv decreased only at pH 7.0. Conclusions: Anti-ADAMTS13 antibody-mediated effects on VWF cleavage differ when comparing standard and more physiologic pH conditions, guiding future investigative directions into iTTP pathophysiology.

Inhibitory anti ADAMTS13 antibodies with a new rapid fully automated CLiA assay

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder characterized by severe ADAMTS13 deficiency. The acquired form is associated with autoantibodies directed against ADAMTS13. Both noninhibitory and inhibitory autoantibodies can be detected by ELISA assay, while only inhibitory autoantibodies are detected by Bethesda assay. Due to its short TAT and good performance, chemiluminescence (CliA) ADAMTS13 activity (HemosIL Acustar) has proven to be a good choice in the diagnosis of TTP in emergency settings. Aim of this study was to analyse the performance of the CliA ADAMTS13 activity assay in detecting inhibitory ADAMTS13 antibodies using the Bethesda assay. A method comparison study was performed on 69 stored samples: 11 acute TTPs, 38 TTP follow-ups, 5 TTP relapses, 1 congenital TTP, 10 HUS, 4 suspected TTPs. We retrieved the results of tests previously run in ELISA for both activity and autoantibodies. At the same time, we reran new tests including ELISA and CliA activity, ELISA autoantibodies, and ELISA and CliA Bethesda assays on thawed frozen samples. Very good correlation was observed between ELISA and CliA activity assay results (r=0.96) and between archived ELISA and CliA activity results (r=0.93). Agreement between the anti-ADAMTS13 assays ranged from good (k=0.63) to very good (k=0.92). CliA and ELISA Bethesda assays showed very good agreement with samples run at the same time using ELISA ADAMTS13-autoantibody assay. Albeit more expensive, the CliA Bethesda assay identified inhibitory anti-ADAMTS13 within almost the same TAT as ELISA, but with better automation and limited operator involvement.

Optimization of the detection of inhibitory autoantibodies against the VWF-cleaving protease ADAMTS13 with an automated chemiluminescent ADAMTS13 activity immunoassay.

Acquired thrombotic thrombocytopenic purpura is a rare disease associated with the production of autoantibodies against the VWF-cleaving protease ADAMTS13. The detection of these antibodies is made difficult by the instability of ADAMTS13 in citrated plasma and the time-consuming ADAMTS13 assays. The aim of our study was to evaluate the optimal conditions for detecting anti-ADAMTS13 inhibitory antibodies with the novel automated chemiluminescent immunoassay HemosILR AcuStar ADAMTS13 Activity assay. The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2hours incubation at 25°C, 37°C, or 37°C after addition of Ca2+ to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosILR AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the TechnozymR ADAMTS13 activity ELISA. The criterion of parallelism was respected for the 3 incubation methods over the range of ADAMTS13 concentrations relevant for the detection of ADAMTS13 inhibitor antibodies in a Bethesda assay. In agreement with this observation, all the incubation methods permitted the accurate detection and quantification of inhibitory anti-ADAMTS13 antibodies in the samples from patients with acquired thrombotic thrombocytopenic purpura. Incubation of plasma samples with normal plasma at 25°C, 37°C, or 37°C after addition of Ca2+ can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosILR AcuStar ADAMTS13 Activity assay.

Bethesda Assay for Detecting Inhibitory Anti-ADAMTS13 Antibodies in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

A diagnosis of thrombotic thrombocytopenic purpura (TTP) is confirmed by a severe deficiency (<10%) of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity. Autoantibodies to ADAMTS13 can be detected with a simplified enzyme-linked immunosorbent assay (ELISA). An alternative methodology is a Bethesda assay, which has never been formally assessed in TTP. This study aimed to investigate the inhibitory anti-ADAMTS13 antibody assay and determine if the Bethesda assay is advantageous compared with the ELISA, measuring total immunoglobulin G (IgG) antibodies to ADAMTS 13. The Bethesda method determines the neutralizing activity of anti-ADAMTS13 antibodies in pooled normal plasma. We selected six immune-mediated TTP (iTTP) patients with ADAMTS13 activity levels <10% and strong ADAMTS13 inhibitors by 50:50 mixing studies and analyzed anti-ADAMTS13 antibodies using the Bethesda and ELISA assays. ADAMTS13 activity was stable at room temperature, while a time-dependent decrease in activity was detected in assay conditions of 37°C. Adding 5 mM Ca 2+ to citrated plasma prevented loss of ADAMTS13 activity with time. There was time dependence to the antibody-mediated inactivation, after 2-hour incubation. Two of the iTTP patients had no detectable ADAMTS13 antibodies by the Bethesda assay, but had high titer of anti-ADAMTS13 antibodies and low ADAMTS13 antigen levels. The Bethesda assay can only detect anti-ADAMTS13 antibodies that functionally inhibit ADAMTS13. The anti-ADAMTS13 IgG ELISA instead allows the rapid identification of total IgG autoantibodies, detecting both inhibitory and noninhibitory antibodies.

