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Pathogenicity of Anti-ADAMTS13 Autoantibodies in Acquired Thrombotic Thrombocytopenic Purpura.

Thomas MR, de Groot R, Scully MA, Crawley JT - EBioMedicine (2015)

Bottom Line: We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen.ADAMTS13 antigen in the lowest quartile at first presentation was associated with increased mortality (odds ratio 5.7).Taken together, our results provide new insights into the pathophysiology of acquired TTP.

View Article: PubMed Central - PubMed

Affiliation: Haemostasis Research Unit, University College London, 51 Chenies Mews, London WC1E 6HX, United Kingdom ; Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom.

ABSTRACT

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease in which anti-ADAMTS13 autoantibodies cause severe enzyme deficiency. ADAMTS13 deficiency causes the loss of regulation of von Willebrand factor multimeric size and platelet-tethering function, which results in the formation of disseminated microvascular platelet microthrombi. Precisely how anti-ADAMTS13 autoantibodies, or antibody subsets, cause ADAMTS13 deficiency (ADAMTS13 activity generally < 10%) has not been formally investigated.

Methods: We analysed 92 acquired TTP episodes at presentation, through treatment and remission/relapse using epitope mapping and functional analyses to understand the pathogenic mechanisms of anti-ADAMTS13 IgG.

Results: 89/92 of TTP episodes had IgG recognising the ADAMTS13 N-terminal domains. The central spacer domain was the only N-terminal antigenic target detected. 38/92 TTP episodes had autoantibodies recognising the N-terminal domains alone; 54/92 TTP episodes also had antibodies against the ADAMTS13 C-terminal domains (TSP2-8 and/or CUB domains). Changes in autoantibody specificity were detected in 9/16 patients at relapse, suggesting a continued development of the disease. Functional analyses on IgG from 43 patients revealed inhibitory IgG were limited to anti-spacer domain antibodies. However, 15/43 patients had autoantibodies with no detectable inhibitory action and as many as 32/43 patients had autoantibodies with inhibitory function that was insufficient to account for the severe deficiency state, suggesting that in many patients there is an alternative pathogenic mechanism. We therefore analysed plasma ADAMTS13 antigen levels in 91 acquired TTP presentation samples. We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen. ADAMTS13 antigen in the lowest quartile at first presentation was associated with increased mortality (odds ratio 5.7).

Conclusions: Anti-spacer domain autoantibodies are the major inhibitory antibodies in acquired TTP. However, depletion of ADAMTS13 antigen (rather than enzyme inhibition) is a dominant pathogenic mechanism. ADAMTS13 antigen levels at presentation have prognostic significance. Taken together, our results provide new insights into the pathophysiology of acquired TTP.

No MeSH data available.


