Optimization of anti-ADAMTS13 antibodies for the treatment of ADAMTS13-related bleeding disorder in patients receiving circulatory assist device support.

ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motif 13)-related bleeding disorder has been frequently observed as a life-threatening clinical complication in patients carrying a circulatory assist device. Currently, treatment modalities for the bleeding disorder are very limited and not always successful. To address the unmet medical need, we constructed humanized antibodies of mouse anti-ADAMTS13 antibody A10 (mA10) by using complementarity-determining region (CDR) grafting techniques with human antibody frameworks, 8A7 and 16E8. The characteristics of the two humanized A10 antibodies, namely A10/8A7 and A10/16E8, were assessed in vitro and in silico. Among the two humanized A10 antibodies, the binding affinity of A10/16E8 to ADAMTS13 was comparable to that of mA10 and human-mouse chimeric A10. In addition, A10/16E8 largely inhibited the ADAMTS13 activity in vitro. The results indicated that A10/16E8 retained the binding affinity and inhibitory activity of mA10. To compare the antibody structures, we performed antibody structure modeling and structural similarity analysis in silico. As a result, A10/16E8 showed higher structural similarity to mA10, compared with A10/8A7, suggesting that A10/16E8 retains a native structure of mA10 as well as its antigen binding affinity and activity. A10/16E8 has great potential as a therapeutic agent for ADAMTS13-related bleeding disorder.

Secretion of von Willebrand Factor and Suppression of ADAMTS-13 Activity by Markedly High Concentration of Ferritin.

Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.

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

INTRODUCTION: 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. METHODS: The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2 hours 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. RESULTS: 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. CONCLUSION: 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.

Thrombotic thrombocytopenic purpura developed during the conservative treatment of anti-phospholipase A2 receptor antibody-positive idiopathic membranous nephropathy: a case report.

BACKGROUND: Idiopathic membranous nephropathy (MN) is one of the major glomerulonephritis that cause nephrotic syndrome. The phospholipase A2 receptor (PLA2R) has recently been identified as an endogenous antigen of idiopathic MN. Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by schistocytes, hemolytic anemia, thrombocytopenia, and organ dysfunction which occurs as a result of thrombi. Patients with acquired TTP have autoantibodies against a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13). These autoantibodies act as an inhibitor and cause ADAMTS13 deficiency. Idiopathic MN and acquired TTP are usually considered as independent autoimmune diseases. We experienced a patient who developed TTP during the conservative treatment of idiopathic MN, with the coexistence of ADAMTS13 inhibitor and anti-PLA2R antibody. CASE PRESENTATION: A 73-year-old man presented with thrombocytopenia, hemolytic anemia, disturbance of consciousness, and acute kidney injury after 4-year course of biopsy-proven idiopathic MN. ADAMTS13 activity was undetectable and the ADAMTS13 inhibitor was identified. Additionally, he was positive for anti-PLA2R antibody. The patient did not have any diseases that could cause secondary thrombotic microangiopathy, and he was diagnosed with acquired TTP. Steroid therapy and plasma exchange were initiated and the acquired TTP resolved. MN achieved remission 3 months after the anti-PLA2R antibody disappeared. CONCLUSIONS: This is the first reported case of acquired TTP developed during conservative treatment of idiopathic MN, with both ADAMTS13 inhibitor and anti-PLA2R antibody positive at the onset of the TTP. The present case suggests that idiopathic MN might be associated with the development of some cases of acquired TTP.

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor.

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWF and inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.

Thrombotic Thrombocytopenic Purpura Cases and Consumption of Large Quantities of Energy Drink: Related or Coincidence?

