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Project

Unravelling the role of ADAMTS13 and anti-ADAMTS13 autoantibodies in the pathophysiology of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening disease, which is caused by a deficiency of the blood protease ADAMTS13. Under normal physiological conditions, ADAMTS13 is responsible for the cleavage of ultra large von Willebrand factor multimers (UL-VWF) into smaller multimers. Following vascular damage, VWF attracts platelets to the place of injury to avoid excessive blood loss as part of haemostasis. However, when ADAMTS13 is deficient, VWF-platelet binding occurs spontaneously resulting in the formation of microthrombi that obstruct the microvasculature causing thrombocytopenia and microangiopathic haemolytic anaemia. ADAMTS13 deficiency can either be congenital in which mutations are present in the ADAMTS13 gene, or acquired, where in most cases anti-ADAMTS13 autoantibodies are the cause of the ADAMTS13 deficiency (immune-mediated TTP). Although major advances have been made in our understanding of the working mechanism of ADAMTS13 and the pathophysiology of TTP, plasma infusion or exchange are  still the first line treatments and the mortality rate remains 10% to 20%. Therefore, further expanding our knowledge on ADAMTS13 and anti-ADAMTS13 autoantibodies is necessary to improve diagnosis and to develop new specific treatment strategies.

Recently it has been shown that ADAMTS13 can adopt either a folded or open conformation, indicating that conformational changes in ADAMTS13 are part of its mode-of-action. In other autoimmune diseases, conformational changes in self antigens can explain the disease pathology. Therefore, we hypothesized that conformational changes in ADAMTS13 could play a role in the pathophysiology of acquired TTP (AIM 1). By first developing a murine anti-ADAMTS13 antibody that can discriminate folded and open ADAMTS13, because it recognizes a cryptic epitope, we were able to show that the conformation of ADAMTS13 is open in acute acquired TTP patients in contrast to healthy people, where ADAMTS13 is folded.

The immune response in immune-mediated TTP patients is polyclonal, but an immune dominant epitope, which is also involved in the interaction with VWF, is targeted in almost all TTP patients by anti-ADAMTS13 autoantibodies. However, how those anti-ADAMTS13 autoantibodies precisely induce ADAMTS13 deficiency is not completely understood. Furthering our understanding of anti-ADAMTS13 autoantibodies from immune-mediated TTP patients by cloning and characterizing them, will help us to better explain their role in the pathophysiology of immune-mediated TTP and ADAMTS13 deficiency (AIM 2). By using B-cell sorting from peripheral blood mononuclear cells of immune-mediated TTP patients, we were able to isolate anti-ADAMTS13 autoantibody sequences and cloned three new anti-ADAMTS13 autoantibodies. Although none of the three anti-ADAMTS13 autoantibodies inhibited ADAMTS13 activity, they were all directed against cryptic epitopes in ADAMTS13, which suggests that exposure of cryptic epitopes (AIM 1) could evoke an immune response and induce autoantibody binding.

It is not always straightforward to discriminate congenital and acquired TTP. Therefore, investigating both the presence of anti-ADAMTS13 autoantibodies, as well as genetic variations in ADAMTS13 can help to unravel the cause of ADAMTS13 deficiency during the disease course, which was investigated in a case of pregnancy-onset TTP (AIM 3). Although the presence of inhibitory anti-ADAMTS13 autoantibodies during the acute phase of the TTP episode indicated immune-mediated TTP, two heterozygous substitutions were also identified. Investigating the influence of the ADAMTS13 substitutions on ADAMTS13 secretion, activity and conformation helped us to explain ADAMTS13 activity levels during the disease course. Indeed, one substitution showed a reduced ADAMTS13 activity and could therefor explain the subnormal ADAMTS13 activity levels found during remission.

About half of the TTP cases do not present as such, but are evoked through a trigger like pregnancy or infection. Obesity has also been suggested as a risk factor for the development of TTP, as the prevalence of obesity is increased in TTP patients. Moreover, macrophages have been associated with thrombocytopenia and are abundantly present in obese people. By using a mouse model in which ADAMTS13 is deficient, we investigated if obesity is indeed a risk factor for the development of TTP and if macrophages could play a role in thrombocytopenia (AIM 4). In mice models an additional trigger, like the injection of VWF is needed to evoke TTP symptoms. To investigate if obese ADAMTS13 deficient mice are more prone to develop TTP, a threshold dose of VWF, which does not induce TTP in lean ADAMTS13 deficient mice, was injected. Obese mice had severe thrombocytopenia and increased lactate dehydrogenase levels in contrast to their lean counterparts, indicating that obesity is indeed a risk factor for the development of TTP. Macrophage depletion through clodronate injection prevented low platelet counts in obese ADAMTS13 deficient mice injected with VWF, showing that platelet depletion via macrophages could be an underlying mechanism of thrombocytopenia.

In this PhD thesis, we further unravelled the role of ADAMTS13 and anti-ADAMTS13 autoantibodies in the pathophysiology of TTP, by showing that: the conformation of ADAMTS13 is changed in acute acquired TTP patients, that anti-ADAMTS13 autoantibodies can recognize cryptic epitopes in ADAMTS13, that ADAMTS13 substitutions can explain ADAMTS13 activity during disease course and that obesity is indeed a risk factor for TTP in ADAMTS13 deficient mice. 

Date:1 Oct 2013 →  14 May 2018
Keywords:ADAMTS13, Thrombotic Thrombocytopenic Purpura/TTP, Autoantibodies
Disciplines:Inorganic chemistry, Organic chemistry, Theoretical and computational chemistry, Other chemical sciences
Project type:PhD project