Recombinant ADAMTS13 for Immune Thrombotic Thrombocytopenic Purpura.

In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

Mechanism underlying severe deficiency of plasma ADAMTS-13 activity in immune thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura is caused by autoantibodies against ADAMTS-13, a plasma enzyme that cleaves von Willebrand factor. However, the mechanism resulting in severe deficiency of plasma ADAMTS-13 activity remains controversial. OBJECTIVES: To determine the mechanism of autoantibody-mediated severe deficiency of plasma ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura. METHODS: Fluorescence resonance energy transfer-VWF73 was used to determine plasma ADAMTS-13 activity. Enzyme-linked immunosorbent assay (ELISA) was used to determine anti-ADAMTS-13 immunoglobulin G. ELISA and capillary electrophoresis-based Western blotting were employed to assess plasma ADAMTS-13 antigen. RESULTS: We showed that plasma ADAMTS-13 antigen levels varied substantially in the samples collected on admission despite all showing plasma ADAMTS-13 activity of <10 IU/dL (or <10% of normal level) using either ELISA or Western blotting. More severe deficiency of plasma ADAMTS-13 antigen (<10%) was detected in admission samples by ELISA than by capillary Western blotting. There was a significant but moderate correlation between plasma ADAMTS-13 activity and ADAMTS-13 antigen by either assay method, suggesting that severe deficiency of plasma ADAMTS-13 activity is not entirely associated with low levels of ADAMTS-13 antigen. CONCLUSION: We conclude that severe deficiency of plasma ADAMTS-13 activity primarily resulted from antibody-mediated inhibition, but the accelerated clearance of plasma ADAMTS-13 antigen via immune complexes may also contribute significantly to severe deficiency of plasma ADAMTS-13 activity in a subset of patients with acute immune-mediated thrombotic thrombocytopenic purpura.

ADAMTS-13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura.

BACKGROUND: Patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti-ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer-CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition. OBJECTIVES: To explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters. METHODS: We developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse. RESULTS: In 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar. CONCLUSION: ADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.

Emerging Concepts in Immune Thrombotic Thrombocytopenic Purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder of which the etiology is not fully understood. Autoantibodies targeting ADAMTS13 in iTTP patients have extensively been studied, the immunological mechanisms leading to the breach of tolerance remain to be uncovered. This review addresses the current knowledge on genetic factors associated with the development of iTTP and the interplay between the patient's immune system and environmental factors in the induction of autoimmunity against ADAMTS13. HLA-DRB1*11 has been identified as a risk factor for iTTP in the Caucasian population. Interestingly, HLA-DRB1*08:03 was recently identified as a risk factor in the Japanese population. Combined in vitro and in silico MHC class II peptide presentation approaches suggest that an ADAMTS13-derived peptide may bind to both HLA-DRB1*11 and HLA-DRB1*08:03 through different anchor-residues. It is apparent that iTTP is associated with the presence of infectious microorganisms, viruses being the most widely associated with development of iTTP. Infections may potentially lead to loss of tolerance resulting in the shift from immune homeostasis to autoimmunity. In the model we propose in this review, infections disrupt the epithelial barriers in the gut or lung, promoting exposure of antigen presenting cells in the mucosa-associated lymphoid tissue to the microorganisms. This may result in breach of tolerance through the presentation of microorganism-derived peptides that are homologous to ADAMTS13 on risk alleles for iTTP.

Conformational plasticity of ADAMTS13 in hemostasis and autoimmunity.

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force-dependent cleavage at a single Tyr-Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13-VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP.

Thrombotic thrombocytopenic purpura: a new menace after COVID bnt162b2 vaccine.

Thrombotic thrombocytopenic purpura (TTP) is a known menace in hematology and is quite rare in practice with known triggers. Lately, in the COVID-19 pandemic, hematology has seen a new pathology amongst which TTP associated with COVID-19 messenger RNA (mRNA) vaccine is unique. We report a case of a 69-year-old male with multiple comorbidities who presented to the hospital with severe fatigue and shortness of breath. Labs were significant for thrombocytopenia, anemia, and hemolysis with schistocytes consistent with TTP with a second dose of BNT162b2 mRNA vaccine as a likely culprit been documented.

Laboratory testing for ADAMTS13: Utility for TTP diagnosis/exclusion and beyond.