Related in: MedlinePlus

Domain organisation of ADAMTS13 and domain specificity of anti-ADAMTS13 antibodies at TTP presentation.A) Domain organisation of ADAMTS13 consisting of the metalloprotease domain (MP), disintegrin-like domain (Dis), eight thrombospondin repeats (1–8, green), Cysteine-rich domain (Cys), spacer domain and two C-terminal CUB domains. Underneath are denoted the domain fragments used in this study — the N-terminal domain fragments, MDTCS, MDTC and MD, and the C-terminal TSP2–8. B) Graph depicting summary of anti-ADAMTS13 domain specificity ELISAs for 92 TTP patient episodes. TTP patient plasmas (diluted 1/50) were analysed for IgG that recognised immobilised full length ADAMTS13 both with and without preincubation of plasmas with 10 nM MDTCS. For each patient, the proportion of full length ADAMTS13 binding that MDTCS could not compete for is plotted in grey (left axis; proportion non-MDTCS Abs, %). Plasma samples were also analysed for the presence of anti-TSP2–8 IgG in a separate ELISA plotted in black (right axis: TSP2–8, mAU). All patients were positive for anti-N-terminal antibodies, except samples labelled with *, denoting the three patients in which MDTCS could not compete at all for full-length ADAMTS13 binding. Patients 1–38 are termed anti-N-terminal alone, as MDTCS competed for > 85% (dotted line) full length ADAMTS13 binding and these samples exhibited no or very low recognition of TSP2–8. Patients 39–64 are positive for anti-TSP2–8 antibodies. Patients 65–92 are termed anti-CUB, as MDTCS competed for < 85% (dotted line) of full length ADAMTS13 binding, consistent with the presence of anti-C-terminal antibodies, but these samples exhibited no or very low recognition of TSP2–8.
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f0010: Domain organisation of ADAMTS13 and domain specificity of anti-ADAMTS13 antibodies at TTP presentation.A) Domain organisation of ADAMTS13 consisting of the metalloprotease domain (MP), disintegrin-like domain (Dis), eight thrombospondin repeats (1–8, green), Cysteine-rich domain (Cys), spacer domain and two C-terminal CUB domains. Underneath are denoted the domain fragments used in this study — the N-terminal domain fragments, MDTCS, MDTC and MD, and the C-terminal TSP2–8. B) Graph depicting summary of anti-ADAMTS13 domain specificity ELISAs for 92 TTP patient episodes. TTP patient plasmas (diluted 1/50) were analysed for IgG that recognised immobilised full length ADAMTS13 both with and without preincubation of plasmas with 10 nM MDTCS. For each patient, the proportion of full length ADAMTS13 binding that MDTCS could not compete for is plotted in grey (left axis; proportion non-MDTCS Abs, %). Plasma samples were also analysed for the presence of anti-TSP2–8 IgG in a separate ELISA plotted in black (right axis: TSP2–8, mAU). All patients were positive for anti-N-terminal antibodies, except samples labelled with *, denoting the three patients in which MDTCS could not compete at all for full-length ADAMTS13 binding. Patients 1–38 are termed anti-N-terminal alone, as MDTCS competed for > 85% (dotted line) full length ADAMTS13 binding and these samples exhibited no or very low recognition of TSP2–8. Patients 39–64 are positive for anti-TSP2–8 antibodies. Patients 65–92 are termed anti-CUB, as MDTCS competed for < 85% (dotted line) of full length ADAMTS13 binding, consistent with the presence of anti-C-terminal antibodies, but these samples exhibited no or very low recognition of TSP2–8.

Mentions: Full-length ADAMTS13 and fragments; metalloprotease to disintegrin-like domain (MD), metalloprotease to cysteine-rich domain (MDTC), metalloprotease to spacer domain (MDTCS), TSP2–8 domains and CUB1/2 domains were expressed in HEK293 cells and purified from conditioned media (Fig. 2A). VWF115 and VWF106 were expressed and purified as previously described (Supplementary Methods) (Pos et al., 2010).


Pathogenicity of Anti-ADAMTS13 Autoantibodies in Acquired Thrombotic Thrombocytopenic Purpura.

Thomas MR, de Groot R, Scully MA, Crawley JT - EBioMedicine (2015)

Domain organisation of ADAMTS13 and domain specificity of anti-ADAMTS13 antibodies at TTP presentation.A) Domain organisation of ADAMTS13 consisting of the metalloprotease domain (MP), disintegrin-like domain (Dis), eight thrombospondin repeats (1–8, green), Cysteine-rich domain (Cys), spacer domain and two C-terminal CUB domains. Underneath are denoted the domain fragments used in this study — the N-terminal domain fragments, MDTCS, MDTC and MD, and the C-terminal TSP2–8. B) Graph depicting summary of anti-ADAMTS13 domain specificity ELISAs for 92 TTP patient episodes. TTP patient plasmas (diluted 1/50) were analysed for IgG that recognised immobilised full length ADAMTS13 both with and without preincubation of plasmas with 10 nM MDTCS. For each patient, the proportion of full length ADAMTS13 binding that MDTCS could not compete for is plotted in grey (left axis; proportion non-MDTCS Abs, %). Plasma samples were also analysed for the presence of anti-TSP2–8 IgG in a separate ELISA plotted in black (right axis: TSP2–8, mAU). All patients were positive for anti-N-terminal antibodies, except samples labelled with *, denoting the three patients in which MDTCS could not compete at all for full-length ADAMTS13 binding. Patients 1–38 are termed anti-N-terminal alone, as MDTCS competed for > 85% (dotted line) full length ADAMTS13 binding and these samples exhibited no or very low recognition of TSP2–8. Patients 39–64 are positive for anti-TSP2–8 antibodies. Patients 65–92 are termed anti-CUB, as MDTCS competed for < 85% (dotted line) of full length ADAMTS13 binding, consistent with the presence of anti-C-terminal antibodies, but these samples exhibited no or very low recognition of TSP2–8.
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f0010: Domain organisation of ADAMTS13 and domain specificity of anti-ADAMTS13 antibodies at TTP presentation.A) Domain organisation of ADAMTS13 consisting of the metalloprotease domain (MP), disintegrin-like domain (Dis), eight thrombospondin repeats (1–8, green), Cysteine-rich domain (Cys), spacer domain and two C-terminal CUB domains. Underneath are denoted the domain fragments used in this study — the N-terminal domain fragments, MDTCS, MDTC and MD, and the C-terminal TSP2–8. B) Graph depicting summary of anti-ADAMTS13 domain specificity ELISAs for 92 TTP patient episodes. TTP patient plasmas (diluted 1/50) were analysed for IgG that recognised immobilised full length ADAMTS13 both with and without preincubation of plasmas with 10 nM MDTCS. For each patient, the proportion of full length ADAMTS13 binding that MDTCS could not compete for is plotted in grey (left axis; proportion non-MDTCS Abs, %). Plasma samples were also analysed for the presence of anti-TSP2–8 IgG in a separate ELISA plotted in black (right axis: TSP2–8, mAU). All patients were positive for anti-N-terminal antibodies, except samples labelled with *, denoting the three patients in which MDTCS could not compete at all for full-length ADAMTS13 binding. Patients 1–38 are termed anti-N-terminal alone, as MDTCS competed for > 85% (dotted line) full length ADAMTS13 binding and these samples exhibited no or very low recognition of TSP2–8. Patients 39–64 are positive for anti-TSP2–8 antibodies. Patients 65–92 are termed anti-CUB, as MDTCS competed for < 85% (dotted line) of full length ADAMTS13 binding, consistent with the presence of anti-C-terminal antibodies, but these samples exhibited no or very low recognition of TSP2–8.
Mentions: Full-length ADAMTS13 and fragments; metalloprotease to disintegrin-like domain (MD), metalloprotease to cysteine-rich domain (MDTC), metalloprotease to spacer domain (MDTCS), TSP2–8 domains and CUB1/2 domains were expressed in HEK293 cells and purified from conditioned media (Fig. 2A). VWF115 and VWF106 were expressed and purified as previously described (Supplementary Methods) (Pos et al., 2010).

