Open ADAMTS-13 conformation index predicts earlier relapse in immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: ADAMTS-13 adopts an open conformation in patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) in acute phase while being closed in healthy donors. We reported that a substantial number of patients with iTTP in remission with restored ADAMTS-13 activity (>50%) still had an open ADAMTS-13 conformation, although a closed conformation is expected given the extent of remission. OBJECTIVES: To investigate whether open ADAMTS-13, represented by a conformation index >0.5, is associated with a risk of earlier ADAMTS-13 and/or clinical relapse. METHODS: We collected follow-up data (ADAMTS-13 parameters, ADAMTS-13 and clinical relapse, and treatment) from 81 patients with iTTP in remission with ADAMTS-13 activity >50%. RESULTS: During follow-up, 19 ADAMTS-13 and 10 clinical relapses were reported (median follow-up period, 20 months). First, open or closed ADAMTS-13 conformation was dichotomized based on the 0.5 conformation index cutoff. Open ADAMTS-13 (conformation index, >0.5) was not identified as a risk factor for ADAMTS-13 and clinical relapse (log-rank test and Cox regression model). In contrast, by identifying the optimal conformation index cutoff for relapse prediction, using classification and regression tree analysis, a conformation index >0.645 and >0.835 was shown to be a risk factor for ADAMTS-13 relapse (hazard ratio, 3.3; 95% CI, 1.3-8.3; P = .01) and clinical relapse (hazard ratio, 4.4; 95% CI, 1.3-15.3; P = .02), respectively. CONCLUSION: Patients with open ADAMTS-13 with a conformation index >0.645 and >0.835 have a >3- and >4-fold higher risk of earlier ADAMTS-13 and clinical relapse, respectively. Hence, ADAMTS-13 conformation index could be used to complement ADAMTS-13 activity monitoring to timely notice ADAMTS-13 relapse and prevent clinical relapse.

A novel von Willebrand factor multimer ratio as marker of disease activity in thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP), an autoantibody-mediated severe ADAMTS13 deficiency, is caused by insufficient proteolytic processing of von Willebrand factor (VWF) multimers (MMs) and microvascular thrombi. Recurrence of acute iTTP is associated with persistence or reappearance of ADAMTS13 deficiency. Some patients remain in remission despite recurring or persisting severe ADAMTS13 deficiency. In a prospective 2-year observational study, we investigated VWF MM patterns and ADAMTS13 in patients with iTTP in remission and at acute episodes. Of the 83 patients with iTTP, 16 suffered 22 acute episodes whereas 67 remained in clinical remission during follow-up, including 13 with ADAMTS13 <10% and 54 with ADAMTS13 ≥10%. High -molecular weight to low-molecular weight VWF MM ratio based on sodium dodecyl sulfate-agarose gel electrophoresis was compared with ADAMTS13 activity. VWF MM ratio was significantly higher in patients in remission with <10% compared with ≥10% ADAMTS13 activity. Fourteen samples obtained from 13 to 50 days (interquartile range; median, 39) before acute iTTP onset (ADAMTS13 <10% in 9 patients and 10%-26% in 5) showed VWF MM ratios significantly higher than those from 13 patients remaining in remission with ADAMTS13 <10%. At acute iTTP onset, VWF MM ratio decreased significantly and was low in all patients despite <10% ADAMTS13. The VWF MM ratio does not depend exclusively on ADAMTS13 activity. The disappearance of high molecular weight VWF MMs resulting in low VWF MM ratio at iTTP onset may be explained by consumption of larger VWF MMs in the microcirculation. The very high VWF MM ratio preceding acute iTTP recurrence suggests that VWF processing is hampered more than in patients remaining in remission.

In vitro characterization of a novel Arg102 mutation in the ADAMTS13 metalloprotease domain.