The vast majority of cases of thrombotic thrombocytopenic purpura (TTP) are the result of acquired antibodies which inhibit the activity of the ADAMTS13 enzyme. Acquired TTP is more frequently seen in young females or in individuals with autoimmune disease. The development of antibodies against ADAMTS13 may also result from the administration or consumption of drugs and other substances. However, specific laboratory tests to identify the pathogenic mechanism of a particular drug may not be available, and the role of a potentially implicated drug or other ingested substance may not be clear. In this report we present 2 acquired TTP cases involving the consumption of a large amount of energy drink.

[Rituximab for treatment of immune thrombocytopenia and thrombotic thrombocytopenic purpura].

Although immune thrombocytopenia (ITP) and thrombotic thrombocytopenic purpura (TTP) appear similar, their symptoms differ. The number of domestic patients diagnosed with ITP and TTP annually has been estimated to be around 24,000 and 400, respectively. Moreover, no major differences in the incidence rate, age of onset, and prognosis have been observed between Europe, the United States (US), and Japan. Both ITP and acquired TTP are autoimmune diseases that require immunosuppressive therapy, though lethal TTP requires the use of plasma exchange in combination with immunosuppression. In Europe and the US, the monoclonal antibody rituximab has been widely used for ITP and TTP since approximately 10 years ago. However, no public health insurance indication has been available for rituximab in Japan. For this reason, investigator-initiated clinical trials were conducted. As a result, rituximab had subsequently been indicated for ITP in 2017. Meanwhile, TTP was designated as an intractable disease in Japan in 2015, and the first clinical practice guidelines were published in 2017. A single-arm study involving rituximab was conducted on high-risk patients in whom treatment with five plasma exchanges was ineffective or ADAMTS13 inhibitor was >2 BU/ml. Approval for the new indication of rituximab for acquired TTP is expected in 2019.

[Thrombotic thrombocytopenic purpura during pregnancy refractory to plasma exchange and rituximab].

A 30-year-old woman who was 14 weeks pregnant was admitted to our hospital due to purpura, nasal bleeding, and abdominal pain. She was diagnosed with acquired thrombotic thrombocytopenic purpura (TTP) based on the presence of hemolytic anemia, thrombocytopenia, decreased ADAMTS 13 activity (<0.01 IU/ml), and high ADAMTS 13 inhibitor levels (4.8 BU/ml). Plasma exchange (PE) and steroid therapy were immediately administered. However, because she did not respond to these therapeutic approaches, rituximab was additionally administered on the sixth day of treatment. The level of ADAMTS 13 inhibitor increased to 12.5 BU/ml on the seventh day. Renal insufficiency, disturbed consciousness, and genital bleeding did not improve in spite of daily PE, steroid therapy, and second dose of rituximab. She finally died after sudden convulsions on the 14th day. Although the treatment outcomes of TTP have remarkably improved, some cases are refractory to therapy. Establishment of adequate treatment strategies for acquired TTP in pregnant women is required.

Unique case of autoantibody mediated inactivation of ADAMTS13 in an Indian TTP patient.

A young Indian female visited hospital as a suspected case of thrombotic thrombocytopenic purpura (TTP) with relapsed thrombotic complications with low platelet counts, infarct in middle cerebral artery and thrombi in microvessels. We first confirmed the deficiency of ADAMTS13 metalloprotease in this patient showing improper cleavage of vWF multimers by her plasma unlike her parents and brother. Although patient had very less ADAMTS13 antigen in plasma, but it did not appear to be the cause of deficiency of the enzyme, because her father had similarly low antigen level and he never had prothrombotic complications. While investigating the genetic change in ADAMTS13, we observed four homozygous-SNPs (g.420T>C, g.1342C>G, g.1716G>A and g.2280T>C) in exon 5, 12, 15 and 19 respectively in patient and her father unlike the heterozygous form of same SNPs in mother and brother. Further to investigate the cause of ADAMTS13 deficiency, we observed an elevated level of antibody against ADAMTS13 in patient unlike her father and other family members. Our study therefore provides the molecular approach of diagnosis of TTP in this patient and also highlights the use of such techniques in India. More importantly, study provides the clue of alternate treatment such as immunosuppressant therapy to this patient.