Anti-ADAMTS13 Antibodies and a Novel Heterozygous p.R1177Q Mutation in a Case of Pregnancy-Onset Immune-Mediated Thrombotic Thrombocytopenic Purpura.

In this study, we investigated a case of pregnancy-onset thrombotic thrombocytopenic purpura (TTP). The patient had severely decreased ADAMTS13 ( a d isintegrin a nd m etalloprotease with t hrombo s pondin type 1 motif, member 13) activity levels during acute phase and the presence of inhibitory anti-ADAMTS13 autoantibodies was demonstrated, which led to the diagnosis of immune-mediated TTP. However, ADAMTS13 activity was only mildly restored during remission, although inhibitory anti-ADAMTS13 antibodies were no longer detected. We hypothesized that genetic abnormalities could account for this discrepancy between ADAMTS13 activity and antigen. Genetic analysis revealed the presence of two heterozygous substitutions on the same allele: a single nucleotide polymorphism (SNP) c.2699C > T (p.A900V), located in the beginning of the T5 domain, and a mutation c.3530G > A (p.R1177Q) located in the third linker region of ADAMTS13. In vitro testing of those substitutions by expression of recombinant proteins revealed a normal secretion but a reduced ADAMTS13 activity by the novel p.R1177Q mutation, which could partially explain the subnormal activity levels found during remission. Although changes in the linker region might induce conformational changes in ADAMTS13, the p.R1177Q mutation in the third linker region of ADAMTS13 did not expose a cryptic epitope in the metalloprotease domain. In conclusion, we report on an immune-mediated pregnancy-onset TTP patient who had inhibitory anti-ADAMTS13 autoantibodies during acute phase, but not during remission. Genetic analysis confirmed the diagnosis of immune-mediated TTP and revealed the novel p.R1177Q mutation which mildly impaired ADAMTS13 activity.

Pharmacodynamic Profile of a Recombinant ADAMTS13 (BAX930) in Hereditary Thrombotic Thrombocytopenic Purpura (Upshaw-Schulman Syndrome (USS))

Pharmacodynamic Profile of a Recombinant ADAMTS13 (BAX930) in Hereditary Thrombotic Thrombocytopenic Purpura (Upshaw-Schulman Syndrome (USS))

Thrombotic thrombocytopenic purpura: basic pathophysiology and therapeutic strategies.

VWF is a multimeric plasma glycoprotein that specifically recruits platelets to sites of vessel injury. VWF multimeric size is central to this function, with larger multimers being more hemostatically active. Regulation of VWF multimeric size is mediated by the plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13). This enzyme can only recognize and cleave VWF when it is unraveled by rheological shear forces of the flowing blood. After the exposure of cryptic exosites, VWF recognition by ADAMTS13 involves multiple interactions that enable the protease to cleave VWF. Loss of VWF multimer size regulation caused by severe ADAMTS13 deficiency (either inherited or acquired) is associated with the microvascular thrombotic disorder thrombotic thrombocytopenic purpura (TTP). The sequelae associated with TTP are widely thought to be linked to hyperreactive circulating VWF that cause unwanted platelet aggregation in the high shear environment of the microvasculature. Diagnosis of TTP is primarily made through a combination of symptoms, analysis of plasma ADAMTS13 activity, and detection of inhibitory anti-ADAMTS13 antibodies. Current frontline treatments for TTP include plasma exchange, which serves to remove inhibitory antibodies (in acquired TTP) and provide a source of functional ADAMTS13, and steroids to treat the autoimmune component of acquired TTP. The use of anti-CD20 therapy has also exhibited encouraging results in the treatment of acquired TTP. Newer therapeutic strategies that are currently being explored or are in development include recombinant ADAMTS13, a hyperreactive ADAMTS13 variant, and anti-VWF therapy. This review discusses the basic biochemistry of VWF and ADAMTS13, their dysfunction in TTP, and therapeutic approaches for the amelioration of TTP.

