Hemophagocytic lymphohistiocytosis is associated with deficiency and closed conformation of ADAMTS-13.

Background: A disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS-13) is the specific von Willebrand factor-cleaving protease and circulates in a closed and latent conformation due to a spacer/CUB1 domain interaction. ADAMTS-13 is allosterically activated after binding of its substrate or antibodies, inducing an open conformation. Recently, we suggested a potential role of plasmin (fibrinolysin) in hemostasis disorders reported in most patients with hemophagocytic lymphohistiocytosis (HLH), a rare and life-threatening condition related to a severe systemic inflammatory state. Most patients with HLH had a partial ADAMTS-13 deficiency, and plasmin could induce a truncation of the C-terminal part of ADAMTS-13 and thus an open conformation. Objectives: To understand the effect of plasmin on ADAMTS-13, our study aimed to investigate ADAMTS-13 conformation in patients with HLH. Methods: Forty-five critically ill patients with HLH were prospectively enrolled between April 2015 and December 2018. ADAMTS-13 activity was measured by fluorescent resonance energy transfer-VWF73 assay, ADAMTS-13 antigen, and conformation with our homemade 3H9-enzyme-linked immunosorbent assay and 1C4-enzyme-linked immunosorbent assay. Results: ADAMTS-13 activity ranged from <10 to 65 IU/dL, and 41 of the 45 patients had a quantitative deficiency in ADAMTS-13 (activity <50 IU/dL). Twenty patients had a severe ADAMTS-13 deficiency (activity <20 IU/dL). ADAMTS-13 conformation was folded in all patients under normal conditions. Surprisingly, the switch of ADAMTS-13 conformation expected with the monoclonal antibody 17G2 (anti-CUB1) was disturbed in 6 patients (activity <20 IU/dL). Conclusion: Our study reported that ADAMTS-13 conformation is closed in HLH and provides an indirect proof that plasmin is not able to massively degrade ADAMTS-13. Further studies on glycosylation and citrullination profiles of ADAMTS-13 are needed to understand their role in HLH.

Toward gene therapy for congenital thrombotic thrombocytopenic purpura.

Congenital thrombotic thrombocytopenic purpura (cTTP) is caused by a severe deficiency in the plasma metalloprotease ADAMTS-13. The current management of cTTP is dependent on the prophylactic administration of ADAMTS-13 via plasma infusion. This is a demanding therapy for patients because transfusions are lifelong and time-consuming and allergic reactions frequently occur. Although current management of cTTP controls acute episodes, it does not provide a long-lasting cure for this disease. The endogenous expression of ADAMTS-13 after gene transfer would provide a curative therapy and ongoing research explores various preclinical gene therapeutic approaches for cTTP. This review focuses on the current state of the literature regarding preclinical gene therapy studies for cTTP and on the challenges of developing a gene therapy medicinal product for cTTP.

Immune-mediated thrombotic thrombocytopenic purpura plasma induces calcium- and IgG-dependent endothelial activation: correlations with disease severity.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 auto-antibodies, with subsequent accumulation of circulating ultra-large von Willebrand factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on the surface of dermal human micro-vascular endothelial cell (HMVEC-d), associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)'2 fragments purified from the immunoglobulin G fraction of iTTP plasma mainly induced endothelial cell activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patients' B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small intering RNA, significantly decreased the stimulating effects of iTTP immunoglobulin G. In conclusion, Ca2+-mediated endothelial cell activation constitutes a "second hit" of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation.

BACKGROUND: Acquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear-induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti-ADAMTS13 monoclonal antibody (mAb) that inhibits the shear-induced proteolysis of VWF by ADAMTS13. METHODS: Human or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti-ADAMTS13 mAb 3H9 or 17C7 (20 µg/ml) or control anti-ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined. RESULTS: We demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella-induced aVWS calf model. CONCLUSION: Hence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

Improvement of recombinant ADAMTS13 production through a more optimal signal peptide or an N-terminal fusion protein.