Temperature-dependent irreversible conformational change of recombinant ADAMTS13 upon metal ion chelation.

BACKGROUND: The catalytic domain of ADAMTS13 possesses one Zn2+ and up to three putative Ca2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion-depleting environment has not been explored. OBJECTIVES: To address the stability of ADAMTS13 in citrated human plasma. METHODS: ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering. RESULTS: ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca2+ or Zn2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn2+ and that its reduced activity correlated with a gradual loss of bound Zn2+ . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures. CONCLUSIONS: Zn2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings.

Beyond plasma exchange: novel therapies for thrombotic thrombocytopenic purpura.

The advent of plasma exchange has dramatically changed the prognosis of acute thrombotic thrombocytopenic purpura (TTP). Recent insights into TTP pathogenesis have led to the development of novel therapies targeting pathogenic anti-ADAMTS13 antibody production, von Willebrand factor (VWF)-platelet interactions, and ADAMTS13 replacement. Retrospective and prospective studies have established the efficacy of rituximab as an adjunct to plasma exchange for patients with acute TTP, either upfront or for refractory disease. Relapse prevention is a major concern for survivors of acute TTP, and emerging data support the prophylactic use of rituximab in patients with persistent or recurrent ADAMTS13 deficiency in clinical remission. Capalcizumab, a nanobody directed against domain A1 of VWF that prevents the formation of VWF-platelet aggregates, recently completed phase 2 (TITAN) and 3 (HERCULES) trials with encouraging results. Compared with placebo, caplacizumab shortened the time to platelet recovery and may protect against microthrombotic tissue injury in the acute phase of TTP, though it does not modify the underlying immune response. Other promising therapies including plasma cell inhibitors (bortezomib), recombinant ADAMTS13, N-acetyl cysteine, and inhibitors of the VWF-glycoprotein Ib/IX interaction (anfibatide) are in development, and several of these agents are in prospective clinical studies to evaluate their efficacy and role in TTP. In the coming years, we are optimistic that novel therapies and international collaborative efforts will usher in even more effective, evidence-based approaches to address refractory acute TTP and relapse prevention.

Multiple centre evaluation study of ADAMTS13 activity and inhibitor assays.

INTRODUCTION: Accurate evaluation of ADAMTS13 activity is required for the diagnosis and clinical management of thrombotic microangiopathies, and commercial kits are available for routine laboratory use. METHODS: Our study compares the results from Technoclone (Technoclone GmbH, Austria) activity and Inhibitor kits with specialist laboratory reference methods (FRETS and ELISA IgG) and the impact of transporting frozen samples and comparison of results. RESULTS: This multicentre study identified differences in Technoclone activity results compared to specialist testing, which could potentially impact diagnosis. A change in the commercial kit during the study period appears to have rectified the detection levels. Frozen samples provided comparable results between sites. CONCLUSION: With close attention to normal ranges, commercial kits are suitable for use in the clinical diagnosis of thrombotic microangiopathies and frozen transportation of samples between sites is a suitable approach. However, a robust external quality control system is essential to provide an independent evaluation of changes in kit production.

Thrombotic thrombocytopenic purpura: The role of ADAMTS13.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon, life-threatening disease requiring prompt diagnosis and initiation of therapeutic plasma exchange to improve patient survival. However, diagnosis is often difficult because of atypical presentations and signs and symptoms that resemble other conditions. Measurements of ADAMTS13 activity, ADAMTS13 inhibitor, and ADAMTS13 autoantibody are useful for diagnosing TTP, guiding therapy, and predicting relapse.

Validation of a panel of ADAMTS13 assays for diagnosis of thrombotic thrombocytopenic purpura: activity, functional inhibitor, and autoantibody test.

INTRODUCTION: Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. METHODS: Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone). RESULTS: A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%. CONCLUSION: ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model.

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.