Patients suffering from acquired thrombotic thrombocytopenic purpura develop autoantibodies directed toward the plasma glycoprotein ADAMTS13. Here, we studied the glycan composition of plasma-derived ADAMTS13. Purified ADAMTS13 was reduced, alkylated, and processed into peptides with either trypsin or chymotrypsin. Glycopeptides were enriched using zwitterionic HILIC zip-tips and analyzed by tandem mass spectrometry employing higher-energy collision dissociation fragmentation. Upon detection of a diagnostic ion of a glycan fragment, electron transfer dissociation fragmentation was performed on the same precursor ion. The majority of N-linked glycans were of the complex type containing terminal sialic acids and fucose residues. A high mannose-containing glycan was attached to Asn614 in the spacer domain. Six O-linked glycans mostly terminating in sialic acid were found dispersed over ADAMTS13. Five O-linked glycans were attached to a Ser and one to Thr. All 6 O-linked glycans contained a terminal sialic acid. O-fucosylation is a common posttranslational modification of thrombospondin type 1 repeats. We identified 7 O-fucosylation sites in the thrombospondin (TSP) type 1 repeats. Unexpectedly, one additional O-fucosylation site was found in the disintegrin domain. This O-fucosylation site did not meet the proposed consensus sequence CSX(S/T)CG. C-mannosylation sites were identified in TSP1, linker TSP4-TSP5, and TSP8. Overall, our findings highlight the complexity of glycan modifications on ADAMTS13, which may have implications for its interaction with immune- or clearance receptors containing carbohydrate recognition domains.

Key Points • CD41 T-cell responses in 2 patients with acquired TTP.• CUB2 domain-derived core peptides are recognized by CD4 1 T cells present in 2 patients with acquired TTP.Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder resulting from the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). HLA-DRB1*11 provides a risk factor for developing acquired TTP. Pulsing of antigen-presenting cells from HLA-DRB1*11-and HLA-DRB1*03-positive individuals with ADAMTS13 resulted in presentation of peptides derived from the CUB2 domain of ADAMTS13 with core sequences FINVAPHAR or ASYILIRD. Here, we assessed whether FINVAPHAR-or ASYILIRD-reactive CD4 IntroductionThe autoimmune disorder thrombotic thrombocytopenic purpura (TTP) is characterized by the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in previously healthy individuals.

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Summary. Background: Acquired deficiency of ADAMTS13 causes a rare and life-threatening disorder called thrombotic thrombocytopenic purpura (TTP). Several studies have shown that aberrant glycosylation can play an important role in the pathogenesis of autoimmune diseases. N-linked glycosylation and putative O-fucosylation sites have been predicted or identified in recombinant ADAMTS13. However, it is not known which of these sites are glycosylated in plasma derived ADAMTS13. Objectives: Here we investigated the presence of putative O-fucosylation, C-mannosylation and N-linked glycosylation sites on plasma derived ADAMTS13. Methods/ Results: Sites of N-linked glycosylation were determined by the use of peptide N-glycosidase-F (PNGase F), which removes the entire carbohydrate from the side chain of asparagines. Nine of the 10 predicted N-linked glycosylation sites were identified in or near the metalloproteinase, spacer, thrombospondin type 1 repeat (TSR1) and the CUB domain of plasma ADAMTS13. Moreover, six putative O-fucosylated sites were identified in the TSR domains of plasma ADAMTS13 by performing searches of the tandem mass spectrometry (MS/MS) data for loss of hexose (162 Da), deoxyhexose (146 Da), or hexosedeoxyhexose (308 Da). The use of electron transfer dissociation (ETD) allowed for unambiguous identification of the modified sites. In addition to putative O-fucosylation and N-linked glycosylation, two putative C-mannosylation sites were identified within the TSR1 and TSR4 domains of ADAMTS13. Conclusions: Our data identify several glycosylation sites on plasma derived ADAMTS13. We anticipate that our findings may be relevant for the initiation of autoimmune reactivity against ADAMTS13 in patients with acquired TTP.

Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies, we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total, 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11, several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an in silico analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T-cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura.

Key Points• Uptake of ADAMTS13 by macrophages is not dependent on the macrophage mannose receptor.• CD163 promotes internalization of ADAMTS13 by macrophages.Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry.Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan;however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13. Recent research suggests that HLA-DRB1*11 provides a risk factor for the development of acquired TTP. Previous work in our department identified ADAMTS13 derived peptides that are presented on MHC class II (Sorvillo et al. 2013). Pulsing of dendritic cells from HLA typed healthy donors showed preferential presentation of the CUB-2 domain derived peptides "FINVAPHAR" and "ASYILIRD" on HLA-DRB1*11 and HLA DRB1*03 respectively. In this study we screened peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with acquired TTP for reactive CD4+ T cells against the previously identified peptides. The presence of CD4+ T cells recognizing FINVAPHAR or the ASYILIRD peptide was addressed by flow cytometry. PBMCs were stimulated for 24 hours and up regulation of CD40L was visualized on CD4+ T cells. Stimulation with Staphylococcus aureus enterotoxin B (SEB) and overlapping peptides from cytomegalovirus (CMV) pp65 protein were used as controls. Several samples derived from patients in the acute phase of the disease were analyzed in this study. A high frequency of FINVAPHAR responsive CD4+ T cells was identified by monitoring the up regulation of CD40L on CD4+ T cells in a sample from a HLA-DRB1*1104 patient. Four percent of the CD4+ T cells were activated in response to the FINVAPHAR peptide. A control peptide in which the anchor-residues of the FINVAPHAR peptide were modified did not induce activation of CD4+ T cells. Incubation with full length ADAMTS13 also induced activation of CD4+ T cells although to a lesser extent. No FINVAPHAR reactive CD4+ T cells were observed after stimulation in HLA-DRB1*1104 positive healthy individuals. PBMC samples from HLA-DRB1*0301 positive patients were screened for CD4+ T cells reactive with the ASYILIRD peptide. ASYILIRD positive CD4+ T cells were detected in samples of 2 DRB1*0301 positive patients. In one patient 0.5% reactive CD4+ T cells were detected after stimulation with the ASYILIRD peptide. In samples derived from a second patient 1.5% of CD4+ T cells reactive against the ASYILIRD peptide were detected. Stimulation with a control peptide in which the anchor residues were modified did not result in activation of CD4+ T cells in both patients. Stimulation with SEB and CMV derived peptides resulted in activation of CD4+ T cells in the samples analyzed. No CD40L+ CD4+ T cells were observed after incubation with full length ADAMTS13, this may be due to insufficient processing of ADAMTS13 under our experimental conditions. Taken together our data provide the first evidence for the presence of ADAMTS13 reactive CD4+ T cells in acquired TTP. Based on our results we propose that FINVAPHAR and ASYILIRD CD4+ T cells are involved in the onset and/or relapse of acquired TTP. Disclosures No relevant conflicts of interest to declare.

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