BACKGROUND: Recombinant human ADAMTS13 (rADAMTS13) is a key protein in fundamental research for investigating its mode of action and the pathophysiology of thrombotic thrombocytopenic purpura (TTP). However, the expression of rADAMTS13 is quite low in mammalian cells, which makes the production of the protein time-consuming and labor-intensive. OBJECTIVES: We aimed at increasing the yield of rADAMTS13 by (1) using a more optimal signal peptide (SP) and (2) constructing an N-terminal fusion protein of ADAMTS13 with human serum albumin domain 1 (AD1-ADAMTS13). METHODS: Six SPs were investigated to select the most optimal SP. Expression plasmids containing the most optimal SP and ADAMTS13 cDNA or the fusion construct AD1-ADAMTS13 were generated and transiently transfected into CHOEBNALT85 cell-line. Expression levels of rADAMTS13 in expression medium were analyzed and compared with the expression level of rADAMTS13 with native SP (nat-SP). RESULTS: Expression of rADAMTS13 with coagulation factor VII (FVII) SP was 3-fold higher (16.00 µg/ml) compared with the expression with nat-SP (5.03 µg/ml). The highest yields were obtained with AD1-ADAMTS13 protein with a 15-fold higher concentration (78.22 µg/ml) compared with the expression with nat-SP. The rADAMTS13 expressed with FVII-SP retained its activity (104.0%) to cleave von Willebrand factor, whereas AD1-ADAMTS13 demonstrated even higher activity (144.3%). CONCLUSION: We succeeded in generating expression vectors that yield (1) rADAMTS13 at higher levels because of more optimal FVII-SP and (2) high levels of AD1-ADAMTS13 N-terminal fusion protein. The highest expression levels were obtained with AD1-ADAMTS13 N-terminal fusion protein, which is paving the way for highly efficient protein production.

Anti-ADAMTS13 autoantibody profiling in patients with immune-mediated thrombotic thrombocytopenic purpura.

Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) autoantibodies cause a severe ADAMTS13 deficiency in immune-mediated thrombotic thrombocytopenic purpura (iTTP). ADAMTS13 consists of a metalloprotease (M), a disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). We recently developed a high-throughput epitope mapping assay based on small, nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2). With this assay, we performed a comprehensive epitope mapping using 131 acute-phase samples and for the first time a large group of remission samples (n = 50). Next, samples were stratified according to their immunoprofiles, a field that is largely unexplored in iTTP. Three dominant immunoprofiles were found in acute-phase samples: profile 1: only anti-CS autoantibodies (26.7%); profile 2: both anti-CS and anti-CUB1-2 autoantibodies (12.2%); and profile 3: anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, and anti-CUB1-2 autoantibodies (8.4%). Interestingly, profile 1 was the only dominant immunoprofile in remission samples (52.0%). Clinical data were available for a relatively small number of patients with acute iTTP (>68), and no correlation was found between immunoprofiles and disease severity. Nevertheless, profile 1 was linked with younger and anti-T2-T5 autoantibodies with older age and the absence of anti-CUB1-2 autoantibodies with cerebral involvement. In conclusion, identifying acute phase and remission immunoprofiles in iTTP revealed that anti-CS autoantibodies seem to persist or reappear during remission providing further support for the clinical development of a targeted anti-CS autoantibody therapy. A large cohort study with acute iTTP samples will validate possible links between immunoprofiles or anti-domain autoantibodies and clinical data.

Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency in ADAMTS13. In healthy individuals, ADAMTS13 has a folded conformation in which the central spacer (S) domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G antibodies (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in patients with iTTP are directed against the different ADAMTS13 domains, but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13, thereby opening ADAMTS13. To this end, we purified anti-CS and anti-CUB autoantibodies from 13 patients with acute iTTP by affinity chromatography. The successfully purified anti-CS (10/13 patients) and anti-CUB (4/13 patients) autoantibody fractions were tested further in our ADAMTS13 conformation enzyme-linked immunosorbent assay to study whether they could open ADAMTS13. Interestingly, all purified anti-CS fractions (10/10 patients) were able to open ADAMTS13. On the other hand, only half of the purified anti-CUB fractions (2/4 patients) opened ADAMTS13. Our finding highlights that anti-CS autoantibodies that open ADAMTS13 are a common feature of the autoimmune response in iTTP.

