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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

Analysis of the inhibitory potential of total IgG isolated from acquired TTP patients.A) and B) 125 pM MDTCS was incubated with increasing concentrations of IgG isolated from either normal or TTP patient plasmas in the absence (solid lines) and presence of preincubation with 10 nM MDTC (dotted lines). A) Normal IgG had no effect on MDTCS activity, and addition of MDTC did not affect the activity detected. Three examples of TTP patients with anti-N-terminal alone antibodies are shown ± MDTC. All samples were inhibitory and this inhibition was not influenced by MDTC. B) as in A, except examples of IgG isolated from patients with both anti-N- and C-terminal antibodies are shown. Note the different x-axis scale highlighting that these IgG preparations are not as inhibitory. IgG from patients #51, #55 and #86 had no inhibitory effect upon MDTCS activity. C) Graph depicting the IgG concentration at which 50% enzyme inhibition was achieved (IC50) for each patient tested with antibodies with different domain specificities. Patients are separated in five groups (Groups I–V) based on their domain specificity and the inhibitory potential of their IgG. D) Inhibition of 125 pM MDTCS by 0.7 μM total IgG isolated from Group I samples, all samples shown inhibit MDTCS appreciably at this concentration. E) Inhibition of 125 pM MDTCS by 1.4 μM total IgG isolated from Group II samples. Samples shown inhibit MDTCS variably at this concentration. F) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group III samples. Samples shown inhibit MDTCS by ~ 50% at 5.6 μM total IgG. G) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group IV samples. At 5.6 μM total IgG, little or no inhibition of MDTCS was detected for these samples.
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f0020: Analysis of the inhibitory potential of total IgG isolated from acquired TTP patients.A) and B) 125 pM MDTCS was incubated with increasing concentrations of IgG isolated from either normal or TTP patient plasmas in the absence (solid lines) and presence of preincubation with 10 nM MDTC (dotted lines). A) Normal IgG had no effect on MDTCS activity, and addition of MDTC did not affect the activity detected. Three examples of TTP patients with anti-N-terminal alone antibodies are shown ± MDTC. All samples were inhibitory and this inhibition was not influenced by MDTC. B) as in A, except examples of IgG isolated from patients with both anti-N- and C-terminal antibodies are shown. Note the different x-axis scale highlighting that these IgG preparations are not as inhibitory. IgG from patients #51, #55 and #86 had no inhibitory effect upon MDTCS activity. C) Graph depicting the IgG concentration at which 50% enzyme inhibition was achieved (IC50) for each patient tested with antibodies with different domain specificities. Patients are separated in five groups (Groups I–V) based on their domain specificity and the inhibitory potential of their IgG. D) Inhibition of 125 pM MDTCS by 0.7 μM total IgG isolated from Group I samples, all samples shown inhibit MDTCS appreciably at this concentration. E) Inhibition of 125 pM MDTCS by 1.4 μM total IgG isolated from Group II samples. Samples shown inhibit MDTCS variably at this concentration. F) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group III samples. Samples shown inhibit MDTCS by ~ 50% at 5.6 μM total IgG. G) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group IV samples. At 5.6 μM total IgG, little or no inhibition of MDTCS was detected for these samples.

Mentions: To examine further the inhibitory potential of the autoantibodies in a larger number of samples, we assayed the ability of isolated total IgG to inhibit proteolysis of FRETS-VWF73 by the ADAMTS13 N-terminal domains, MDTCS. Total IgG from 43 patients (29 first presentation and 14 relapse samples) was isolated and titrated into FRETS-VWF73 activity assays to estimate the IgG concentration at which 50% enzyme inhibition was achieved (IC50) (Fig. 4A–G).


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

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

Analysis of the inhibitory potential of total IgG isolated from acquired TTP patients.A) and B) 125 pM MDTCS was incubated with increasing concentrations of IgG isolated from either normal or TTP patient plasmas in the absence (solid lines) and presence of preincubation with 10 nM MDTC (dotted lines). A) Normal IgG had no effect on MDTCS activity, and addition of MDTC did not affect the activity detected. Three examples of TTP patients with anti-N-terminal alone antibodies are shown ± MDTC. All samples were inhibitory and this inhibition was not influenced by MDTC. B) as in A, except examples of IgG isolated from patients with both anti-N- and C-terminal antibodies are shown. Note the different x-axis scale highlighting that these IgG preparations are not as inhibitory. IgG from patients #51, #55 and #86 had no inhibitory effect upon MDTCS activity. C) Graph depicting the IgG concentration at which 50% enzyme inhibition was achieved (IC50) for each patient tested with antibodies with different domain specificities. Patients are separated in five groups (Groups I–V) based on their domain specificity and the inhibitory potential of their IgG. D) Inhibition of 125 pM MDTCS by 0.7 μM total IgG isolated from Group I samples, all samples shown inhibit MDTCS appreciably at this concentration. E) Inhibition of 125 pM MDTCS by 1.4 μM total IgG isolated from Group II samples. Samples shown inhibit MDTCS variably at this concentration. F) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group III samples. Samples shown inhibit MDTCS by ~ 50% at 5.6 μM total IgG. G) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group IV samples. At 5.6 μM total IgG, little or no inhibition of MDTCS was detected for these samples.
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Related In: Results  -  Collection

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f0020: Analysis of the inhibitory potential of total IgG isolated from acquired TTP patients.A) and B) 125 pM MDTCS was incubated with increasing concentrations of IgG isolated from either normal or TTP patient plasmas in the absence (solid lines) and presence of preincubation with 10 nM MDTC (dotted lines). A) Normal IgG had no effect on MDTCS activity, and addition of MDTC did not affect the activity detected. Three examples of TTP patients with anti-N-terminal alone antibodies are shown ± MDTC. All samples were inhibitory and this inhibition was not influenced by MDTC. B) as in A, except examples of IgG isolated from patients with both anti-N- and C-terminal antibodies are shown. Note the different x-axis scale highlighting that these IgG preparations are not as inhibitory. IgG from patients #51, #55 and #86 had no inhibitory effect upon MDTCS activity. C) Graph depicting the IgG concentration at which 50% enzyme inhibition was achieved (IC50) for each patient tested with antibodies with different domain specificities. Patients are separated in five groups (Groups I–V) based on their domain specificity and the inhibitory potential of their IgG. D) Inhibition of 125 pM MDTCS by 0.7 μM total IgG isolated from Group I samples, all samples shown inhibit MDTCS appreciably at this concentration. E) Inhibition of 125 pM MDTCS by 1.4 μM total IgG isolated from Group II samples. Samples shown inhibit MDTCS variably at this concentration. F) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group III samples. Samples shown inhibit MDTCS by ~ 50% at 5.6 μM total IgG. G) Inhibition of 125 pM MDTCS by 5.6 μM total IgG isolated from Group IV samples. At 5.6 μM total IgG, little or no inhibition of MDTCS was detected for these samples.
Mentions: To examine further the inhibitory potential of the autoantibodies in a larger number of samples, we assayed the ability of isolated total IgG to inhibit proteolysis of FRETS-VWF73 by the ADAMTS13 N-terminal domains, MDTCS. Total IgG from 43 patients (29 first presentation and 14 relapse samples) was isolated and titrated into FRETS-VWF73 activity assays to estimate the IgG concentration at which 50% enzyme inhibition was achieved (IC50) (Fig. 4A–G).

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