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Background: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total., Methods: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes., Results: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels ( GCKR , BAZ1B , and HP-TXNL4B ) and 1 with PS levels ( ORM1 - ORM2 ). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level ( FCGRT ), 1 with PC ( GOLM2 ), and 1 with PS ( MYL7 ). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR , SNX17 , and HP genes in antithrombin regulation., Conclusions: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels., Competing Interests: Disclosures None.

The development of algorithms for remote sensing of water quality (RSWQ) requires a large amount of in situ data to account for the bio-geo-optical diversity of inland and coastal waters. The GLObal Reflectance community dataset for Imaging and optical sensing of Aquatic environments (GLORIA) includes 7,572 curated hyperspectral remote sensing reflectance measurements at 1 nm intervals within the 350 to 900 nm wavelength range. In addition, at least one co-located water quality measurement of chlorophyll a, total suspended solids, absorption by dissolved substances, and Secchi depth, is provided. The data were contributed by researchers affiliated with 59 institutions worldwide and come from 450 different water bodies, making GLORIA the de-facto state of knowledge of in situ coastal and inland aquatic optical diversity. Each measurement is documented with comprehensive methodological details, allowing users to evaluate fitness-for-purpose, and providing a reference for practitioners planning similar measurements. We provide open and free access to this dataset with the goal of enabling scientific and technological advancement towards operational regional and global RSWQ monitoring., (© 2023. The Author(s).)

Background: Recombinant human ADAMTS13 (rADAMTS13) is a key protein in fundamental research for investigating its mode of action and the pathophysiology of thrombotic thrombocytopenic purpura (TTP). However, the expression of rADAMTS13 is quite low in mammalian cells, which makes the production of the protein time-consuming and labor-intensive., Objectives: We aimed at increasing the yield of rADAMTS13 by (1) using a more optimal signal peptide (SP) and (2) constructing an N-terminal fusion protein of ADAMTS13 with human serum albumin domain 1 (AD1-ADAMTS13)., Methods: Six SPs were investigated to select the most optimal SP. Expression plasmids containing the most optimal SP and ADAMTS13 cDNA or the fusion construct AD1-ADAMTS13 were generated and transiently transfected into CHOEBNALT85 cell-line. Expression levels of rADAMTS13 in expression medium were analyzed and compared with the expression level of rADAMTS13 with native SP (nat-SP)., Results: Expression of rADAMTS13 with coagulation factor VII (FVII) SP was 3-fold higher (16.00 μg/ml) compared with the expression with nat-SP (5.03 μg/ml). The highest yields were obtained with AD1-ADAMTS13 protein with a 15-fold higher concentration (78.22 μg/ml) compared with the expression with nat-SP. The rADAMTS13 expressed with FVII-SP retained its activity (104.0%) to cleave von Willebrand factor, whereas AD1-ADAMTS13 demonstrated even higher activity (144.3%)., Conclusion: We succeeded in generating expression vectors that yield (1) rADAMTS13 at higher levels because of more optimal FVII-SP and (2) high levels of AD1-ADAMTS13 N-terminal fusion protein. The highest expression levels were obtained with AD1-ADAMTS13 N-terminal fusion protein, which is paving the way for highly efficient protein production., (© 2022 International Society on Thrombosis and Haemostasis.)

The high number of mutations in the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes its immune escape. We report a longitudinal analysis of 111 vaccinated individuals for their antibody levels up to 6 months after the third dose of the BNT162b2 vaccine. After the third dose, the antibody levels decline but less than after the second dose. The booster dose remarkably increases the serum ability to block wild-type or Omicron variant spike protein's receptor-binding domain (RBD) interaction with the angiotensin-converting enzyme 2 (ACE2) receptor, and these protective antibodies persist 3 months later. Three months after the booster dose, memory CD4 + and CD8 + T cells to the wild-type and Omicron variant are detectable in the majority of vaccinated individuals. Our data show that the third dose restores the high levels of blocking antibodies and enhances T cell responses to Omicron., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2-specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum immediately before calving. Here, we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks the interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persisted on the nasal mucosa for at least 4 hours, as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for rapid and versatile adaptation for preparing prophylactic treatments against other diseases using the defined characteristics of antibody movement into the colostrum., Competing Interests: The use of bovine colostrum as a prophylactic agent against SARS-CoV-2 has been patented (US patent application no 63/160,833) by Mario Plaas, K. Kogermann, E. Žusinaite, T. Tiirats, B. Aasmäe, A. Kavak, V. Poikalainen, L. Lepasalu, S. Piiskop, S. Rom, R. Oltjer, K. Kangro, E. Sankovski, J. M. Gerhold, R. Pert, A. Männik, A. Planken, A. Tover, M. Kurašin, M. Ustav and M. Ustav Jr.

