Open ADAMTS13 Conformation in Immune-Mediated Thrombotic Thrombocytopenic Purpura Is Induced By Anti-ADAMTS13 Autoantibodies and Corresponds with an Ongoing ADAMTS13 Pathology

Background. Deficient ADAMTS13 activity (TS13:act <10%) induced by anti-ADAMTS13 autoantibodies (autoAbs) causes immune-mediated thrombotic thrombocytopenic purpura (iTTP). Recently we showed that an open ADAMTS13 conformation is characteristic for acute iTTP patients, while folded ADAMTS13 was found in 78% of iTTP patients in remission with an TS13:act >50%. However, also iTTP patients in remission with a persistent (<10%) or moderately restored (10-50%) TS13:act have been described, but their ADAMTS13 conformation is unknown. Intriguingly, the factor responsible for inducing open ADAMTS13 in iTTP patients remains elusive. Identifying the cause of open ADAMTS13 in iTTP will help better understand the pathophysiology of iTTP and could help appreciate the prognosis and better manage the prevention of subsequent relapses.

Aim. Determine ADAMTS13 conformation in plasma of iTTP patients during acute TTP and remission when TS13:act is <10%, moderately restored (10-50%) or >50% and investigate if anti-ADAMTS13 autoAbs induce conformational changes in ADAMTS13.

Methods. TS13:act was determined in 120 iTTP plasma samples from 4 different centers (Marseille, Milan, Budapest, Utrecht). Samples were categorized according to the presence of clinical symptoms (acute versusremission) and their TS13:act in remission (>50%, 10-50%,<10%). Next, ADAMTS13 conformation was determined in all samples using our ADAMTS13 conformation ELISA. Additionally, presence of anti-ADAMTS13 autoAbs was also determined via ELISA. Finally, IgG's from 18 acute iTTP plasma samples were purified and added to folded ADAMTS13 from healthy donor (HD) plasma to test whether iTTP IgG's are able to induce the open HD ADAMTS13 conformation.

Results. Of the 120 iTTP plasma samples, 46 were obtained during the acute (clinical signs present) and 74 during the remission phase (clinical signs absent). Further subdividing remission samples showed that TS13:act was >50% in 41, 10-50% in 14 and <10% in 19 samples. ADAMTS13 was open in 98% (45/46) of the acute samples and folded in 71% (29/41) of the remission samples with TS13:act >50%, confirming our previous results. Interestingly, ADAMTS13 was open in 93% and 89% of remission samples with TS13:act 10-50% and <10%, respectively (chi square, P<0.0001). Since anti-ADAMTS13 autoAbs influence TS13:act in iTTP patients, we next could demonstrate that open ADAMTS13 conformation was linked with presence of anti-ADAMTS13 autoAbs (chi square, P<0.0001) suggesting that anti-ADAMTS13 autoAbs could be a factor able to induce an open ADAMTS13 conformation in iTTP patients. To further test this hypothesis, we purified IgG's from 18 acute iTTP plasma's with open ADAMTS13 and added them to HD plasma containing closed ADAMTS13, where 14 of the 18 patient IgG pools (78%) did induce the open conformation in HD ADAMTS13, indicating that patient anti-ADAMTS13 autoAbs indeed can induce conformational changes in ADAMTS13.

Conclusion. We show that ADAMTS13 is not only in the open conformation in iTTP patient plasma during the acute phase but also in remission when TS13:act is <10% or 10-50%. Hence, the presence of open ADAMTS13 in those remission patients indicates that the underlying pathophysiology is still ongoing, emphasizing the need for a close monitoring of those patients. In addition, anti-ADAMTS13 autoAbs were identified as a factor responsible for the change in conformation in ADAMTS13 in iTTP.