ADAMTS13 Conformation and Immunoprofiles in Japanese Patients with Immune-Mediated Thrombotic Thrombocytopenic Purpura

Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare thrombotic disease caused by severe ADAMTS13 deficiency due to autoantibody (autoAb) production. Immunoprofiles of anti-ADAMTS13 autoAbs have been well-studied in mainly the Caucasian population. In the same population, ADAMTS13 with an open conformation was identified as a novel biomarker for iTTP. These data led to a more precise diagnosis in the acute phase and a profound understanding of the pathophysiology of iTTP. However, these results are based on mainly Caucasian iTTP cohorts, and little information is available for other ethnic cohorts. To validate these novel findings in other ethnic cohorts, we investigated plasma samples from the Japanese iTTP registry.

Materials and Methods This study enrolled 195 Japanese iTTP patients, diagnosed based on the 2017 diagnostic and treatment guidelines for TTP in Japan: (i) severe thrombocytopenia and microangiopathic hemolytic anemia, (ii) a severe deficiency in ADAMTS13 activity (below 10 % of healthy individuals) and (iii) detectable inhibitory anti-ADAMTS13 autoAbs. ADAMTS13 antigen levels were measured using our in-house developed ELISA as previously described. Subsequently, ADAMTS13 conformation ELISA (1C4 ELISA) was applied to the samples in which ADAMTS13 antigen was detectable (>0.02 µg/mL), and Open ADAMTS13 was determined when the conformation index was >0.5. The amount of anti-ADAMTS13 IgG and its IgG subclasses were identified using an in-house ELISA. Immunoprofiling of patient anti-ADAMTS13 autoAbs was done by our high throughput ELISA using non-overlapping ADAMTS13 fragments containing an N-terminal albumin domain 1 (AD1) as a fusion partner. This study was approved by the ethics committee of Nara Medical University.

Results Of the 195 acute phase plasma samples, 81 were eligible for ADAMTS13 conformation determination (ADAMTS13 antigen levels were > 0.02 µg/mL). Open ADAMTS13 was detected in 72 samples and closed ADAMTS13 in 4 samples. ADAMTS13 conformation could not be determined in the remaining 5 samples as their OD values were below the OD corresponding to the detection limit in 1C4 ELISA. In summary, 94.7% (72/76) of the investigated Japanese patients in the acute phase had an open ADAMTS13 conformation, which is in agreement with our previous reports where mainly Caucasian patients were studied.

Next, the median level of anti-ADAMTS13 IgG autoAbs was 68.9 % TTP03 equivalents (IQR: 29.8-139.3). Only 2 samples did not show any autoAbs. As expected, there was a positive correlation between total anti-ADAMTS13 IgG autoAb levels and ADAMTS13 inhibitor (r=0.432, p<0.0001). IgG₁, IgG₂, IgG₃, and IgG₄ were present in 103 (56.3%), 68 (37.2%), 153 (83.6%), and 158 (86.3%) samples respectively. The percentage Japanese patients with IgG₁ autoAbs was not as high as in Caucasian cohorts.

Finally, using an immunoprofiling ELISA, it was shawn that more than 70 % of the patients had anti-Cysteine-rich/Spacer (CS) autoAbs. In addition, we identified that profile 1 with anti-CS autoAbs alone was the most common immunoprofile in the Japanese patients as was observed in the Caucasian patients. While profile 1 did not impact the one-year TTP-related mortality rate, patients with autoAbs against all 6 ADAMTS13 fragments had a higher risk for TTP-related death compared to the other patients (p=0.02).

Conclusion We here validated open ADAMTS13 as a novel biomarker for acute iTTP and determined the dominant immunoprofile in the Japanese cohort, contributing to setting up the diagnosis and managing guidelines across different ethinc cohorts and developing ADAMTS13 variants that do not bind to the anti-CS autoAbs.

Sakai:Takeda Japan Medical Office: Research Funding. Matsumoto:Takeda Pharmaceutical Co. Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Asahi Kase Pharma Coeporation: Honoraria, Research Funding; Chugai Pharmaceutical Co. Ltd.: Research Funding; Alexion Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alfresa Pharma Corporation: Patents & Royalties: ADAMTS13 activity ELISA kit. Vanhoorelbeke:Takeda: Membership on an entity's Board of Directors or advisory committees.