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., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: SARS-CoV-2 mRNA vaccines have proven high efficacy, however, limited data exists on the duration of immune responses and their relation to age and side effects., Methods: We studied the antibody and memory T cell responses after the two-dose BNT162b2 vaccine in 122 volunteers up to 6 months and correlated the findings with age and side effects., Findings: We found a robust antibody response to Spike protein after the second dose. However, the antibody levels declined at 12 weeks and 6 months post-vaccination, indicating a waning of the immune response over time. At 6 months after the second dose, the Spike antibody levels were similar to the levels in persons vaccinated with one dose or in COVID-19 convalescent individuals. The antibodies efficiently blocked ACE2 receptor binding to SARS-CoV-2 Spike protein of five variants of concern at one week but this was decreased at three months. 87% of individuals developed Spike-specific memory T cell responses, which were lower in individuals with increased proportions of immunosenescent CD8 + TEMRA cells. We found antibody response to correlate negatively with age and positively with the total score of vaccination side effects., Interpretation: The mRNA vaccine induces a strong antibody response to SARS-CoV-2 and five VOCs at 1 week post-vaccination that decreases thereafter. T cell responses, although detectable in the majority, were lower in individuals with higher T cell immunosenescence. The deterioration of vaccine response suggests the need to monitor for the potential booster vaccination., Competing Interests: The authors have nothing to disclose., (© 2021 The Authors.)

The frequency of heatwave events in Europe is increasing as a result of climate change. This can have implications for the water quality and ecological functioning of aquatic systems. We deployed three spectroradiometer WISPstations at three sites in Europe (Italy, Estonia, and Lithuania/Russia) to measure chlorophyll-a at high frequency. A heatwave in July 2019 occurred with record daily maximum temperatures over 40 °C in parts of Europe. The effects of the resulting storm that ended the heatwave were more discernable than the heatwave itself. Following the storm, chlorophyll-a concentrations increased markedly in two of the lakes and remained high for the duration of the summer while at one site concentrations increased linearly. Heatwaves and subsequent storms appeared to play an important role in structuring the phenology of the primary producers, with wider implications for lake functioning. Chlorophyll-a peaked in early September, after which a wind event dissipated concentrations until calmer conditions returned. Synoptic coordinated high frequency monitoring needs to be advanced in Europe as part of water management policy and to improve knowledge on the implications of climate change. Lakes, as dynamic ecosystems with fast moving species-succession, provide a prism to observe the scale of future change.

Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) autoantibodies cause a severe ADAMTS13 deficiency in immune-mediated thrombotic thrombocytopenic purpura (iTTP). ADAMTS13 consists of a metalloprotease (M), a disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). We recently developed a high-throughput epitope mapping assay based on small, nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2). With this assay, we performed a comprehensive epitope mapping using 131 acute-phase samples and for the first time a large group of remission samples (n = 50). Next, samples were stratified according to their immunoprofiles, a field that is largely unexplored in iTTP. Three dominant immunoprofiles were found in acute-phase samples: profile 1: only anti-CS autoantibodies (26.7%); profile 2: both anti-CS and anti-CUB1-2 autoantibodies (12.2%); and profile 3: anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, and anti-CUB1-2 autoantibodies (8.4%). Interestingly, profile 1 was the only dominant immunoprofile in remission samples (52.0%). Clinical data were available for a relatively small number of patients with acute iTTP (>68), and no correlation was found between immunoprofiles and disease severity. Nevertheless, profile 1 was linked with younger and anti-T2-T5 autoantibodies with older age and the absence of anti-CUB1-2 autoantibodies with cerebral involvement. In conclusion, identifying acute phase and remission immunoprofiles in iTTP revealed that anti-CS autoantibodies seem to persist or reappear during remission providing further support for the clinical development of a targeted anti-CS autoantibody therapy. A large cohort study with acute iTTP samples will validate possible links between immunoprofiles or anti-domain autoantibodies and clinical data., (© 2021 by The American Society of Hematology.)

