27 results found
Shin J-S, Subhan M, Cambridge G, et al., 2022, Alterations in B- and circulating T-follicular helper cell subsets in immune thrombotic thrombocytopenic purpura., Blood Adv
T follicular helper (Tfh) cells regulate development of antigen-specific B-cell immunity. We prospectively investigated B-cell and cTfh subsets in 45 immune TTP patients at presentation and longitudinally after rituximab (RTX). B-cell phenotype was altered at acute iTTP presentation with decreased transitional cells and postgerminal centre (post-GC) memory B cells and increased plasmablasts compared to healthy controls. A higher percentage of plasmablasts was associated with higher anti-ADAMTS13 IgG and lower ADAMTS13 antigen levels. In asymptomatic patients with ADAMTS13 relapse, there were increased naïve B cells and a global decrease in memory subsets, with a trend to increased plasmablasts. Total circulating Tfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased at iTTP presentation. CD80 expression was decreased on IgD+ memory cells and double negative memory cells in acute iTTP. Longitudinal analysis: at repopulation after B cell depletion in de novo iTTP, post-GC and double negative memory B cells were reduced compared to pre-RTX. RTX did not cause alteration in cTfh frequency. The subsequent kinetics of naïve, transitional, memory B cells and plasmablasts did not differ significantly between patients who went on to relapse vs those who remained in remission. In summary, acute iTTP is characterised by dysregulation of B- and cTfh-cell homeostasis with depletion of post-GC memory cells and cTfh cells and increased plasmablasts. Changes in CD80 expression on B cells further suggest altered interactions with T cells.
Yamamoto K, de Groot R, Scilabra SD, et al., 2021, Editorial: ADAM, ADAMTS and Astacin Proteases: Challenges and Breakthroughs in the -Omics Era, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 8
The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC50 values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-β–binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.
Santamaria S, Cuffaro D, Nuti E, et al., 2021, Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide, Scientific Reports, Vol: 11, ISSN: 2045-2322
ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly linked to the etiology of osteoarthritis (OA). For this reason, ADAMTS-5 is a pharmaceutical target for the treatment of OA. ADAMTS-5 shares high structural and functional similarities with ADAMTS-4, which makes the design of selective inhibitors particularly challenging. Here we exploited the ADAMTS-5 binding capacity of β-N-acetyl-d-glucosamine to design a new class of sugar-based arylsulfonamides. Our most promising compound, 4b, is a non-zinc binding ADAMTS-5 inhibitor which showed high selectivity over ADAMTS-4. Docking calculations combined with molecular dynamics simulations demonstrated that 4b is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Furthermore, the interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533). Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. This positively-charged cluster of ADAMTS-5 represents a previously unknown substrate-binding site (exosite) which is critical for substrate recognition and can therefore be targeted for the development of selective ADAMTS-5 inhibitors.
Santamaria S, de Groot R, 2020, ADAMTS proteases in cardiovascular physiology and disease, Open Biology, Vol: 10, Pages: 1-18, ISSN: 2046-2441
The a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) family comprises 19 proteases that regulate the structure and function of extracellular proteins in the extracellular matrix and blood. The best characterized cardiovascular role is that of ADAMTS-13 in blood. Moderately low ADAMTS-13 levels increase the risk of ischeamic stroke and very low levels (less than 10%) can cause thrombotic thrombocytopenic purpura (TTP). Recombinant ADAMTS-13 is currently in clinical trials for treatment of TTP. Recently, new cardiovascular roles for ADAMTS proteases have been discovered. Several ADAMTS family members are important in the development of blood vessels and the heart, especially the valves. A number of studies have also investigated the potential role of ADAMTS-1, -4 and -5 in cardiovascular disease. They cleave proteoglycans such as versican, which represent major structural components of the arteries. ADAMTS-7 and -8 are attracting considerable interest owing to their implication in atherosclerosis and pulmonary arterial hypertension, respectively. Mutations in the ADAMTS19 gene cause progressive heart valve disease and missense variants in ADAMTS6 are associated with cardiac conduction. In this review, we discuss in detail the evidence for these and other cardiovascular roles of ADAMTS family members, their proteolytic substrates and the potential molecular mechanisms involved.
