Characterization of autoantibodies to ADAMTS13 in HIV-associated thrombotic thrombocytopenic purpura
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Date
2020-01
Authors
Khemisi, Mmakgabu Martha
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Publisher
University of the Free State
Abstract
Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal thrombotic microangiopathic disorder that can occur secondary to human immunodeficiency virus (HIV) infection and is prevalent in sub-Saharan Africa. The pathogenesis involves deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13 and the presence of anti-ADAMTS13 autoantibodies. Insufficient information is however available regarding epitope specificity and reactivity of the ADAMTS13 autoantibodies present in HIV-associated TTP. In this study, epitope-mapping analysis was performed to provide novel insight into the specific antigenic regions (epitopes) on ADAMTS13 domains affected by autoantibodies in patients with HIV-associated TTP. Anti-ADAMTS13 IgG autoantibodies are also present in HIV positive individuals, and their binding specificities were analysed. Methods: A total of 59 HIV-associated TTP plasma samples with severe ADAMTS13 deficiency of less than 10% were collected prior to plasma therapy and analysed. Hundred (100) plasma samples from HIV positive patients without TTP were included as a control group. We compared the ADAMTS13 parameters i.e. ADAMTS13 antigen and activity levels and autoantibody titers and VWF parameters i.e. antigen levels, propeptide and multimeric patterns between the HIV-associated TTP and the control cohort. To understand the pathogenic mechanisms of anti-ADAMTS13 IgG autoantibodies, a synthetic peptide library comprising of ADAMTS13 proximal domains was used to map potential epitope regions that bind to purified anti-ADAMTS13 IgG antibodies isolated from 53 individual HIV-associated TTP patient samples and 18 control cohort samples using a newly developed Peptide ELISA-based assay. Results: The HIV-associated TTP patient plasma samples had severely reduced ADAMTS13 antigen (<50%) and activity (<10%) levels compared to the HIV positive control samples (ADAMTS13 antigen and activity levels >25% but <150%), with a statistically significance difference (p<0.05%). AntiADAMTS13 IgG autoantibodies were detected in 90% of the HIV-associated TTP patient samples, and only in 18% of the HIV infected control cohort plasma samples. About 90% of the HIV-associated TTP patient samples were found to contain clinically significant ADAMTS13 autoantibodies of which 64% were inhibitory as demonstrated with mixing studies. Furthermore, high anti-ADAMTS13 IgG autoantibody titers (≥50µg/mL) were detected in samples with a low median ADAMTS13 antigen level of ~4.5% and low anti-ADAMTS13 IgG autoantibody titers (<50µg/mL) in samples with a high median ADAMTS13 antigen level of ~12.5%. Additional anti-ADAMTS13 autoantibodies that were detected in the HIV-associated TTP patient samples were IgM (30%) and IgA (64%) isotypes in combination with IgG isotype autoantibodies. In 18 out of the 100 HIV positive control patient samples positive for anti-ADAMTS13 IgG autoantibodies, 28% were positive for IgM and 22% for IgA autoantibody isotypes. Both groups presented with normal to significantly increased VWF:Ag and VWFpp levels (>200%), and no statistically significant difference between them (p>0.05). The epitope mapping results revealed that the Metalloprotease, Cysteine-rich and Spacer domains were constantly (100%) involved in binding anti-ADAMTS13 IgG antibodies isolated from the 53 patients with HIV-associated TTP samples. 58% of these samples contained anti-ADAMTS13 IgG antibodies that bind to the C-terminal part of ADAMTS13 Disintegrin-like domain. However, in the HIV positive plasma samples, the Metalloprotease and Disintegrin-like domains were the primary targets (100%) for anti-ADAMTS13 IgG antibody binding, while only 61% of samples with IgG antibodies showed binding to the Cysteine-rich and Spacer domains of ADAMTS13. The IgG autoantibodies detected in the control cohort samples shared linear epitopes at various regions of the ADAMTS13 proximal domains investigated with anti-ADAMTS13 IgG antibodies detected in HIV-associated TTP patient samples. Conclusions: Most (90%) of patients diagnosed with HIV-associated TTP with severe ADAMTS13 activity levels of less than 10% have anti-ADAMTS13 autoantibodies. Thus, highlighting that ADAMTS13 autoantibody-mediated deficiency may be involved in HIV-associated TTP. Both inhibitory and non-inhibitory anti-ADAMTS13 autoantibodies are present in these patients, suggesting that different pathogenic mechanisms may be involved in HIV-associated TTP. The Metalloprotease, Cysteine-rich and Spacer domains are the primary target for anti-ADAMTS13 IgG autoantibodies in patients with HIV-associated TTP. In contrast, HIV positive patients without TTP may have anti-ADAMTS13 IgG autoantibodies, which may even share linear epitopes with those detected in patients with HIV-associated TTP, but their pathological relevance has not been elucidated. The results of this study provides new insight into the pathophysiology of HIV-associated TTP. HIV-associated TTP patients have anti-ADAMTS13 antibodies potentially affecting the proteolytic activity of this enzyme.
National Health Laboratory Service (NHLS)
National Health Laboratory Service (NHLS)
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Keywords
Thesis (Ph.D. (Haematology and Cell Biology))--University of the Free State, 2020, Human immunodeficiency virus, Thrombotic thrombocytopenic purpura, ADAMTS13, ADAMTS13 autoantibodies