Peak ADAMTS13 activity to assess ADAMTS13 conformation and risk of relapse in immune-mediated thrombotic thrombocytopenic purpura.

ABSTRACT: Previous studies have demonstrated that >38% of patients with immune-mediated thrombotic thrombocytopenic purpura in remission with activity >50% had an open ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) conformation. We assessed ADAMTS13 conformation in remission (ADAMTS13 activity >60%), focusing on peak ADAMTS13 activity levels and longitudinal assessment in 420 samples across 157 patients. Fewer cases had an open conformation at peak ADAMTS13 activity than unselected remission samples with ADAMTS13 activity >60% (23% vs 43%). Patients with a closed ADAMTS13 conformation at peak ADAMTS13 activity had an eightfold lower relapse rate in the subsequent year (9% vs 46%) and a fivefold lower relapse rate within 2 years (23% vs 62%) compared with cases with an open conformation. Patients with an open conformation at peak ADAMTS13 activity required preemptive anti-CD20 treatment earlier than those with a closed conformation (median, 10 vs 25 months). Longitudinally, an open conformation was evident at, and often preceded relapse. When the conformation was already open before relapse, an increase in the conformation index at relapse was seen despite the undetectable anti-ADAMTS13 immunoglobulin G (IgG) antibody. In cases with detectable anti-ADAMTS13 IgG antibody, these became undetectable before achieving a closed conformation, highlighting the relapse risk even with undetectable anti-ADAMTS13 IgG antibody and the clinical utility of open/closed during monitoring. To our knowledge, this is the first study to show an association between relapse risk and ADAMTS13 conformation when activity levels are at a peak. The open conformation identifies antibody-mediated subclinical disease that is not detectable by the current ADAMTS13 testing.

ADAMTS13 activity testing: evaluation of commercial platforms for diagnosis and monitoring of thrombotic thrombocytopenic purpura.

Background: ADAMTS13 activity is one of the key investigations needed to diagnose thrombotic thrombocytopenic purpura, and there are a number of different assays available to measure it. HemosIL AcuStar, a chemiluminescent immunoassay, was developed and used as a quicker, automated test. In clinical practice, discrepancies between AcuStar and the gold standard FRETS-VWF73 have been documented in a manner that would affect diagnosis and treatment. Objectives: We aimed to identify and highlight clinical situations where this discrepancy occurs and to attempt to determine the cause. Method: Therefore, we undertook a study to compare the FRETS-VWF73 assay with AcuStar, the Technozym Activity ELISA, and Ceveron FRET assays using a mixture of 94 retrospective and prospective patient samples. Results: We found that although the concordance between FRETS-VWF73 and the other methods was generally very good, discrepancies were found in a small number tested on AcuStar affecting diagnosis (5 of 32) and follow-up (7 of 51). A Wilcoxon test comparing FRETS-VWF73 to the AcuStar results suggested that the AcuStar results were significantly lower in 42 samples tested on all 4 platforms. We investigated potential causes for this difference by testing the impact of high vWF levels and addition of a monoclonal ADAMTS13 autoantibody (3H9) to samples. We found no impact of high vWF levels on interassay variability but found that 3H9 reduced ADAMTS13 activity levels much more in AcuStar and ELISA assays than in FRETS assays. Conclusion: Based on our findings, we would suggest that when AcuStar is used upfront to guide management, a second testing method should be used in patients with an atypical thrombotic thrombocytopenic purpura presentation or unexpectedly slow ADAMTS13 recovery.

Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome.

BACKGROUND: Post-COVID syndrome (PCS) affects millions of people worldwide, causing a multitude of symptoms and impairing quality of life months or even years after acute COVID-19. A prothrombotic state has been suggested; however, underlying mechanisms remain to be elucidated. OBJECTIVES: To investigate thrombogenicity in PCS using a microfluidic assay, linking microthrombi, thrombin generation, and the von Willebrand factor (VWF):a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13 (ADAMTS13) axis. METHODS: Citrated blood was perfused through microfluidic channels coated with collagen or an antibody against the VWF A3 domain, and thrombogenicity was monitored in real time. Thrombin generation assays were performed and α(2)-antiplasmin, VWF, and ADAMTS13 activity levels were also measured. RESULTS: We investigated thrombogenicity in a cohort of 21 patients with PCS with a median time following symptoms onset of 23 months using a dynamic microfluidic assay. Our data show a significant increase in platelet binding on both collagen and anti-VWF A3 in patients with PCS compared with that in controls, which positively correlated with VWF antigen (Ag) levels, the VWF(Ag):ADAMTS13 ratio (on anti-VWF A3), and inversely correlated with ADAMTS13 activity (on collagen). Thrombi forming on collagen presented different geometries in patients with PCS vs controls, with significantly increased thrombi area mainly attributable to thrombi length in the patient group. Thrombi length positively correlated with VWF(Ag):ADAMTS13 ratio and thrombin generation assay results, which were increased in 55.5% of patients. α(2)-Antiplasmin levels were normal in 89.5% of patients. CONCLUSION: Together, these data present a dynamic assay to investigate the prothrombotic state in PCS, which may help unravel the mechanisms involved and/or establish new therapeutic strategies for this condition.

Evaluation of N-aryl-ß-alanine derivatives as anticancer agents in triple-negative breast cancer and glioblastoma in vitro models.

Synthesis of ß-amino acid derivatives containing hydrazone and azole moieties is described. For this purpose, the appropriate hydrazide was treated with aromatic aldehydes, ketones and phenyl iso(thio)cyanates to obtain the desired outcome. The synthesized target compounds were evaluated for their anticancer properties. The assay displayed 3,3'-((2,6-diethylphenyl)azanediyl)bis(N'-(benzylidene)propanehydrazide) to possess the convincing anticancer effect against triple-negative breast cancer cells in vitro. To further study the anticancer properties of compounds containing a hydrazone moiety in breast cancer, series of previously and newly prepared dihydrazones were investigated. It was determined that derivatives with the bis(N'-(4-bromobenzylidene) fragment in the structure are exclusively cytotoxic to cancer cells. The most active compounds against both cell lines were those containing electron withdrawing 4-BrPh or 4-ClPh moieties, together with either chlorine, bromine or iodine groups in para position of phenyl ring. Selected two representative compounds showed migrastatic activity in MDA-MB-231 cell line, where both of them reduced the growth of breast cancer and glioblastoma cell 3D cultures and inhibited cell colony formation. 2009 Elsevier Ltd. All rights reserved.