The metalloproteinase ADAMTS13 (a disintegrin with a thrombospondin type 1 motif, member 13), also known as VWF (von Willebrand factor) protease, may be assessed in a vast array of clinical conditions. Notably, a severe deficiency of ADAMTS13 characterizes TTP (thrombotic thrombocytopenic purpura), a rare but potentially fatal disorder associated with thrombosis due to accumulation of prothrombotic ultra-large VWF multimers. Although prompt identification/exclusion of TTP can be facilitated by rapid ADAMTS13 testing, the most commonly utilized assays are based on ELISA (enzyme linked immunosorbent assay) and require long turnaround time and have relatively limited throughput. Nevertheless, several rapid ADAMTS13 assays are now available, at least in select geographies. The current mini-review discusses these issues, as well as the potential utility of ADAMTS13 testing in a range of other conditions, including coronavirus disease 2019 (COVID-19).

N-glycan-mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared with WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of von Willebrand factor, positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP.

Identification of Biomarkers in Patients with Thrombotic Thrombocytopenic Purpura Presenting with Large and Small Ischemic Stroke.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder resulting in organ damage including ischemic strokes. We sought to characterize the neuroimaging patterns of stroke in a large cohort of patients with immune-mediated TTP (iTTP) and determined their associations with clinical and laboratory parameters and outcomes. METHODS: We analyzed the Alabama TTP Registry who had laboratory confirmation of acute iTTP. We reviewed the neuroimaging patterns of those with ischemic stroke on MRI, clinical information, and laboratory results. Small ischemic strokes were ≤20 mm in their maximum diameter in the axial plane. Large ischemic strokes were >20 mm. Student t test, Mann-Whitney U test, and χ2 test were all used for data analysis. RESULTS: Of 108 iTTP patients, 21 had ischemic stroke on neuroimaging. The median platelet count in these patients was 12 × 109/L (interquartile range, IQR, 8.8-21 × 109/L), plasma ADAMTS13 activity 1.8 U/dL (IQR 0-4.5 U/dL), and the mean plasma level of anti-ADAMTS13 IgG was 6,595.8 U/mL (SD 3,448.9 U/mL). Comparison between patients with large ischemic strokes (n = 10) and small ischemic strokes (n = 11) revealed that patients with small stroke were older (p = 0.043) and had higher plasma levels of citrullinated histone 3 (p = 0.006) and histone/DNA complex (p = 0.014) than those with large strokes. There were no significant differences between 2 stroke groups in mortality or exacerbation. CONCLUSIONS: iTTP patients can present with large ischemic strokes and are usually younger. Further research should be performed in assessing different etiologies of iTTP-associated stroke based on neutrophil extracellular trap formation biomarkers (e.g., histone markers) seen in small ischemic stroke.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Immunogenic hotspots in the spacer domain of ADAMTS13 in immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 autoantibodies inducing a severe deficiency of ADAMTS13. Epitope mapping studies on samples obtained during acute iTTP episodes have shown that the iTTP immune response is polyclonal, with almost all patients having autoantibodies targeting the spacer domain of ADAMTS13. OBJECTIVES: To identify the immunogenic hotspots in the spacer domain of ADAMTS13. PATIENTS/METHODS: A library of 11 full-length ADAMTS13 spacer hybrids was created in which amino acid regions of the spacer domain of ADAMTS13 were exchanged by the corresponding region of the spacer domain of ADAMTS1. Next, the full-length ADAMTS13 spacer hybrids were used in enzyme-linked immunosorbent assay to epitope map anti-spacer autoantibodies in 138 samples from acute and remission iTTP patients. RESULTS: Sixteen different anti-spacer autoantibody profiles were identified with a similar distribution in acute and remission patients. There was no association between the anti-spacer autoantibody profiles and disease severity. Almost all iTTP samples contained anti-spacer autoantibodies against the following three regions: amino acid residues 588-592, 602-610, and 657-666 (hybrids E, G, and M). Between 31% and 57% of the samples had anti-spacer autoantibodies against amino acid regions 572-579, 629-638, 667-676 (hybrids C, J, and N). In contrast, none of the samples had anti-spacer autoantibodies against amino acid regions 556-563, 564-571, 649-656, and 677-685 (hybrids A, B, L, and O). CONCLUSION: We identified three hotspot regions (amino acid regions 588-592, 602-610, and 657-666) in the spacer domain of ADAMTS13 that are targeted by anti-spacer autoantibodies found in a large cohort of iTTP patients.

Absolute Immature Platelet Counts Suggest Platelet Production Suppression during Complicated Relapsing Thrombotic Thrombocytopenic Purpura.