BACKGROUND: Congenital thrombotic thrombocytopenic purpura is caused by defects in the ADAMTS13 gene. ADAMTS13 is normally preactivated by conformational changes of the Metalloprotease (M) domain. Studying a novel congenital thrombotic thrombocytopenic purpura p.R102S mutation in the M domain, which results in undetectable ADAMTS13 activity in the patient, could help to explain the patients' phenotype and to elucidate the currently unclear mechanism of allosteric preactivation. OBJECTIVES: To investigate the in vitro effect of p.R102S mutation on ADAMTS13 secretion, activity, and allosteric preactivation. METHODS: Molecular modeling was used to study the effect of the mutation on the stability of ADAMTS13. Recombinant mutant ADAMTS13 was generated by transient and stable transfection of, respectively, CHO K1 and HEK293-T cells. ADAMTS13 antigen was measured in enzyme-linked immunosorbent assay. ADAMTS13 activity was measured in a FRETS-VWF73 assay. Allosteric preactivation was assessed in FRETS-VWF73 assay, using monoclonal antibody (mAb) 17G2 that normally induces a ∼2-fold increase in activity, and in enzyme-linked immunosorbent assay using mAb 6A6 recognizing a cryptic epitope in the M domain that becomes exposed after binding of 17G2. RESULTS: p.R102S mutation destabilizes the interactions between the M and Disintegrin-like (D) domain. p.R102S mutant secretion was impaired (35% of wild type) and activity was severely reduced (12% of wild type). p.R102S mutant could still be activated and the cryptic epitope of 6A6 was still fully exposed by 17G2 addition. CONCLUSION: p.R102S mutation destabilizes the M-D domain interactions, causing impaired ADAMTS13 secretion and activity, which explains the patients' phenotype. Allosteric preactivation of ADAMTS13 remains conserved in the presence of the p.R102S mutation.

ADAMTS13 conformation and immunoprofiles in Japanese patients with immune-mediated thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultrarare thrombotic disease caused by autoantibody-induced ADAMTS13 deficiency. Open ADAMST13 conformation, induced by autoantibodies, was identified as a novel biomarker for iTTP. Determining immunoprofiles in patients with iTTP has been shown to guide the development of novel targeted therapies. However, these studies were done in mainly Caucasian iTTP cohorts. To validate those findings across other ethnic cohorts, we investigated 195 acute TTP plasma samples from the Japanese iTTP registry. Seventy-six of the 195 samples had detectable ADAMTS13 antigen levels, of which 94.7% were shown to have an open ADAMTS13 conformation. A positive correlation was observed between ADAMTS13 inhibitor titers (a diagnostic parameter in Japan) and anti-ADAMTS13 immunoglobulin G autoantibody titers. Studying anti-M, anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, anti-CUB1-2 autoantibodies and the corresponding immunoprofile showed that 73% of the patients had anti-CS autoantibodies and 25.8% had anti-M autoantibodies, with the latter being higher than in Caucasians. Stratifying patients according to their immunoprofiles revealed that the profile with only anti-CS autoantibodies was the most common immunoprofile similar to that in Caucasians (28.9%). Although this profile did not affect the 1-year TTP-related mortality rate, patients with autoantibodies against all 6 ADAMTS13 fragments had a higher risk for TTP-related death than other patients (P = .02). We here validated open ADAMTS13 as a novel biomarker for acute iTTP and determined the dominant immunoprofiling in the Japanese cohort, contributing to setting up the diagnosis and managing guidelines across different ethnic cohorts and developing ADAMTS13 variants that do not bind to the anti-CS autoantibodies.

Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency in ADAMTS13. In healthy individuals, ADAMTS13 has a folded conformation in which the central spacer (S) domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G antibodies (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in patients with iTTP are directed against the different ADAMTS13 domains, but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13, thereby opening ADAMTS13. To this end, we purified anti-CS and anti-CUB autoantibodies from 13 patients with acute iTTP by affinity chromatography. The successfully purified anti-CS (10/13 patients) and anti-CUB (4/13 patients) autoantibody fractions were tested further in our ADAMTS13 conformation enzyme-linked immunosorbent assay to study whether they could open ADAMTS13. Interestingly, all purified anti-CS fractions (10/10 patients) were able to open ADAMTS13. On the other hand, only half of the purified anti-CUB fractions (2/4 patients) opened ADAMTS13. Our finding highlights that anti-CS autoantibodies that open ADAMTS13 are a common feature of the autoimmune response in iTTP.

First report of a de novo iTTP episode associated with an mRNA-based anti-COVID-19 vaccination.

Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially life-threatening thrombotic microangiopathy, characterized by disseminated thrombus formation in the microvasculature, causing severe organ failure. Immune-mediated TTP (iTTP) is occasionally described after vaccination, especially against viral agents. We report a case of a 38-year-old woman with a de novo iTTP after exposure to the mRNA-based anti-coronavirus disease 2019 (COVID-19) vaccine produced by Pfizer-BioNTech. She presented with increased bruising and petechiae starting 2 weeks after receiving the first dose of the anti-COVID-19 vaccine. Laboratory data revealed a severe ADAMTS13-deficiency in combination with a very high autoantibody titer against ADAMTS13. She was successfully treated with plasma exchange, corticosteroids, rituximab, and caplacizumab. To our knowledge, this is the first case report of iTTP after mRNA-based COVID-19 vaccination in a previously TTP-naïve patient.