ADAMTS13-RELATED ASSAYS IN ACQUIRED THROMBOTIC THROMBOCYTOPENIC PURPURA

Thrombotic thrombocytopenic purpura (TTP) is a rare but life-threatening microangiopathic disorder, due to the persistence of highly thrombogenic ultra large (UL) von Willebrand factor (VWF) multimers in the microcirculation. TTP is associated with the severe deficiency of ADAMTS13 (activity <10%), the metalloprotease responsible for the proteolytic regulation of VWF multimers size. In the acquired form of the disease, the severe deficiency of ADAMTS13 is caused by anti-ADAMTS13 antibodies that inhibit ADAMTS13 activity (neutralizing anti-ADAMTS13 antibodies or inhibitors) and/or increase its clearance from the circulation (non-neutralizing anti-ADAMTS13 antibodies). In the absence of ADAMTS13, highly adhesive ULVWF, that spontaneously aggregate platelets, accumulates promoting platelet thrombi formation and eventually leading to the clinical features typical of TTP. In the past decade, the increasing knowledge regarding the molecular mechanisms involved in the pathogenesis of TTP, yielded to a proliferation of assays for the measurement of ADAMTS13 and anti-ADAMTS13 antibodies. Despite the numerous studies conducted to assess the clinical utility of ADAMTS13-related markers, their prognostic value is still controversial. The finding that severe ADAMTS13 deficiency and the presence of anti-ADAMTS13 antibody is associated with an increased risk of relapse is rather consistent, but studies on larger cohort of patients are needed. More uncertain is the predictive value of immunoglobulin (Ig) class subtype of anti-ADAMTS13 antibodies, whether or not inhibitory. Controversial results may be partially due to differences in the assays used to measure ADAMTS13 activity and anti-ADAMTS13 antibodies, which require further standardization. With this background, the present research had two main objectives: to evaluate, in a large cohort of acquired TTP patients enrolled in the international Milan TTP Registry (developed and curated at the The Hemophilia and Thrombosis Centre of Milan), the clinical utility of ADAMTS13 related biomarkers and to investigate which ADAMTS13 assay may be more reliable in the management of acquired TTP. In the first part of this study, we analyzed, during both acute and remission phase, all the biomarkers associated with ADAMTS13 (activity, antigen and class, subclass and titre of anti ADAMTS13 autoantibodies) in acquired TTP patients referred to our centre and we correlated them to episode severity and recurrence. We found that both the Ig class and subclass are of predictive value for acute episode severity in patients with TTP. Disease recurrence seemed to be associated with the presence of IgG antibodies during the acute phase, while alterations in several ADAMTS13 related biomarkers (ADAMTS13 activity or antigen levels <10%, presence of ADAMTS13 inhibitor or IgG) could predict recurrence risk when measured during disease remission. The results of this study confirmed and extended previous data on the prognostic value of ADAMTS13 testing, providing valuable information for the identification of patients at higher risk requiring closer follow up. The studies conducted in the second part of this doctorate intended to investigate the performance of two widely used methods, the collagen-binding assay (CBA) and the more recent fluorescence resonance energy transfer (FRET) assay, for the measurement of ADAMTS13 activity and anti ADAMTS13 inhibitors. Given the prognostic value of ADAMTS13-related markers (as demonstrated in the first part of this thesis), it is essential to determine which ADAMTS13 assay is more reliable in the management of patients suffering from this rare but life-threatening disease. CBA and FRET assay strongly differ from a methodological point of view: they use different substrates (full-length VWF VS peptide VWF), different experimental conditions (urea VS none) and different detection methods (indirect detection of cleavage products through absorbance measurement VS direct detection of cleavage products through fluorescence measurement). Several studies have been performed to evaluate the performance and utility of the two assays for the measurement of ADAMTS13 activity, but they have never been compared for the detection of anti ADAMTS13 inhibitors in mixing studies. Moreover, despite the good correlation in the quantification of ADAMTS13 activity in normal plasmas, important discrepancies have been described in a small but relevant percentage of TTP cases. First, we decided to compare anti-ADAMTS13 inhibitors titres measured by CBA and FRET assays in acquired TTP patients enrolled in the Milan TTP Registry. We found that the two assays performed similarly and concluded that FRET, which is more rapid and easier to perform than CBA, could hence be the assay of choice for inhibitor quantification. Secondly, we investigated whether CBA or FRET assay results reflect ADAMTS13 activity level in vivo in samples presenting circulating anti-ADAMTS13 antibodies and discordant results between the two assays. We performed an analysis of the multimeric pattern of VWF and measured ADAMTS13 activity using an additional assay which employs a full-length VWF substrate under flow conditions. We found that FRET assay results, despite the use of a non-physiologic peptide-based VWF substrate, more closely resemble ADAMTS13 activity level in these particular group of samples. Moreover, we showed that the presence of denaturing agents in CBA is a likely cause of the observed discrepancies. On the whole, these findings allow us to affirm that FRET rather than CBA should be considered the assay of choice for the measurement of both ADAMTS13 activity and ADAMTS13 inhibitors. In conclusion, the research conducted during the doctorate further established the clinical utility of ADAMTS13-related assays in acquired TTP and provided methodological guidelines for the choice of such assays when assessing the severe deficiency of ADAMTS13 or the presence of anti-ADAMTS13 inhibitors.