Anti-ADAMTS13 antilichamen: Essentiële hulpmiddelen om de ADAMTS13 structuur/functie te onderzoeken en thrombotische thrombocytopenische purpura te diagnosticeren

By regulating the proteolysis of von Willebrand factor (VWF), the plasma enzyme ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type-1 repeats, member 13) supports the balance between hemostasis and thrombosis. Hereto, the activity of ADAMTS13 is strictly regulated by structural features that appear unique within the ADAMTS enzyme family. Upon severe ADAMTS13 deficiency, persistent ultra-large VWF multimers can spontaneously bind platelets to cause microthrombi formation. Given its life-threatening pathophysiology, this thrombotic thrombocytopenic purpura (TTP) disorder requires prompt diagnosis to initiate the appropriate acute treatment regimen and manage patients correctly during follow-up upon remission. As essential information on the structural autoinhibition and allosteric activation of ADAMTS13 is still missing, computational simulations predicted the ADAMTS13 structure and thereby provided novel perspectives to explore the conformational dynamics that regulate the ADAMTS13 protease. Within the predicted, more compact ADAMTS13 structure, the proximal ADAMTS13 substrate exosites are structurally shielded by the distal ADAMTS13 domains that could thereby significantly contribute to the ADAMTS13 autoinhibition. Therefore, we aimed to explore circulating plasma ADAMTS13 for the existence of conformationally sensitive epitopes in multiple ADAMTS13 domains that could reflect the structural ADAMTS13 autoinhibition, and to characterize the accessibility of such epitopes in healthy donors and immune-mediated TTP (iTTP) patients (Aim 1). In this PhD project, we validated the existence of cryptic epitopes, recognized by our unique monoclonal antibodies (mAbs) 6A6, 1D5, 1C4, 9C12 and 19H4, in five domains of closed, autoinhibited ADAMTS13 that become exposed upon mAb-mediated allosteric ADAMTS13 activation. Sub-physiological pH conditions revealed the allosteric ADAMTS13 activation to be pH dependent and here, cryptic epitope exposure at low pH validated these conformational changes to be identical as observed for the mAb-induced allostery. Thereby, the non-covalent nature of these conformational changes underscores the feasibility of interdomain protein-protein interactions to structurally shield these cryptic ADAMTS13 epitopes. Moreover, in nearly all healthy donors, the closed, autoinhibited ADAMTS13 conformation was reflected by the concealing of all five cryptic epitopes whereas iTTP patients represented a resemblant open ADAMTS13 conformation since all cryptic epitopes were exposed. As a clinically relevant biomarker, we validated patient-derived autoantibodies as the trigger for the structural ADAMTS13 opening (Chapter 4). As the characterization of the closed, autoinhibited ADAMTS13 conformation raises new questions on the molecular mechanism that is involved in the structural ADAMTS13 opening, we wanted to explore the structural dynamics within the ADAMTS13 CUB domains that uncouple the spacer-CUB domain interactions upon mAb binding (Aim 2). Using hydrogen to deuterium exchange mass spectrometry (HDX MS), we identified and mapped the mobile and stable CUB domain regions onto the static CUB domain crystal structure. Upon conformational opening using the anti-CUB1 mAb 17G2, HDX differences stratified the conformational mAb binding epitope to the CUB1 L3 and L9 loops. Structural analysis of ADAMTS13 CUB mutants verified this mAb epitope and thereby suggested the indirect spacer-CUB domain uncoupling as the CUB1 contiguous surface residues that dock to the spacer domain were not targeted. Moreover, binding of the anti-CUB1 mAb also induced extensive structural dynamics within the CUB2 domain that could possibly be transferred via the short linker and unusually large interdomain interface to slightly reposition both CUB domains and thereby reorient the residues of the contiguous surface that engage with the spacer domain. However, the structural impact of our single residue ADAMTS13 CUB mutants was found to be insufficient to verify the role of these CUB domain features in the disruption of the ADAMTS13 global latency when compared to the extensive structural dynamics that occur upon substrate or mAb binding (Chapter 5). To answer the unmet need for automated ADAMTS13 testing in clinical context and enable prompt TTP treatment initiation, we aimed to transfer our in-house ADAMTS13 activity (Aim 3) as well as our ADAMTS13 antigen and conformation (Aim 4) assays onto an easy-to-use, automated fiber-optic surface plasmon resonance (FO-SPR) platform. Analytical characterization of our step-by-step optimized FO-SPR ADAMTS13 activity assay illustrated a detection limit of 6.8% with an inter-assay variation of 7.2%. Given this analytical sensitivity, plasma samples of acute iTTP were retrospectively confirmed to have a severely deficient ADAMTS13 activity below the diagnostic cut-off of 10%, which discriminated acute iTTP patient plasma samples from healthy donors, patients with alternative thrombotic microangiopathies and sepsis patients. Our FO-SPR ADAMTS13 activity assay revealed strong correlation with our reference FRET-based assay and no systematic bias was observed between both diagnostic strategies (Chapter 6). Similarly, the optimized FO-SPR ADAMTS13 antigen assay revealed a sensitive detection limit of 0.001 µg/mL ADAMTS13 with a satisfactory inter-assay variation of 14.4%. Strong correlation was found for the ADAMTS13 antigen levels of healthy donors and acute iTTP patients determined using both our FO-SPR and reference ELISA assay, whereas no systematic bias was indicated by Bland-Altman analysis. However, the modest proportional bias suggested our ELISA reference assay to measure slightly higher ADAMTS13 antigen titers. Following the stepwise optimization of the transferred ADAMTS13 conformation assay, all healthy donor plasma samples indicated a closed conformation that could be opened by means of our opening, activating anti-CUB1 mAb 17G2. As expected, the acute iTTP patient cohort indicated an open ADAMTS13 conformation in absence as well as in presence of our opening mAb (Chapter 7). By transferring all relevant immunoassays for ADAMTS13 biomarker testing, this FO-SPR platform represents the first automated device for the on-demand testing of all four iTTP diagnostic biomarkers.