N-glycan-mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP.

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.

Conformational plasticity of ADAMTS13 in hemostasis and autoimmunity.

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force-dependent cleavage at a single Tyr-Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13-VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP.

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear.

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

Dissecting the pathophysiology of immune thrombotic thrombocytopenic purpura: interplay between genes and environmental triggers.

Although outstanding progress has been made in understanding the pathophysiology of thrombotic thrombocytopenic purpura (TTP), knowledge of the immunopathogenesis of the disease is only at an early stage. Anti-ADAMTS13 auto-antibodies were shown to block proteolysis of von Willebrand factor and/or induce ADAMTS13 clearance from the circulation. However, it still remains to identify which immune cells are involved in the production of anti-ADAMTS13 autoantibodies, and therefore account for the remarkable efficacy of the B-cell depleting agents in this disease. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factors of the human leukocyte antigen class II locus DRB1*11 and DQB1*03 alleles as well as the protective allele DRB1*04, and modifying factors such as ethnicity, sex and obesity. Future studies have to identify why these identified genetic risk factors are also frequently to be found in the healthy population although the incidence of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is extremely low. Moreover, the development of recombinant ADAMTS13 opens a new therapeutic era in the field. Interactions of recombinant ADAMTS13 with the immune system of iTTP patients will require intensive investigation, especially for its potential immunogenicity. Better understanding of iTTP immunopathogenesis should, therefore, provide a basis for the development of novel therapeutic approaches to restore immune tolerance towards ADAMTS13 and thereby better prevent refractoriness and relapses in patients with iTTP. In this review, we address these issues and the related challenges in this field.

Mass spectrometry-assisted identification of ADAMTS13-derived peptides presented on HLA-DR and HLA-DQ.

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.

Preparation and application of anti-peptide antibodies for detection of orphan cytochromes P450.

OBJECTIVES: Cytochromes P450 (CYP) are monooxygenases, which metabolize mostly hydrophobic endogenous and exogenous compounds. CYPs without any clear connection to metabolism are called "orphans". Interestingly, these "orphan" CYPs are over-expressed in tumor tissues. Thus, the main aim of the paper is the development of antibodies for immunodetection of these CYPs as potential malignancy markers. METHODS: Unique sequences of CYP2S1 and 2W1 were selected and peptides synthesized. Chickens were immunized with peptides bound to hemocyanin (KLH). The antibodies were isolated from egg yolks and their reactivity was tested by ELISA. Antibodies were further affinity purified on immobilized peptides. Western blots containing CYP2S1 and 2W1 standards were developed with purified antibodies. RESULTS: Using unique peptide immunogens of CYP2S1 and 2W1 the antibodies were developed. As judged from ELISA all chickens produced specific antibodies against the respective peptides. Both affinity purified antibodies against CYP2S1 peptide recognized the CYP2S1 standard on Western blots, but only one of four anti-peptide antibodies against CYP2W1 reacted with CYP2W1 standard. The antibodies were used for the detection of CYPs in cancer cell lines and human tissues samples. Although both CYPs were frequently co-expressed in cancer cells, CYP2S1 was solely induced in the cell line BxPC3, while CYP2W1 was predominantly present in cell lines MCF7 and HeLa. Our data show that anti-peptide antibodies are an indispensable tool for detection of homologous CYPs. CONCLUSIONS: The anti-peptide antibodies successfully recognized CYP2S1 and 2W1 in the cancer cell lines and tissue samples.

Rational modulator design by exploitation of protein-protein complex structures.

The horizon of drug discovery is currently expanding to target and modulate protein-protein interactions (PPIs) in globular proteins and intrinsically disordered proteins that are involved in various diseases. To either interrupt or stabilize PPIs, the 3D structure of target protein-protein (or protein-peptide) complexes can be exploited to rationally design PPI modulators (inhibitors or stabilizers) through structure-based molecular design. In this review, we present an overview of experimental and computational methods that can be used to determine 3D structures of protein-protein complexes. Several approaches including rational and in silico methods that can be applied to design peptides, peptidomimetics and small compounds by utilization of determined 3D protein-protein/peptide complexes are summarized and illustrated.

The class I scavenger 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.