Abstract: ADAMTS13 contains complex type N-linked glycans, which contain terminal mannose, sialic acids, and fucose residues. TSP1 repeats are modified by O-fucosylation and C-mannosylation; O-fucosylation was also observed in the disintegrin domain.

Abstract: Von Willebrand Factor (VWF) cleaving protease ADAMTS13 is responsible for proteolysis of ultra large VWF multimers in the circulation. ADAMTS13 is synthesized by hepatic stellate cells in the liver. Also endothelial cells have been suggested to contribute to the synthesis of ADAMTS13. In patients with acquired thrombotic thrombocytopenic purpura auto-reactive antibodies are formed primarily against the spacer domain of ADAMTS13. Previously we have shown that monocyte-derived dendritic cells were able to endocytose ADAMTS13 via the macrophage mannose receptor and subsequently process it into peptides and present it on MHC class II. However, it is currently unclear which receptor contributes to the clearance of ADAMTS13 from the circulation. The half-life of ADAMTS13 was measured following plasma infusion in patients with congenital TTP and was found to vary between 2.1 and 3.3 days. Internalization of ADAMTS13 by tissue-resident macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms contributing to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages (MDMs) were used to monitor the uptake of fluorescently labelled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin-inhibitor dynasore. Partial blockage of ADAMTS13 internalization was observed employing mannan; uptake however was not affected by a blocking antibody directed towards the macrophage mannose receptor CD206. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the hemoglobin scavenger receptor CD163 as a candidate receptor for ADAMTS13. CD163 is primarily expressed by the monocyte-macrophage lineage and is highly expressed on type-2 macrophages present in the bone marrow, the red pulp of the spleen and in the liver on Kupffer cells. Blocking experiments with a monoclonal anti-CD163 antibody EDHu-1 resulted in a decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in macrophages in which the expression of CD163 was boosted upon incubation with IL-10. In agreement with these findings CD163-expressing CHO cells but not CHO CD163-/- cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed high affinity binding of ADAMTS13 to soluble CD163 containing scavenger receptor cysteine-rich (SRCR) domain 1-9. Our results position CD163 as a novel binding partner for ADAMTS13 and suggest that binding of ADAMTS13 to CD163 promotes its internalization by macrophages. Disclosures No relevant conflicts of interest to declare.

Abstract: Background: Patients’, parents’ and providers’ preferences with regard to medical interventions may have a major impact on the implementation of innovations, often delaying initiation significantly. Illustratively, as early as 1997, Carlsson et al. suggested that a 30% reduction of consumption of clotting factor concentrate in prophylactic treatment could be attained by dosing based on an individual pharmacokinetic (PK) profile, with a concomitant cost reduction. Therefore, we aim to evaluate do’s and don’ts in hemophilia patients, parents and professionals with regard to individualized dosing according to PK-profile of prophylaxis with clotting factor concentrate. This in order to successfully implement this intervention when imperative. Methods: In this study we included patientswith hemophilia currently or previously on prophylactic treatment with clotting factor concentrate (n=114) and parents of patients aged 12-18 years (n=19) from five Dutch Academic Hemophilia Treatment Centers, and hemophilia professionals attending the World Federation of Haemophia congress 2012 from throughout the world (n=91). Data were analysed using a Discrete Choice Experiment. Patients’, parents’ and professionals’ preferences with regard to the intervention, are measured by specific attributes with varying levels: ‘number of blood samples necessary to construct individual PK-profile’, ‘advised frequency of prophylactic infusions’, ‘frequency of repetitive PK-profiling’, ‘risk of bleeding’, ‘estimated cost reduction of treatment with benefit for society’. Results: For patients and parents (response rate 64%), a higher dosing frequency e.g. daily dosing was an important barrier. They were however willing to infuse more frequently, if bleeding was consequently reduced. ‘Reduction of costs for society’ by implementation of individualized dosing according to PK profile was found relevant and motivating to implement PK-guided dosing. For professionals the most important attributes driving implementation were an acceptable ‘advised frequency of prophylactic infusions’ and reduction of ‘risk of bleeding’. Conclusions: When anticipating implementation of a medical intervention, defining of preferences of those involved is of importance. In case of PK-guided prophylactic dosing in hemophilia conclusions are: realise the impact of daily dosing of clotting factor concentrate, use frequent bleeding as a motivator to initiate PK-guided dosing and actively discuss costs of treatment with those undergoing treatment and the cost reduction that may result from PK-guided dosing. Identification of these preferences will secure successful implementation in the near future. Disclosures Lock: ZonMW: Research Funding; Baxter: Research Funding. Laros-van Gorkum:Sanquin: speakers fee Other; Baxter: Unrestricted educational grant was provided to the Hemophilia Treatment Center of the Radboud university medical center, Unrestricted educational grant was provided to the Hemophilia Treatment Center of the Radboud university medical center Other; CSL Behring: Unrestricted educational grant was provided to the Hemophilia Treatment Center of the Radboud university medical center, Unrestricted educational grant was provided to the Hemophilia Treatment Center of the Radboud university medical center Other. Driessens:Baxter: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other; Bayer Schering Pharma: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work, unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other; CSL Behring: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work, unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other; Eurocept: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work, unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other; Novo Nordisk: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other; Pfizer: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other; Sanquin: unrestricted grant for meetings and educational courses with hemophilia patients and members of the Netherlands Hemophilia Patient Society, outside the submitted work Other. Fijnvandraat:Baxter: Member of the European Hemophilia Treatment and Standardisation Board sponsored by Baxter Other; CSL Behring: Research Funding; Pfizer: Has given lectures at educational symposiums organised by Pfizer, Has given lectures at educational symposiums organised by Pfizer Other, Research Funding; Bayer Schering Pharma: Has given lectures at educational symposiums organised by Bayer Schering Pharma, Has given lectures at educational symposiums organised by Bayer Schering Pharma Other, Research Funding. Leebeek:CSL Behring: has served on advisory boards of CSL Behring, outside the submitted work Other, Research Funding; Baxter: has served on advisory boards of Baxter, outside the submitted work, has served on advisory boards of Baxter, outside the submitted work Other. Cnossen:Pfizer: Educational funding Other, Research Funding; Bayer Schering Pharma: Educational funding and travel support, Educational funding and travel support Other, Research Funding; Baxter: Educational funding and travel support, Educational funding and travel support Other, Research Funding; Novo Nordisk: Educational funding, Educational funding Other, Research Funding; Novartis: Educational funding and travel support Other, Research Funding.

Abstract: Activated neutrophils can suppress T-cell proliferation in a CD11b-dependent multistep process involving ROS production and degranulation. MDSC activity results in nonapoptotic T-cell damage.

Abstract: Development of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 domain of FVIII. We created a C1 domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4+ T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.

Abstract: Hemophilia is a congenital bleeding disorder caused by low levels of clotting factor VIII or IX. The life expectancy of people with hemophilia (PWH) has increased with the availability of clotting factor concentrates. At the same time, the incidence of cardiovascular disease (CVD) has increased; in retrospective studies, there are conflicting data regarding if, despite this increase, the incidence is still lower than in the general population. We prospectively compared the incidence of CVD in PWH vs the predicted incidence. This prospective, multicenter, observational study included adult PWH (aged & gt;30 years) from The Netherlands and United Kingdom. They were followed up for a 5-year period, and CVD incidence was compared with a predicted event rate based on the QRISK2-2011 CVD risk model. The primary end point was the observed fatal and nonfatal CVD incidence after 5 years compared with the estimated events and in relation to severity of hemophilia. The study included 709 patients, of whom 687 (96.9%) completed 5 years’ follow-up or reached an end point. For 108 patients, the QRISK score could not be calculated at inclusion. For the remaining 579, fewer CVD events were observed than predicted: 9 vs 24 (relative risk, 0.38; 95% confidence interval, 0.18-0.80; P = .01), corresponding with an absolute risk reduction of 2.4%. Severe hemophilia treated on demand had the highest risk reduction. There was no statistically significant relation between severity of hemophilia and incidence of CVD. In hemophilia, a lower-than-predicted CVD incidence was found, supporting the theory that hemophilia protects against CVD. The study is registered at www.clinicaltrials.gov as #NCT01303900.