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by inhibitory and/or clearing anti-ADAMTS-13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13) autoantibodies. To determine the presence and total level of anti-ADAMTS-13 autoantibodies, commercial and in-house developed ELISAs are performed. However, different ELISA methods vary in relation to the presentation of recombinant (r)ADAMTS-13 and the detection method of the anti-ADAMTS-13 autoantibodies. Currently, the influence of those different approaches on anti-ADAMTS-13 autoantibody titers is not known., Objectives: To assess the influence of different ADAMTS-13 presentation- and autoantibody detection methods on anti-ADAMTS-13 autoantibody titers in ELISA., Materials/methods: Anti-ADAMTS-13 autoantibody titers from 18 iTTP patients were determined using four different set-ups of anti-ADAMTS-13 autoantibody ELISAs. The ELISAs varied in the used presentation of rADAMTS-13 (directly coated full-length rADAMTS-13, directly coated rMDTCS and rT2C2, or antibody-captured full-length rADAMTS-13) and the detection antibodies (polyclonal anti-human IgG or monoclonal anti-human IgG 1-4 antibodies)., Results: Strong correlations between the different anti-ADAMTS-13 autoantibody ELISA approaches were observed, when using polyclonal anti-human IgG detection antibodies recognizing all IgG subclasses similarly, independent of the method of rADAMTS-13 presentation. Anti-ADAMTS-13 autoantibody titers correlated less when using a mixture of monoclonal anti-human IgG 1-4 , because not all IgG subclasses were recognized with similar affinities., Conclusion: Anti-ADAMTS-13 autoantibody levels using different methods of rADAMTS-13 presentation strongly correlate. However, the levels of anti-ADAMTS-13 autoantibodies are highly dependent on the detection antibody used, which should detect all IgG subclasses (IgG 1-4 ) equally well., (© 2021 International Society on Thrombosis and Haemostasis.)

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., (© 2021 by The American Society of Hematology.)

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., (© 2020 International Society on Thrombosis and Haemostasis.)

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated 'MDTCS' variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.

Following an acute thrombotic thrombocytopenic purpura (TTP) episode, patients are at risk for relapse, and a careful long-term follow-up is needed. Adherence to the follow-up by patients implies a good understanding of the disease. However, TTP literacy in patients is currently unknown. To explore the TTP literacy in patients and identify factors associated with poor disease understanding, a questionnaire was developed focusing on patient's characteristics, knowledge about TTP and patients' actions in an emergency. The questionnaire was presented to 120 TTP patients in remission from the French National Registry for Thrombotic Microangiopathies. TTP literacy was low in 24%, intermediate in 43% and high in 33% of the patients. Low TTP literacy was associated with older age and low education level. Among the knowledge gaps identified, few patients knew that plasma exchange in acute phase is mandatory and has to be done daily (39%), 47% of participants did not consider themselves at risk for relapse, and 30% of women did not know that pregnancy exposes them to a greater risk of relapse. Importantly, few patients responded about life-saving actions in an emergency. Hence, the design of educational material should pay special attention to the age and education level of the target population focusing on the events leading to TTP, the importance of the emergency treatment, controllable predisposing factors for TTP development and patient attitude in an emergency., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Background: In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments., Objectives: We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients., Methods: A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples., Results: Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2-T5, T6-T8, and CUB1-2 were sufficient to accurately determine the antibody-binding sites., Conclusion: We have developed a tool to profile patients with iTTP according to their anti-ADAMTS13 antibodies for a better insight in their immune response., (© 2020 Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains., Competing Interests: The authors declare no conflict of interest.