Santamaria S, De Groot R, 2020, ADAMTS proteases in cardiovascular physiology and disease: ADAMTS, cardiovascular roles, Open Biology, Vol: 10
The a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) family comprises 19 proteases that regulate the structure and function of extracellular proteins in the extracellular matrix and blood. The best characterized cardiovascular role is that of ADAMTS-13 in blood. Moderately low ADAMTS-13 levels increase the risk of ischeamic stroke and very low levels (less than 10%) can cause thrombotic thrombocytopenic purpura (TTP). Recombinant ADAMTS-13 is currently in clinical trials for treatment of TTP. Recently, new cardiovascular roles for ADAMTS proteases have been discovered. Several ADAMTS family members are important in the development of blood vessels and the heart, especially the valves. A number of studies have also investigated the potential role of ADAMTS-1,-4 and-5 in cardiovascular disease. They cleave proteoglycans such as versican, which represent major structural components of the arteries. ADAMTS-7 and-8 are attracting considerable interest owing to their implication in atherosclerosis and pulmonary arterial hypertension, respectively. Mutations in the ADAMTS19 gene cause progressive heart valve disease and missense variants in ADAMTS6 are associated with cardiac conduction. In this review, we discuss in detail the evidence for these and other cardiovascular roles of ADAMTS family members, their proteolytic substrates and the potential molecular mechanisms involved.
de Groot R, 2020, ADAMTS7: Recombinant Protein Expression and Purification., Methods Mol Biol, Vol: 2043, Pages: 63-73
ADAMTS7 is a secreted protease that is predominantly expressed in tissues of the cardiovascular system and tendon. Although recent evidence suggests that it may play a role in the etiology of coronary artery disease, its physiological function and substrates are unknown. The enzyme undergoes extensive posttranslational modifications, including chondroitin sulfate attachment, N and O-linked glycosylation, and a two-step activation process. For the benefit of scientists who study the function of ADAMTS7 and its role in disease, this chapter provides an introduction to the chemical and functional properties of the various ADAMTS7 domains, as well as a protocol for the recombinant expression and purification of ADAMTS7.
Petri A, Kim HJ, Xu Y, et al., 2019, Crystal structure and substrate-induced activation of ADAMTS13., Nat Commun, Vol: 10
Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.
Santamaria S, Yamamoto Y, Teraz-Orosz A, et al., 2019, Exosites in hypervariable loops of ADAMTS dpacer domains control substrate recognition and proteolysis, Scientific Reports, Vol: 9, ISSN: 2045-2322
ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M−1 s−1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M−1 sec−1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739–744 and 837–844) and ADAMTS-4 (residues 717–724 and 788–795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.
Colige A, Monseur C, Crawley J, et al., 2019, Proteomic discovery of substrates of the cardiovascular protease ADAMTS7, Journal of Biological Chemistry, Vol: 294, Pages: 8037-8045, ISSN: 0021-9258
The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729–Val-730 and Glu-732–Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β–binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1–binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.
Santamaria S, de Groot R, 2019, Monoclonal antibodies against metzincin targets, British Journal of Pharmacology, Vol: 176, Pages: 52-66, ISSN: 1476-5381
The metzincin clan of metalloproteinases includes the MMP, disintegrin and metalloproteinase (ADAM) and ADAM with thrombospondin motifs families, which cleave extracellular targets in a wide range of (patho)physiological processes. Antibodies constitute a powerful tool to modulate the activity of these enzymes for both therapeutic and research purposes. In this review, we give an overview of monoclonal antibodies (mAbs) that have been tested in preclinical disease models, human trials and important studies of metzincin structure and function. Initial attempts to develop therapeutic small molecule inhibitors against MMPs were hampered by structural similarities between metzincin active sites and, consequently, off‐target effects. Therefore, more recently, mAbs have been developed that do not bind to the active site but bind to surface‐exposed loops that are poorly conserved in closely related family members. Inhibition of protease activity by these mAbs occurs through a variety of mechanisms, including (i) barring access to the active site, (ii) disruption of exosite binding, and (iii) prevention of protease activation. These different modes of inhibition are discussed in the context of the antibodies' potency, selectivity and, importantly, the effects in models of disease and clinical trials. In addition, various innovative strategies that were used to generate anti‐metzincin mAbs are discussed.