Bottom Line: We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen.ADAMTS13 antigen in the lowest quartile at first presentation was associated with increased mortality (odds ratio 5.7).Taken together, our results provide new insights into the pathophysiology of acquired TTP.

View Article: PubMed Central - PubMed

Affiliation: Haemostasis Research Unit, University College London, 51 Chenies Mews, London WC1E 6HX, United Kingdom ; Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom.

ABSTRACT

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease in which anti-ADAMTS13 autoantibodies cause severe enzyme deficiency. ADAMTS13 deficiency causes the loss of regulation of von Willebrand factor multimeric size and platelet-tethering function, which results in the formation of disseminated microvascular platelet microthrombi. Precisely how anti-ADAMTS13 autoantibodies, or antibody subsets, cause ADAMTS13 deficiency (ADAMTS13 activity generally < 10%) has not been formally investigated.

Methods: We analysed 92 acquired TTP episodes at presentation, through treatment and remission/relapse using epitope mapping and functional analyses to understand the pathogenic mechanisms of anti-ADAMTS13 IgG.

Results: 89/92 of TTP episodes had IgG recognising the ADAMTS13 N-terminal domains. The central spacer domain was the only N-terminal antigenic target detected. 38/92 TTP episodes had autoantibodies recognising the N-terminal domains alone; 54/92 TTP episodes also had antibodies against the ADAMTS13 C-terminal domains (TSP2-8 and/or CUB domains). Changes in autoantibody specificity were detected in 9/16 patients at relapse, suggesting a continued development of the disease. Functional analyses on IgG from 43 patients revealed inhibitory IgG were limited to anti-spacer domain antibodies. However, 15/43 patients had autoantibodies with no detectable inhibitory action and as many as 32/43 patients had autoantibodies with inhibitory function that was insufficient to account for the severe deficiency state, suggesting that in many patients there is an alternative pathogenic mechanism. We therefore analysed plasma ADAMTS13 antigen levels in 91 acquired TTP presentation samples. We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen. ADAMTS13 antigen in the lowest quartile at first presentation was associated with increased mortality (odds ratio 5.7).

Conclusions: Anti-spacer domain autoantibodies are the major inhibitory antibodies in acquired TTP. However, depletion of ADAMTS13 antigen (rather than enzyme inhibition) is a dominant pathogenic mechanism. ADAMTS13 antigen levels at presentation have prognostic significance. Taken together, our results provide new insights into the pathophysiology of acquired TTP.

No MeSH data available.


Related in: MedlinePlus