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Novel insights into conformational changes of ADAMTS13

Boek - Dissertatie

The plasma enzyme ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13) has a critical role in balancing hemostasis and thrombosis by regulating the size of the platelet-binding protein von Willebrand Factor (VWF). A deficiency of ADAMTS13 results in the persistence of ultra-large VWF multimers that bind platelets in an uncontrolled manner and form microthrombi. This potentially life-threatening pathophysiology is known as thrombotic thrombocytopenic purpura (TTP) and can either be caused by anti-ADAMTS13 autoantibodies that clear and/or inhibit ADAMTS13 (immune-mediated, iTTP) or by mutations in the ADAMTS13 gene (congenital, cTTP). Recent studies revealed a unique mode-of-action of ADAMTS13 for VWF cleavage. Indeed, ADAMTS13 circulates as a latent enzyme and is activated by conformational changes that are induced by binding of the VWF substrate. ADAMTS13 consists of 14 domains: the Metalloprotease (M) domain, which contains the active site, the Disintegrin-like (D), first Thrombospondin type 1 repeat (T1), Cysteine-rich (C) and spacer (S) domain (N-terminal MDTCS domains), 7 additional Thrombospondin type 1 repeat domains (T2-T8) and two CUB domains (CUB1-CUB2) (C-terminal T2-C2 domains). Using our previously developed murine anti-ADAMTS13 antibodies (Abs), we showed that ADAMTS13 circulates in a so-called closed conformation which is maintained by interactions between its S and CUB domains. Binding of VWF disrupts the S-CUB interactions thereby opening the ADAMTS13 conformation, and induces a conformational change in the M domain which preactivates ADAMTS13. In this PhD project, we aimed at developing novel murine Abs to further unravel the conformational changes of ADAMTS13 and the structure of full-length plasma ADAMTS13. We successfully developed novel anti-MDT Abs and showed that besides conformational changes in the M domain, also conformational changes in the D and T1 domain are involved in the mechanism of allosteric preactivation of ADAMTS13. In addition, we identified mAbs that indicate general differences between the conformation of the MDT domains in full-length plasma ADAMTS13 versus in MDTCS. Studying ADAMTS13 mutations found in cTTP patients could help to elucidate the mechanism of allosteric preactivation in ADAMTS13 and to identify the regions that are involved in this mechanism. Therefore, we also expressed an ADAMTS13 mutant with a mutation in the M domain, and showed that the mutation, which destabilizes the interactions between the M and D domain, causes reduced ADAMTS13 secretion and activity, but does not affect allosteric preactivation of ADAMTS13. Intriguingly, whereas in healthy individuals ADAMTS13 circulates in its closed conformation, we recently showed that in iTTP, anti-ADAMTS13 autoantibodies open the ADAMTS13 conformation. The autoimmune response in iTTP is polyclonal with the majority of the iTTP patients having autoantibodies against the C/S domains and the CUB domains. However, it was not clear yet against which ADAMTS13 domains the opening anti-ADAMTS13 autoantibodies are directed. We showed that anti-CS autoantibodies that open ADAMTS13 are a common feature in acute iTTP. Additionally, we showed that anti-CUB autoantibodies from some acute iTTP patients can also open ADAMTS13. In remission iTTP, anti-ADAMTS13 autoantibodies disappear and ADAMTS13 activity restores. However, approximately one third of the iTTP patients in remission with restored ADAMTS13 activity >50% still has an open ADAMTS13 conformation indicating that the subclinical disease is still ongoing. Therefore, we aimed to investigate whether open ADAMTS13 in remission iTTP patients with ADAMTS13 activity >50%, can be a biomarker to predict relapse. Open ADAMTS13 was identified to be associated with earlier ADAMTS13 relapse (drop in activity to <20%) in the latter group of remission patients and hence can be a warning to monitor the patient more often.
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