Mechanism underlying severe deficiency of plasma ADAMTS-13 activity in immune thrombotic thrombocytopenic purpura

Abstract

Background: Immune-mediated thrombotic thrombocytopenic purpura is caused by autoantibodies against ADAMTS-13, a plasma enzyme that cleaves von Willebrand factor. However, the mechanism resulting in severe deficiency of plasma ADAMTS-13 activity remains controversial.

Objectives: To determine the mechanism of autoantibody-mediated severe deficiency of plasma ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura.

Methods: Fluorescence resonance energy transfer-VWF73 was used to determine plasma ADAMTS-13 activity. Enzyme-linked immunosorbent assay (ELISA) was used to determine anti-ADAMTS-13 immunoglobulin G. ELISA and capillary electrophoresis-based Western blotting were employed to assess plasma ADAMTS-13 antigen.

Results: We showed that plasma ADAMTS-13 antigen levels varied substantially in the samples collected on admission despite all showing plasma ADAMTS-13 activity of <10 IU/dL (or <10% of normal level) using either ELISA or Western blotting. More severe deficiency of plasma ADAMTS-13 antigen (<10%) was detected in admission samples by ELISA than by capillary Western blotting. There was a significant but moderate correlation between plasma ADAMTS-13 activity and ADAMTS-13 antigen by either assay method, suggesting that severe deficiency of plasma ADAMTS-13 activity is not entirely associated with low levels of ADAMTS-13 antigen.

Conclusion: We conclude that severe deficiency of plasma ADAMTS-13 activity primarily resulted from antibody-mediated inhibition, but the accelerated clearance of plasma ADAMTS-13 antigen via immune complexes may also contribute significantly to severe deficiency of plasma ADAMTS-13 activity in a subset of patients with acute immune-mediated thrombotic thrombocytopenic purpura.

Keywords: ADAMTS-13; TTP/HUS; autoantibodies; capillary Western blotting; von Willebrand factor.

Fig. 1.. The laboratory parameters in patients with iTTP during acute episode and in remission.

A. Platelet count; B. Serum LDH levels; C. Plasma ADAMTS13 activity levels; D. Plasma ADAMTS13 antigen levels. A paired t-test determined the statistical significance of laboratory value differences between acute and remission. Here, **** indicates p value < 0.0001.

Fig. 2.. Detection of plasma ADAMTS13 antigen by capillary electrophoresis plus Western blotting analysis.

A. A representative whole image of CEWB results with molecular weight maker, serially diluted normal plasma (NHP) for calibration, and three iTTP samples. Area with an arrowhead (←) or a star (**) indicates the ADAMTS13 (A13) and non-specific IgG in plasma, respectively. B. The % of cases found to have <10% (purple bars) or ≥10% (blue bars) of plasma ADAMTS13 antigen in acute episode and during remission by ELISA vs CEWB. C-F. Representative images showing the concordant or discordant results of various iTTP patients for their plasma levels of ADAMTS13 antigen (%) by ELISA vs. CEWB, comparing to their proteolytic activity (%) by FRETS-73 (underneath the gel image).

Fig. 3.. Spearman correlation coefficients.

A. correlation between ADAMTS13 antigen by CEWB and that by ELISA; B or C. A positive or a negative correlation between plasma levels of ADAMTS13 activity or anti-ADAMTS13 IgG and ADAMTS13 antigen by ELISA. C or D. A positive or negative correlation between ADAMTS13 activity or anti-ADAMTS13 IgG and ADAMTS13 antigen by CEWB. Here, rho indicates Spearman correlation coefficient. P<0.05 and P<0.01 are statistically significant and highly significant, respectively.