de Groot R, Monseur C, Colige A, et al., 2018, ADAMTS7 substrate and cleavage site specificity, Matrix Biology Europe Meeting, Publisher: WILEY, Pages: A50-A51, ISSN: 0959-9673
Santamaria S, Crawley JTB, Yamamoto K, et al., 2017, A comparison of COMP (TSP5) proteolysis by ADAMTS7 and ADAMTS4, Autumn Meeting of the British-Society-for-Matrix-Biology (BSMB), Publisher: WILEY, Pages: A3-A4, ISSN: 0959-9673
Thomas MR, de Groot R, Scully MA, et al., 2015, Pathogenicity of anti-ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura, EBioMedicine, Vol: 2, Pages: 942-952, ISSN: 2352-3964
BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease in which anti-ADAMTS13 autoantibodies cause severe enzyme deficiency. ADAMTS13 deficiency causes the loss of regulation of von Willebrand factor multimeric size and platelet-tethering function, which results in the formation of disseminated microvascular platelet microthrombi. Precisely how anti-ADAMTS13 autoantibodies, or antibody subsets, cause ADAMTS13 deficiency (ADAMTS13 activity generally < 10%) has not been formally investigated. METHODS: We analysed 92 acquired TTP episodes at presentation, through treatment and remission/relapse using epitope mapping and functional analyses to understand the pathogenic mechanisms of anti-ADAMTS13 IgG. RESULTS: 89/92 of TTP episodes had IgG recognising the ADAMTS13 N-terminal domains. The central spacer domain was the only N-terminal antigenic target detected. 38/92 TTP episodes had autoantibodies recognising the N-terminal domains alone; 54/92 TTP episodes also had antibodies against the ADAMTS13 C-terminal domains (TSP2-8 and/or CUB domains). Changes in autoantibody specificity were detected in 9/16 patients at relapse, suggesting a continued development of the disease. Functional analyses on IgG from 43 patients revealed inhibitory IgG were limited to anti-spacer domain antibodies. However, 15/43 patients had autoantibodies with no detectable inhibitory action and as many as 32/43 patients had autoantibodies with inhibitory function that was insufficient to account for the severe deficiency state, suggesting that in many patients there is an alternative pathogenic mechanism. We therefore analysed plasma ADAMTS13 antigen levels in 91 acquired TTP presentation samples. We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen. ADAMTS13 antigen in the lowest quartile at first presentation was associated with increa
Nowak AA, de Groot R, Laffan MA, et al., 2015, N-linked glycosylation is a modulator of ADAMTS13 expression, structure and function, Journal of Thrombosis and Haemostasis, Vol: 13, Pages: 64-64, ISSN: 1538-7933
de Groot R, Lane DA, Crawley JTB, 2015, The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis, Blood, Vol: 125, Pages: 1968-1975, ISSN: 0006-4971
ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain–charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.
Crawley JTB, de Groot R, 2012, Cardiovascular string theory, BLOOD, Vol: 119, Pages: 2181-2182, ISSN: 0006-4971
Crawley JTB, de Groot R, Xiang Y, et al., 2011, Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor, BLOOD, Vol: 118, Pages: 3212-3221, ISSN: 0006-4971
Xiang Y, de Groot R, Crawley JTB, et al., 2011, Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 11602-11607, ISSN: 0027-8424
de Groot R, Ryu J-Y, Jaring M, et al., 2011, VWF proteolysis by adamts13 is dependent on cooperation between the ADAMTS13 cysteine-rich domain loop Q456-Q478 and the spacer domain, Publisher: WILEY-BLACKWELL, Pages: 307-307, ISSN: 1538-7933
Xiang Y, de Groot R, Crawley JTB, et al., 2011, Essential role of the P3 residue (Leu1603) of von Willebrand factor in scissile bond cleavage by ADAMTS13, Publisher: WILEY-BLACKWELL, Pages: 909-909, ISSN: 1538-7933
de Groot R, Lane DA, Crawley JTB, 2010, The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity, BLOOD, Vol: 116, Pages: 3064-3072, ISSN: 0006-4971
Abuknesha RA, Jeganathan F, DeGroot R, et al., 2010, Detection of proteases using an immunochemical method with haptenylated-gelatin as a solid-phase substrate, ANALYTICAL AND BIOANALYTICAL CHEMISTRY, Vol: 396, Pages: 2547-2558, ISSN: 1618-2642
Crawley JTB, de Groot R, Luken BM, 2009, Circulating ADAMTS-13-von Willebrand factor complexes: an enzyme on demand, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 7, Pages: 2085-2087, ISSN: 1538-7933
de Groot R, Bardhan A, Ramroop N, et al., 2009, Essential role of the disintegrin-like domain in ADAMTS13 function, BLOOD, Vol: 113, Pages: 5609-5616, ISSN: 0006-4971
Gardner MD, Chion C, de Groot R, et al., 2009, A functional calcium-binding site in the metalloprotease domain of ADAMTS13, Blood, Vol: Vol. 113, Pages: 1149-1157
de Groot R, Lane DA, 2008, Shear tango: dance of the ADAMTS13/VWF complex, Blood, Vol: 112, Pages: 1548-1549
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