Anti-ADAMTS13 autoantibody immunoprofiles in the pathophysiology of thrombotic thrombocytopenic purpura

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by a severe deficiency of ADAMTS13 due to the presence of anti-ADAMTS13 autoantibodies. The lack of a functional enzyme leads to the formation of platelet-rich microthrombi, since the proteolytic cleavage of hemostatically active ultra-large von Willebrand factor (VWF) multimers into smaller multimers is hindered. The formation of microthrombi leads to organ failure, severe thrombocytopenia and hemolytic anemia. Without prompt treatment, this condition is lethal and even with timely treatment, the mortality rate still remains up to 20%. In order to better understand the pathophysiology of iTTP, we aimed at investigating the binding sites of anti-ADAMTS13 autoantibodies, and therefore developed an epitope mapping assay (Aim 1). This assay facilitates to fine-map the anti-ADAMTS13 autoantibodies more accurately since it employs small nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2), in contrast to the relatively large overlapping ADAMTS13 fragments that have been used in previous studies. Furthermore, an easy-to-perform epitope mapping ELISA (enzyme-linked immunosorbent assay) which requires small volumes of patient plasma or serum, enables to screen large cohorts of iTTP patients. Moreover, to improve the management of iTTP patients there is an increasing interest in biomarkers which could predict an imminent relapse or disease outcome, therefore immunoprofiles could be an interesting biomarker, as seen in other autoimmune disorders. Hence we fine-mapped the epitopes of anti-ADAMTS13 autoantibodies in both remission (n=50) and acute phase (n=131) samples from a large multicenter iTTP patient cohort, which allowed to determine the most prevalent immunoprofiles (Aim 2). Interestingly, in our iTTP patient cohort, there were only 74.8% of samples with detectable anti-CS autoantibodies, although the previous epitope mapping studies have found nearly 98% of patients having anti-CS autoantibodies. The most prevalent immunoprofile was the same in both phases and contained patients with only anti-CS autoantibodies (Profile 1). We next investigated whether certain immunprofiles could be linked with clinical data (Aim 2). Although we could not find a strong linkage between immunoprofiles and disease severity, we could determine a correlation between the most prevalent immunoprofile (Profile 1) and age, as well as between the presence of anti-T2-T5 autoantibodies and age. Moreover, we also observed a correlation between the absence of anti-CUB1-2 autoantibodies and cerebral involvement. However, the number of iTTP patient samples with available clinical data was still rather small, thus a study involving a larger amount of iTTP patient samples would be needed for identifying a potential link between disease severity and immunoprofiles. In addition, large volumes of recombinant ADAMTS13 (rADAMTS13) are needed in fundamental research to study the mode of action of ADAMTS13 as well as to further investigate the pathophysiology of iTTP. However, since rADAMTS13 is generally expressed at a relatively low expression level, we aimed to enhance the rADAMTS13 protein production by either using a more optimal signal peptide or employing an N-terminal fusion protein in combination with the QMCF Technology (Aim 3). Although rADAMTS13 expression was already increased 3-fold using the factor VII signal peptide compared to the native signal peptide, developing an N-terminal fusion protein of ADAMTS13 with albumin domain 1 (AD1-ADAMTS13) further increased the expression to 15-fold. Moreover, the AD1-ADAMTS13 protein retained its activity to cleave VWF which supports the employment of this fusion protein in fundamental research. In this PhD thesis we developed and validated an in-depth epitope mapping ELISA and deciphered the immunoprofiles of iTTP patients during remission and acute phase in a large multicenter iTTP patient cohort. Additionally, the rADAMTS13 expression could be strongly enhanced by generating an AD1-ADAMTS13 fusion protein.

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