Thrombotic Thrombocytopenic Purpura At the Cleveland Clinic 2000–2012: Review of 100 Cases and Identification of Prognostic Factors

AbstractAbstract 3325 BACKGROUND AND OBJECTIVE:The advent of plasma exchange has led to a dramatic improvement in the survival of patients with Thrombotic Thrombocytopenic Purpura (TTP). However 10–20% of patients do not respond to plasma exchange and up to a third suffer relapses. Recent studies suggest that Rituximab as an adjunct to plasma exchange and corticosteroids may be effective in refractory or relapsing disease, although clinical factors that identify patients at high risk for poor outcomes have not been clearly defined. This concern prompted a retrospective review of all patients with TTP treated at the Cleveland Clinic over the last 12 years in an attempt to identify factors associated with poor prognosis. Records from all patients were reviewed from the date of initial presentation until at least two years afterwards to determine the incidence of refractory disease and relapse. STUDY AND METHODS:A total of 284 patients who were diagnosed with a first episode of thrombotic microangiopathy at the Cleveland Clinic from January 2000 to March 2012 were identified. Records from these patients were reviewed and individuals with other explanations for thrombocytopenia and hemolytic anemia such as DIC, hypertensive crisis or HELLP were excluded. One hundred patients were included in the final analysis. Fischer exact test and t- test were used to compare variables. A p value of <0.05 was considered significant. RESULTS:Of the 100 patients with TTP, 73% were female, with an age range of 16 to 79 years (median 49 years). Fifty percent of patients were Caucasian, 45% African American and 2% Hispanic. Sixty seven percent of cases occurred without predisposing conditions while 12% were associated with autoimmune disease (6 with SLE, 2 with rheumatoid arthritis, one with SLE and rheumatoid arthritis, and one each with Sjogren’s syndrome, dermatomyositis and mixed connective tissue disorder), 8% with pregnancy or the postpartum state, 6% each with cancer and solid organ transplantation and 2% each with bone marrow and stem cell transplant. ADAMTS13 activity was tested in 57% of cases of which 36 (63%) had <5% and 21 (37%) had 8% to 56% activity, respectively. All patients were treated with plasma exchange, and all but 17 received corticosteroids, while some received additional therapies including vincristine (10), rituximab (15) and splenectomy (7) for refractory disease. Mortality after a first episode of TTP was 8%, while 13% of patients had exacerbations (within 30 days) and 18% had relapses (11 patients had a single relapse, 6 patients had 2 relapses and 1 had three relapses).Non survivors were older (p=0.042) with this association particularly striking for patients greater than age 60 (OR 8.75, 95% CI 2.32–33.01, p=0.002). Non-survivors also presented more frequently with severe neurological symptoms including obtundation, focal deficits and seizures (p=0.001). A higher LDH level after 1 or 2 cycles of plasma exchange, i.e. LDH on day 3, 4 or 5 of admission was also strongly associated with mortality (p<0.01) as well as with prolonged duration of plasma exchange.ADAMTS13 activity and levels of inhibitory anti-ADAMTS13 antibodies were comparable between survivors and non-survivors. However, undetectable ADAMTS13 levels were associated with a lower incidence of adverse renal outcomes including need for dialysis during the acute episode (p=0.007) and the development of chronic kidney disease (p=0.033) and/or end stage renal disease at 2 years (p=0.015). CONCLUSION:The most significant independent variables predicting death in TTP were increasing age, especially age>60, severe neurological symptoms at presentation and a persistently high LDH level the second day after diagnosis and initiation of plasma exchange. These variables could be used to identify patients who would benefit from close monitoring and potentially from early institution of adjunctive therapy. Treatment of high risk patients in this manner could limit the duration of plasma exchange, improve outcomes, and decrease associated morbidity and costs. Disclosures:No relevant conflicts of interest to declare.

Bilateral serous retinal detachment as a complication of acquired peripartum thrombotic thrombocytopenic purpura bout

We report a case of a 26-year-old primigravid woman, believed to have HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count) syndrome, which turned out to be a thrombotic thrombocytopenic purpura (TTP) bout. At the 40th gestational week, based on the clinical picture of HELLP syndrome, a cesarean section was performed and a dysmature male newborn was delivered. Afterwards, clinical symptoms and laboratory abnormalities persisted. Severe ADAMTS13 deficiency with the presence of inhibitory anti-ADAMTS13 antibodies revealed acquired thrombotic thrombocytopenic purpura bout, which was complicated with bilateral vision decrease due to bilateral retinal detachment. At the first ophthalmological examination, ultrasonography and binocular indirect ophthalmoscopy confirmed the diagnosis of the serous retinal detachment. After the diagnosis of acquired TTP bout, the patient was treated with multiple plasmapheresis and intravenous immunoglobulin with rapid improvement of the clinical and laboratory parameters. The ophthalmologic complications disappeared later without sequelae. At the 18-month examination, substantial visual acuity improvement without serous retinal detachment and full best corrected visual acuity were observed.

Microangiopatias trombóticas: púrpura trombocitopênica trombótica e síndrome hemolítico-urêmica

Thrombotic microangiopathies (TMAs) are pathological conditions characterized by generalized microvascular occlusion by platelet thrombi, thrombocytopenia, and microangiopathic hemolytic anemia. Two typical phenotypes of TMAs are hemolytic- uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Other disorders occasionally present with similar manifestations. Depending on whether renal or brain lesions prevail, two pathologically indistinguishable but somehow clinically different disorders have been described: HUS and TTP. Injury to the endothelial cell is the central and likely inciting factor in the sequence of events leading to TMA. Loss of physiological thromboresistance, leukocyte adhesion to damaged endothelium, complement consumption, abnormal von Willebrand factor release and fragmentation, and increased vascular shear stress may then sustain and amplify the microangiopathic process. Intrinsic abnormalities of the complement system and of the von Willebrand factor pathway may account for a genetic predisposition to the disease that may play a paramount role in particular in familial and recurrent forms. In the case of diarrhea-associated HUS (D+HUS), renal endothelial damage is mediated (at least in large part) by the bacterial agent Shigatoxin (Stx), which is actually a family of toxins elaborated by certain strains of Escherichia coli and Shigella dysenteriae. Outcome is usually good in childhood, Shiga toxin-associated HUS, whereas renal and neurological sequelae are more frequently reported in adult, atypical, and familial forms of HUS and in TTP. Recent studies have demonstrated that deficiency in the von Willebrand factor cleaving protease ADAMTS13, due to deficiency of ADAMTS13 can be genetic or more common, acquired, resulting from autoimmune production of inhibitory anti-ADAMTS13 antibodies, that causes TTP. During the last decade, atypical HUS (aHUS) has been demonstrated to be a disorder of the complement alternative pathway dysregulation, as there is a growing list of mutations and polymorphisms in the genes encoding the complement regulatory proteins that alone or in combination may lead to aHUS. Approximately 60% of aHUS patients have so-called 'loss-of-function' mutations in the genes encoding the complement regulatory proteins, which normally protect host cells from complement activation: complement factor H (CFH), factor I (CFI) and membrane cofactor protein (MCP or CD46), or have 'gain-of-function' mutations in the genes encoding the complement factor B or C3. In addition, approximately 10% of aHUS patients have a functional CFH deficiency due to anti-CFH antibodies. Although TMAs are highly heterogeneous pathological conditions, one-third of TMA patients have severe deficiency of ADAMTS13. Platelet transfusions are contraindicated. Plasma infusion or exchange (PE) is the only treatment of proven efficacy.

Prognostic value of inhibitory anti‐ADAMTS13 antibodies in adult‐acquired thrombotic thrombocytopenic purpura

In order to assess the prognostic value of inhibitory anti-ADAMTS13 antibodies in thrombotic thrombocytopenic purpura (TTP), we performed a multicentre prospective study of 33 adult patients with idiopathic acquired TTP. Patients were treated with high-dose plasma infusion and therapeutic plasma exchange. Patients without (group 1, n = 12) and with (group 2, n = 21) detectable inhibitory anti-ADAMTS13 antibodies were compared for clinical presentation, treatment and outcome. Both groups were comparable for clinical presentation. All patients in group 1 achieved a sustained complete remission within a median of 7 d [95% confidence interval (CI), 4-18], which required a median plasma volume of 235 ml/kg (range, 131-1251). In group 2, 17 patients achieved a durable complete remission within a median of 23 d (95% CI, 11-32) (P = 0.001). Median plasma volume was 718 ml/kg (range, 219-3107) (P = 0.02). In group 2, there was a trend for more episodes of flare-up than in group 1 (13 vs. 3, respectively, P = 0.07). Four patients, all from group 2, died (P = not significant). The relapse rate was comparable between both groups. We suggest that TTP with detectable inhibitory anti-ADAMTS13 antibodies displays a worse prognosis, relative to a delayed platelet count recovery, a higher plasma volume requirement to achieve complete remission, and a trend for more frequent episodes of flare-up.