Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in southern Africa has been characterised by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, whilst the second and third waves were driven by the Beta and Delta variants, respectively1-3. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng Province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, predicted to influence antibody neutralization and spike function4. Here, we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity

ADAMTS13's tail tale: Developing antibodies to reveal novel insights

ADAMTS13 is an essential proteolytic enzyme that prevents spontaneous thrombus formation. When an injury takes place in a blood vessel, the exposed subendothelial collagen is able to attract von Willebrand factor (VWF) multimers. Adherence of this multimeric plasma glycoprotein to collagen induces thrombus formation by the recruitment and activation of platelets, which subsequently both limits blood loss and facilitates repair of the injury. This process is highly dependent on the conformation of VWF, which relies on the size of the multimers. VWF is constitutively expressed as dimers or small multimers by the endothelial cells. Stimulation (e.g. injury) however induces in the release of ultra-large (UL) VWF multimers, which are the most reactive in binding platelets, from the endothelial cells and megakaryocytes. Due to their size, UL-VWF multimers are most prone to flow-induced conformational changes, which expose their hidden platelet binding sites. In the absence of an injury, these UL-VWF multimers are highly dangerous as they are able to induce spontaneous microthrombus formation. Therefore, UL-VWF multimers are cleaved into smaller, less reactive multimers by ADAMTS13. ADAMTS13 is a multidomain metalloprotease consisting of a catalytic metalloprotease (M), a disintegrin-like (D), thrombospondin type-1 repeat (T), cysteine-rich (C), spacer (S), seven additional T and two CUB domains. It is expressed as a constitutive active enzyme by mainly the hepatic stellate cells of the liver. Despite this constitutive activity, flow-dependent conformational changes in VWF also regulate proteolysis of VWF by ADAMTS13. The intrinsic property of the folded VWF A2 domain of hiding the ADAMTS13 cleavage site consequently restricts proteolysis. Depending on the attachment and size of the VWF multimers and the shear rate, a threshold could be reached to unfold the A2 domain, thereby allowing binding and cleavage by the catalytic amino(N)-terminal (proximal) MDTCS domains. The well-documented binding of MDTCS to the A2 domain initiates at the spacer domain which recruits the remaining MDTC domains to bind like a molecular zipper to finally cleave VWF. Unraveling the complete mode-of-action of the carboxyl(C)-terminal (distal) T2 to CUB2 (T2 to CUB2) domains proved to be more challenging. Distal domains were shown to be important in docking of ADAMTS13 on VWF (with a folded or unfolded A2 domain) under flow conditions. Ambiguities between different studies did not only illustrate the limited knowledge on the role of the distal domains in ADAMTS13 function, but did also indicate the existence of additional properties on top of docking onto VWF. Hence, we aimed at further elucidating this complex mode-of-action of the distal domains of ADAMT13 using the strategy of antigen-specific antibody development (AIM 1). A deficiency in ADAMTS13 due to autoantibody formation (acquired, ~95% of all cases) or genetic mutations (congenital, ~5% of all cases) leads to the life-threatening microangiopathic disease thrombotic thrombocytopenic purpura (TTP) due to the spontaneous development of VWF-rich microthrombi. TTP is often misdiagnosed and current therapies fail to help 10 to 20% of all patients. In addition, ~30 to 40% of the TTP patients encounter one or multiple relapses within 10 years after obtaining remission. Hence, in order to improve both treatment and patient care, novel treatment therapies are currently being developed. Suitable animal models are crucial in the further development and validation of novel treatment therapies before they are transferred to the patient level. For congenital TTP, small animal models (mice) have already been developed but still require further investigation. In this thesis, we focused on the availability of animal models for acquired TTP. Previously, our lab reported a large animal model for acquired TTP. Baboons (Papio ursinus) were injected with an inhibitory anti-metalloprotease domain antibody (3H9), resulting in the development of TTP symptoms (thrombocytopenia, anemia, presence of schistocytes [fragmented red blood cells], increased lactate dehydrogenase [LDH] activity). Despite the availability of this model and the physiological correlation with the early stages of the acute phase of the disease, this large animal model encounters certain technical (difficulty of high throughput screenings, difficult to handle and house, long gestation time, high maintenance cost …) and ethical limitations. Consequently, a small animal model (e.g. mouse) is required to facilitate both the development and validation of novel treatment strategies for acquired TTP (AIM 2). In this thesis, the powerful technique of antigen-specific antibody development was used to reach the objectives of both aims. A large pool of monoclonal antibodies was developed, directed against either the human ADAMTS13 variant (hADAMTS13) and the murine ADAMTS13 variant (mADAMTS13). The antibodies directed against hADAMTS13 were used to further elucidate the interaction of the distal ADAMTS13 domains with VWF and their functional role (AIM 1), while the antibodies directed against mADAMTS13 were used to generate a novel mouse model of acquired TTP (AIM 2). The anti-hADAMTS13 monoclonal antibodies proved to be crucial in the discovery of a new mechanism of ADAMTS13 activity regulation and consequently in further elucidation of the complex mode-of-action of the distal domains (AIM 1). A large proportion of the antibodies directed against the distal domains of ADAMTS13 individually increased the proteolytic activity of ADAMTS13 up to two-fold under both static and shear conditions. Furthermore, a subset of the anti-T2-CUB2 antibodies also increased the binding of ADAMTS13 to folded VWF up to three-fold. Activation of the functionality of ADAMTS13 was attributed to conformational changes in ADAMTS13. We found that the flexible conformation of ADAMTS13 (mainly annotated to flexible linker regions between the T2-CUB2 domains) enables an interaction between the proximal MDTCS and the distal T2-CUB2 domains. This interaction consequently shields the catalytic proximal domains and thereby negatively regulates the proteolytic activity of ADAMTS13. Anti-T2-CUB2 antibodies or docking of the distal domains onto VWF abrogates these interactions and allosterically activates ADAMTS13. Supplemental to docking onto VWF under flow conditions, binding of the distal ADAMTS13 domains to VWF also results in the formation of circulating ADAMTS13-VWF complexes. In this thesis, a specific ELISA that measures the binding capacity of ADAMTS13 to folded VWF was used to screen the effect of all anti-T2-CUB2 antibodies. This revealed a major role of the T8 domain in this interaction. Remarkably, additional screening of the anti-MDTCS antibodies in this ELISA revealed specific antibodies inhibiting the interaction of ADAMTS13 with folded VWF, indicating destabilization of the A2 domain. The use of specific ADAMTS13 variants (with mutations in the spacer domain) and VWF variants (with mutations in the A2 domain) furthermore confirmed the mechanism of ADAMTS13-induced destabilization of the A2 domain. Further in vitro analysis confirmed the crucial role of the distal domains but however indicated that this is a slow process. The anti-mADAMTS13 monoclonal antibodies proved to be very valuable in the development of a novel small animal model (mouse) for acquired TTP (AIM 2). In vitro screening of the inhibitory effect of this large panel of anti-mADAMTS13 monoclonal mouse antibodies revealed full inhibition of plasma mADAMTS13 activity by combining two specific anti-MDTCS antibodies. The intravenous injection of both mouse antibodies achieved an acquired ADAMTS13 deficiency for at least 7 days in wild type mice (WT, Adamts13+/+). In line with the human disease pattern and the congenital TTP mouse models, administration of a secondary trigger (recombinant human VWF [hVWF]) resulted in TTP symptoms (thrombocytopenia, elevated LDH activity …). This novel acquired TTP mouse model is very helpful in the validation of novel treatment therapies for acquired TTP. Regardless of the applicability of these inhibitory antibodies in the validation of novel treatment therapies, these antibodies are also carry great potential in the discovery of novel therapies for acquired TTP (e.g. plasmin) and determination of the role of ADAMTS13 in other diseases like atherosclerosis, myocardial infarction, ischemic stroke, preeclampsia and cerebral malaria.nrpages: 220status: publishe

FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.status: publishe

FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and 6520% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pat tern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences

Conformational activation of ADAMTS13

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.status: publishe

Development of a pre‐ and postoperative physical activity promotion program integrated in the electronic health system of patients with bladder cancer (The POPEYE study) : an intervention mapping approach

Introduction: Uptake of sufficient physical activity before and after radical cystectomy is important to improve physical and psychosocial outcomes in bladder cancer (BC) patients. Methods: In this paper, we describe the development of an evidence-based and theory-informed intervention, guided by the steps of the Intervention Mapping approach, to promote physical activity before and after radical cystectomy in patients with BC. Results: The intervention is a home-based physical activity program. The preoperative timeframe of the intervention is 4 or 12 weeks, depending on administration of neoadjuvant chemotherapy. Postoperatively, the intervention will last for 12 weeks. The intervention consists of a digital oncological platform (DOP), several consultations with healthcare professionals, personal booklet and follow-up phone calls. DOP includes information, diaries, visual representation of progress, mailbox, videos of peers and treating physician explaining the benefits of physical activity, photo material of exercises and a walking program with an activity tracker. Individual goals will be set and will be self-monitored by the patient through DOP. Patients will receive alerts and regular feedback. Conclusions: Intervention Mapping ensures transparency of all intervention components and offers a useful approach for the development of behaviour change interventions for cancer patients and for translation of theories into practice

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13

Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF).status: publishe

Anti-ADAMTS13 Antibodies and a Novel Heterozygous p.R1177Q Mutation in a Case of Pregnancy-Onset Immune-mediated Thrombotic Thrombocytopenic Purpura

status: publishe

Allosteric activation of ADAMTS13 by von Willebrand factor

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties