Search

Immune thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare, life-threatening disorder, mediated through severe ADAMTS13 deficiency causing multi-system micro-thrombi formation, and has specific human leukocyte antigen associations. We undertook a large genome-wide association study to investigate additional genetically distinct associations in iTTP. We compared two iTTP patient cohorts with controls, following standardized genome-wide quality control procedures for single-nucleotide polymorphisms and imputed HLA types. Associations were functionally investigated using expression quantitative trait loci (eQTL), and motif binding prediction software. Independent associations consistent with previous findings in iTTP were detected at the HLA locus and in addition a novel association was detected on chromosome 3 (rs9884090, P=5.22x10-10, odds ratio 0.40) in the UK discovery cohort. Meta-analysis, including the French replication cohort, strengthened the associations. The haploblock containing rs9884090 is associated with reduced protein O-glycosyltransferase 1 (POGLUT1) expression (eQTL P<0.05), and functional annotation suggested a potential causative variant (rs71767581). This work implicates POGLUT1 in iTTP pathophysiology and suggests altered post-translational modification of its targets may influence disease susceptibility.

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.

Thrombotic thrombocytopenic purpura (TTP) is caused by severe deficiency of ADAMTS13 (A13), a plasma metalloprotease that cleaves endothelium-derived von Willebrand factor (VWF). However, severe A13 deficiency alone is often not sufficient to cause an acute TTP; additional factors may be required to trigger the disease. Using CRISPR/Cas9, we created and characterized several novel zebrafish lines carrying a null mutation in a13 -/- , vwf , and both. We further used these zebrafish lines to test the hypothesis that inflammation that results in neutrophil activation and release of histone/DNA complexes may trigger TTP. As shown, a13 -/- zebrafish exhibit increased levels of plasma VWF antigen, multimer size, and ability of thrombocytes to adhere to a fibrillar collagen-coated surface under flow. The a13 -/- zebrafish also show an increased rate of occlusive thrombus formation in the caudal venules after FeCl 3 injury. More interestingly, a13 -/- zebrafish exhibit ~30% reduction in the number of total, immature, and mature thrombocytes with increased fragmentation of erythrocytes. Administration of a lysine-rich histone results in more severe and persistent thrombocytopenia and a significantly increased mortality rate in a13 -/- zebrafish than in wildtype ( wt ) ones. However, both spontaneous and histone-induced TTP in a13 -/- zebrafish are rescued by the deletion of vwf These results demonstrate a potentially mechanistic link between inflammation and the onset of TTP in light of severe A13 deficiency; the novel zebrafish models of TTP may help accelerate our understanding of pathogenic mechanisms and the discoveries of novel therapeutics for TTP and perhaps other arterial thrombotic disorders., (Copyright© 2020 Ferrata Storti Foundation.)

Congenital thrombotic thrombocytopenic purpura is an autosomal recessive inherited disease with a clinically heterogeneous course and an incompletely understood genotype-phenotype correlation. In 2006, the Hereditary TTP Registry started recruitment for a study which aimed to improve the understanding of this ultra-rare disease. The objective of this study is to present characteristics of the cohort until the end of 2017 and to explore the relationship between overt disease onset and ADAMTS13 activity with emphasis on the recurring ADAMTS13 c.4143&#95;4144dupA mutation. Diagnosis of congenital thrombotic thrombocytopenic purpura was confirmed by severely deficient ADAMTS13 activity (≤10% of normal) in the absence of a functional inhibitor and the presence of ADAMTS13 mutations on both alleles. By the end of 2017, 123 confirmed patients had been enrolled from Europe (n=55), Asia (n=52, 90% from Japan), the Americas (n=14), and Africa (n=2). First recognized disease manifestation occurred from around birth up to the age of 70 years. Of the 98 different ADAMTS13 mutations detected, c.4143&#95;4144dupA (exon 29; p.Glu1382Argfs*6) was the most frequent mutation, present on 60 of 246 alleles. We found a larger proportion of compound heterozygous than homozygous carriers of ADAMTS13 c.4143&#95;4144dupA with overt disease onset at < 3 months of age (50% vs 37%), despite the fact that ADAMTS13 activity was <1% in 18 of 20 homozygous, but in only 8 of 14 compound heterozygous carriers. An evaluation of overt disease onset in all patients with an available sensitive ADAMTS13 activity assay (n=97) shows that residual ADAMTS13 activity is not the only determinant of age at first disease manifestation. Registered at clinicaltrials.gov identifier NCT01257269 ., (Copyright© 2019 Ferrata Storti Foundation.)

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13 th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4 + T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4 + T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4 + T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4 + T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS13 1239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4 + T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4 + T cells from Sure-L1 mice and was recognized by CD4 + T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS13 1239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4 + T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS13 1239-1253 -loaded HLA-DR tetramers., (Copyright© Ferrata Storti Foundation.)

The inherited deficiency of ADAMTS13 is usually associated with severe forms of thrombotic thrombocytopenic purpura. Among the mutations identified in the ADAMTS13 gene, none have been described on the TSP1-6 repeat domain. We investigated an Iranian family with a history of chronic recurrent thrombotic thrombocytopenic purpura, severe ADAMTS13 deficiency and a heterogeneous pattern of clinical symptoms among affected members. Genetic analysis revealed a homozygous deletion of nucleotides 2930-2935 (GTGCCC) in exon 23 of ADAMTS13, leading to the replacement of Cys977 by a Trp and the deletion of Ala978 and Arg979 in the TSP1-6 repeat domain. To explore the mechanism of ADAMTS13 deficiency, in vitro expression studies were performed. Western blotting, pulse-chase labeling and immunofluorescence studies demonstrated a secretion pathway defect of the mutant protein, with no intracellular accumulation. This finding is consistent with the severe ADAMTS13 deficiency but does not explain the heterogeneous clinical picture of the 3 siblings carrying the same mutation.