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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

ADAMTS13 inhibition is mediated by anti-spacer antibodies.ADAMTS13 activity assays using VWF115 (top) and VWF106 (bottom) in the presence and absence of isolated IgG samples. 2 nM ADAMTS13 proteolysed VWF115 into 10 kDa and 6.9 kDa fragments (− IgG), within 60 min. 17 μM normal human IgG (NH IgG) did not inhibit this reaction, whereas 7 μM rabbit polyclonal anti-ADAMTS13 led to complete inhibition. Identical reactions containing isolated total IgG (17 μM) from TTP patient samples #16, #81 (remission sample) and #61 are shown (* denotes contaminating band from IgG extraction). In parallel, proteolysis of VWF106, which lacks 9 residues that are critical to ADAMTS13 spacer domain binding, was investigated using 3.5 nM ADAMTS13 and 2 hour reaction times. Under these conditions, VWF106 was only partially proteolysed by ADAMTS13 after 120 min (− IgG). Cleavage was unaffected by normal IgG (NH IgG), but completely inhibited by rabbit polyclonal anti-ADAMTS13. Reactions containing isolated total IgG (29 μM) from TTP patient samples #16, #81 and #61 are shown. TTP patient IgG does not inhibit proteolysis of VWF106.
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f0015: ADAMTS13 inhibition is mediated by anti-spacer antibodies.ADAMTS13 activity assays using VWF115 (top) and VWF106 (bottom) in the presence and absence of isolated IgG samples. 2 nM ADAMTS13 proteolysed VWF115 into 10 kDa and 6.9 kDa fragments (− IgG), within 60 min. 17 μM normal human IgG (NH IgG) did not inhibit this reaction, whereas 7 μM rabbit polyclonal anti-ADAMTS13 led to complete inhibition. Identical reactions containing isolated total IgG (17 μM) from TTP patient samples #16, #81 (remission sample) and #61 are shown (* denotes contaminating band from IgG extraction). In parallel, proteolysis of VWF106, which lacks 9 residues that are critical to ADAMTS13 spacer domain binding, was investigated using 3.5 nM ADAMTS13 and 2 hour reaction times. Under these conditions, VWF106 was only partially proteolysed by ADAMTS13 after 120 min (− IgG). Cleavage was unaffected by normal IgG (NH IgG), but completely inhibited by rabbit polyclonal anti-ADAMTS13. Reactions containing isolated total IgG (29 μM) from TTP patient samples #16, #81 and #61 are shown. TTP patient IgG does not inhibit proteolysis of VWF106.

Mentions: To explore the inhibitory potential of TTP patient autoantibodies, we performed functional analyses using isolated total IgG (Fig. 3). Purified total IgG (17 μM) from a TTP patient with high titre (105% by comparison to reference plasma (18, 20)) anti-ADAMTS13 IgG directed against the N-terminal domains alone (#16 in Fig. 2B) appreciably inhibited VWF115 proteolysis, although specific cleavage products were still detected at this IgG concentration, suggesting that inhibition was not complete under these conditions. Total IgG (17 μM) from another patient (#81, remission sample) with similar anti-ADAMTS13 titre but only anti-C-terminal antibodies, had no inhibitory effect upon VWF115 proteolysis. Patient #61 at presentation (anti-ADAMTS13 IgG titre 99%) with antibodies against both anti-N- and C-terminal domains partially inhibited VWF115 cleavage at this IgG concentration.


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

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

ADAMTS13 inhibition is mediated by anti-spacer antibodies.ADAMTS13 activity assays using VWF115 (top) and VWF106 (bottom) in the presence and absence of isolated IgG samples. 2 nM ADAMTS13 proteolysed VWF115 into 10 kDa and 6.9 kDa fragments (− IgG), within 60 min. 17 μM normal human IgG (NH IgG) did not inhibit this reaction, whereas 7 μM rabbit polyclonal anti-ADAMTS13 led to complete inhibition. Identical reactions containing isolated total IgG (17 μM) from TTP patient samples #16, #81 (remission sample) and #61 are shown (* denotes contaminating band from IgG extraction). In parallel, proteolysis of VWF106, which lacks 9 residues that are critical to ADAMTS13 spacer domain binding, was investigated using 3.5 nM ADAMTS13 and 2 hour reaction times. Under these conditions, VWF106 was only partially proteolysed by ADAMTS13 after 120 min (− IgG). Cleavage was unaffected by normal IgG (NH IgG), but completely inhibited by rabbit polyclonal anti-ADAMTS13. Reactions containing isolated total IgG (29 μM) from TTP patient samples #16, #81 and #61 are shown. TTP patient IgG does not inhibit proteolysis of VWF106.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563118&req=5

f0015: ADAMTS13 inhibition is mediated by anti-spacer antibodies.ADAMTS13 activity assays using VWF115 (top) and VWF106 (bottom) in the presence and absence of isolated IgG samples. 2 nM ADAMTS13 proteolysed VWF115 into 10 kDa and 6.9 kDa fragments (− IgG), within 60 min. 17 μM normal human IgG (NH IgG) did not inhibit this reaction, whereas 7 μM rabbit polyclonal anti-ADAMTS13 led to complete inhibition. Identical reactions containing isolated total IgG (17 μM) from TTP patient samples #16, #81 (remission sample) and #61 are shown (* denotes contaminating band from IgG extraction). In parallel, proteolysis of VWF106, which lacks 9 residues that are critical to ADAMTS13 spacer domain binding, was investigated using 3.5 nM ADAMTS13 and 2 hour reaction times. Under these conditions, VWF106 was only partially proteolysed by ADAMTS13 after 120 min (− IgG). Cleavage was unaffected by normal IgG (NH IgG), but completely inhibited by rabbit polyclonal anti-ADAMTS13. Reactions containing isolated total IgG (29 μM) from TTP patient samples #16, #81 and #61 are shown. TTP patient IgG does not inhibit proteolysis of VWF106.
Mentions: To explore the inhibitory potential of TTP patient autoantibodies, we performed functional analyses using isolated total IgG (Fig. 3). Purified total IgG (17 μM) from a TTP patient with high titre (105% by comparison to reference plasma (18, 20)) anti-ADAMTS13 IgG directed against the N-terminal domains alone (#16 in Fig. 2B) appreciably inhibited VWF115 proteolysis, although specific cleavage products were still detected at this IgG concentration, suggesting that inhibition was not complete under these conditions. Total IgG (17 μM) from another patient (#81, remission sample) with similar anti-ADAMTS13 titre but only anti-C-terminal antibodies, had no inhibitory effect upon VWF115 proteolysis. Patient #61 at presentation (anti-ADAMTS13 IgG titre 99%) with antibodies against both anti-N- and C-terminal domains partially inhibited VWF115 cleavage at this IgG concentration.

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