Abstract: Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13 disrupting the binding of ADAMTS13 to von Willebrand factor and thereby preventing the proteinase activity and/or increasing the clearance from the circulation. Previous research from our department identified 9 O-linked glycosylation, 6 O-fucosylation and 2 C-mannosylation sites on plasma derived ADAMTS13. One of the N-linked glycosylation sites (N1354) is close to one of the previously identified peptides preferentially presented on HLA-DRB1*0301 and HLA-DRB1*1501 (ASYILIRD amino acid A1355-D1362) and also close to the HLA-DRB1*1101 peptide (FINVAPHAR amino acid F1328-R1336) suggesting a possible role for the glycosylation in the onset of acquired TTP. To study the glycosylation and glycan trees ADAMTS13 purified from cryosupernatant was reduced with dithiothreitol, alkylated with iodoacetamide and subsequently processed into peptides overnight with either trypsin or chymotrypsin. The peptides were then purified using ZIC-HILIC proteatips and finally analyzed by tandem mass spectrometry employing both higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD). The data files were analyzed using the BYONIC software package as well as manually. Using this approach we identified the glycan structure on 10 N-linked glycosylation. Nine out of 10 glycans contained complex carbohydrate structures terminating in sialic acid. The glycans at these N-linked sites were identified both with or without a fucose on the primary GlcNAc. We were unable to identify a GalNAc residue in the glycan linked to N614 in the spacer domain. This suggest that the glycan on N614 consist primarily of high mannose structures. Binding of ADAMTS13 to the mannose receptor on dendritic cells is most likely facilitated by the high mannose glycan on N614. Furthermore we identified 6 O-linked glycosylation sites either on a serine of a threonine. One O-linked glycan is located in the spacer domain, 2 were found in the thrombospondin type 1 repeat-6 (TSP6), another one was found in TSP8 and 1 O-linked glycosylation site was found in both of the CUB domains. Four out of 6 O-glycans contained terminal sialic acid of which 2 also contained a fucose attached to the GlcNAc. Several O-glycans contained a terminal galactose residue; one O-glycan in TSP6 terminated in both a GlcNAc and a GalNAc residue. O-fucosylation is a common post-translational modification of thrombospondin type 1 repeats. We identified 9 O-fucosylation sites in the TSP repeats. Seven out of 9 sites adhered to the consensus sequence previously defined for O-fucosylation. TSP1 and 2 contained an additional O-fucosylation site at residues T407 and S724; these sites did not match the consensus sequence for O-fucosylation. Interestingly, two additional O-fucosylation sites were identified in cysteine rich and spacer domain at residue S553 and S698. All these residues were predicted to contain a glucose-fucose modification. Next to these glucose-fucose modifications we also identified 2 fucose modification in both of the CUB domains at residues S1170 and T1344. These results show that ADAMTS13 is extensively modified by O-fucosylation. Evidence for C-mannosylation of 8 different tryptophans was obtained. In accordance with previous findings the W387 or W390 (TSP1) and W884 (TSP4) were found to be C-mannosylated. We also found C-mannosylated tryptophans at position and W730 (TSP2) and W1081 (TSP8). Four additional C-mannosylated tryptophans were detected at position W208 (metallo proteinase domain), W1307 (CUB1 domain) and W1379 and W1406 (CUB2 domain). These results show that C-mannosylation is a common post translational modification in ADAMTS13 that is also found outside the TSP domains. Taken together these findings highlight the extensive post translational modification of ADAMTS13 by diverse carbohydrate structures. We anticipate that our findings might be relevant for the clearance and/or immune recognition of ADAMTS13. Disclosures No relevant conflicts of interest to declare.

Abstract: To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a+ subsets were identified as most likely skin-derived CD11cint Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11chi dermal DCs with variable expression of langerin. Two other CD1a− LN-residing cDC subsets were characterized as CD14−BDCA3hiCD103− and CD14+BDCA3loCD103+, respectively. Whereas the CD1a+ skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFNγ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a− cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies.

Abstract: The inhibitor incidence in nonsevere hemophilia A patients with certain F8 mutations approaches the inhibitor incidence in severe patients. These findings are highly relevant for clinical practice, as they facilitate identification of high-risk patients based on F8 genotype.