Absolute immature platelet counts (A-IPC) aid in diagnosis and treatment follow-up in thrombotic thrombocytopenic purpura (TTP). A-IPC was used to follow a patient on mycophenolate mofetil (MMF) maintenance therapy treated with a prolonged therapeutic plasma exchange (TPE) regimen for relapsing TTP. On admission, the platelet (PLT) count was 95 × 109/L declining to 14 × 109/L in 5 days. Daily TPE was initiated for suspected TTP, and MMF was discontinued. A-IPC and PLT count were 1 × 109/L and 14 × 109/L, respectively, prior to first TPE. A-IPC improved to 3.2 × 109/L with 1 TPE, and on day 5, A-IPC and PLT count were 7.5 × 109/L and 218 × 109/L, respectively. On day 6, A-IPC and PLT count decreased to 4.8 × 109/L and 132 × 109/L further worsening to 0.4 × 109/L and 13 × 109/L, respectively. ADAMTS13 activity remained <5% with an inhibitor; counts did not recover. Initial improvement followed by rapidly declining A-IPC despite therapy suggested production suppression. In TTP, A-IPC may aid in establishing early therapy effects over PLT production.

Exome Sequencing Identifies Abnormalities in Glycosylation and ANKRD36C in Patients with Immune-Mediated Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). However, the mechanism underlying anti-ADAMTS13 autoantibody formation is not known, nor it is known how genetic aberrations contribute to the pathogenesis of iTTP. METHODS: Here we performed whole exome sequencing (WES) of DNA samples from 40 adult patients with iTTP and 15 local healthy subjects with no history of iTTP and other hematological disorders. RESULTS: WES revealed variations in the genes involved in protein glycosylation, including O-linked glycosylation, to be a major pathway affected in patients with iTTP. Moreover, variations in the ANKRD gene family, particularly ANKRD36C and its paralogs, were also more prevalent in patients with iTTP than in the healthy controls. The ANKRD36 family of proteins have been implicated in inflammation. Mass spectrometry revealed a dramatic alternation in plasma glycoprotein profile in patients with iTTP compared with the healthy controls. CONCLUSION: Altered glycosylation may affect the disease onset and progression in various ways: it may predispose patients to produce ADAMTS13 autoantibodies or affect their binding properties; it may also alter clearance kinetics of hemostatic and inflammatory proteins. Together, our findings provide novel insights into plausible mechanisms underlying the pathogenesis of iTTP.

Insights Into Immunothrombosis: The Interplay Among Neutrophil Extracellular Trap, von Willebrand Factor, and ADAMTS13.

Both neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) are essential for thrombosis and inflammation. During these processes, a complex series of events, including endothelial activation, NET formation, VWF secretion, and blood cell adhesion, aggregation and activation, occurs in an ordered manner in the vasculature. The adhesive activity of VWF multimers is regulated by a specific metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Increasing evidence indicates that the interaction between NETs and VWF contributes to arterial and venous thrombosis as well as inflammation. Furthermore, contents released from activated neutrophils or NETs induce the reduction of ADAMTS13 activity, which may occur in both thrombotic microangiopathies (TMAs) and acute ischemic stroke (AIS). Recently, NET is considered as a driver of endothelial damage and immunothrombosis in COVID-19. In addition, the levels of VWF and ADAMTS13 can predict the mortality of COVID-19. In this review, we summarize the biological characteristics and interactions of NETs, VWF, and ADAMTS13, and discuss their roles in TMAs, AIS, and COVID-19. Targeting the NET-VWF axis may be a novel therapeutic strategy for inflammation-associated TMAs, AIS, and COVID-19.

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.

A critical evaluation of caplacizumab for the treatment of acquired thrombotic thrombocytopenic purpura.

Introduction: Acquired thrombotic thrombocytopenic purpura (aTTP) is a thrombotic microangiopathy caused by inhibitory autoantibodies against ADAMTS13 protein. Until recently, the combination of plasma exchange (PEX) and immunosuppression has been the standard front-line treatment in this disorder. However, aTTP-related mortality, refractoriness, and relapse are still a matter of concern. Areas covered: The better understanding of the pathophysiological mechanisms of aTTP has allowed substantial improvements in the diagnosis and treatment of this disease. Recently, the novel anti-VWF nanobody caplacizumab has been approved for acute episodes of aTTP. Caplacizumab is capable to block the adhesion of platelets to VWF, therefore inhibiting microthrombi formation in the ADAMTS13-deficient circulation. In this review, the characteristics of caplacizumab together with the available data of its efficacy and safety in the clinical setting will be analyzed. Besides, the current scenario of aTTP treatment will be provided, including the role of other innovative drugs. Expert opinion: With no doubt, caplacizumab is going to change the way we treat aTTP. In combination with standard treatment, caplacizumab can help to significantly reduce aTTP-related mortality and morbidity and could spare potential long-term consequences by minimizing the risk of exacerbation.