Determination of anti-ADAMTS-13 autoantibody titers in ELISA: Influence of ADAMTS-13 presentation and autoantibody detection.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by inhibitory and/or clearing anti-ADAMTS-13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13) autoantibodies. To determine the presence and total level of anti-ADAMTS-13 autoantibodies, commercial and in-house developed ELISAs are performed. However, different ELISA methods vary in relation to the presentation of recombinant (r)ADAMTS-13 and the detection method of the anti-ADAMTS-13 autoantibodies. Currently, the influence of those different approaches on anti-ADAMTS-13 autoantibody titers is not known. OBJECTIVES: To assess the influence of different ADAMTS-13 presentation- and autoantibody detection methods on anti-ADAMTS-13 autoantibody titers in ELISA. MATERIALS/METHODS: Anti-ADAMTS-13 autoantibody titers from 18 iTTP patients were determined using four different set-ups of anti-ADAMTS-13 autoantibody ELISAs. The ELISAs varied in the used presentation of rADAMTS-13 (directly coated full-length rADAMTS-13, directly coated rMDTCS and rT2C2, or antibody-captured full-length rADAMTS-13) and the detection antibodies (polyclonal anti-human IgG or monoclonal anti-human IgG1-4 antibodies). RESULTS: Strong correlations between the different anti-ADAMTS-13 autoantibody ELISA approaches were observed, when using polyclonal anti-human IgG detection antibodies recognizing all IgG subclasses similarly, independent of the method of rADAMTS-13 presentation. Anti-ADAMTS-13 autoantibody titers correlated less when using a mixture of monoclonal anti-human IgG1-4 , because not all IgG subclasses were recognized with similar affinities. CONCLUSION: Anti-ADAMTS-13 autoantibody levels using different methods of rADAMTS-13 presentation strongly correlate. However, the levels of anti-ADAMTS-13 autoantibodies are highly dependent on the detection antibody used, which should detect all IgG subclasses (IgG1-4 ) equally well.

Immunogenic hotspots in the spacer domain of ADAMTS13 in immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 autoantibodies inducing a severe deficiency of ADAMTS13. Epitope mapping studies on samples obtained during acute iTTP episodes have shown that the iTTP immune response is polyclonal, with almost all patients having autoantibodies targeting the spacer domain of ADAMTS13. OBJECTIVES: To identify the immunogenic hotspots in the spacer domain of ADAMTS13. PATIENTS/METHODS: A library of 11 full-length ADAMTS13 spacer hybrids was created in which amino acid regions of the spacer domain of ADAMTS13 were exchanged by the corresponding region of the spacer domain of ADAMTS1. Next, the full-length ADAMTS13 spacer hybrids were used in enzyme-linked immunosorbent assay to epitope map anti-spacer autoantibodies in 138 samples from acute and remission iTTP patients. RESULTS: Sixteen different anti-spacer autoantibody profiles were identified with a similar distribution in acute and remission patients. There was no association between the anti-spacer autoantibody profiles and disease severity. Almost all iTTP samples contained anti-spacer autoantibodies against the following three regions: amino acid residues 588-592, 602-610, and 657-666 (hybrids E, G, and M). Between 31% and 57% of the samples had anti-spacer autoantibodies against amino acid regions 572-579, 629-638, 667-676 (hybrids C, J, and N). In contrast, none of the samples had anti-spacer autoantibodies against amino acid regions 556-563, 564-571, 649-656, and 677-685 (hybrids A, B, L, and O). CONCLUSION: We identified three hotspot regions (amino acid regions 588-592, 602-610, and 657-666) in the spacer domain of ADAMTS13 that are targeted by anti-spacer autoantibodies found in a large cohort of iTTP patients.

Insights into ADAMTS13 structure: impact on thrombotic thrombocytopenic purpura diagnosis and management.

PURPOSE OF REVIEW: Fundamental knowledge on the role of a disintegrin and metalloprotease with thrombospondin type one repeats, member 13 (ADAMTS13) has been crucial to better understand the pathophysiology of the rare and life-threatening disease thrombotic thrombocytopenic purpura (TTP). RECENT FINDINGS: ADAMTS13 works through a molecular zipper mechanism to proteolyze its substrate von Willebrand factor (VWF). Recent insights into the structure and function of ADAMTS13 led to the identification of an allosteric activation mechanism. Therefore, ADAMTS13 is roughly folded in two in which the N-terminal spacer (S) domain and C-terminal T7-CUB2 domains interact to adopt a closed conformation. Upon substrate binding, ADAMTS13 adopts an open conformation in which the S-T7-CUB2 interaction is abrogated to further position VWF towards the catalytic cleft, inducing activation of the latent metalloprotease domain and resulting in cleavage of VWF. Unravelling the structure function relationship of ADAMTS13 helped identifying open ADAMTS13 as a novel and unique biomarker for immune-mediated TTP (iTTP). This novel biomarker has potential in the diagnosis, treatment and follow-up of iTTP. SUMMARY: In this review, the most recent findings on the structure and working mechanism of ADAMTS13 are addressed. In addition, how those findings led to the identification of a novel biomarker, and how this novel biomarker could have an impact on the diagnosis, management and follow-up of iTTP patients are discussed.

Current and Future Perspectives on ADAMTS13 and Thrombotic Thrombocytopenic Purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare, relapsing, and life-threatening disorder with an annual incidence of 10 cases per million people. TTP is a thrombotic microangiopathy characterized by severe thrombocytopenia, microangiopathic hemolytic anemia, and organ ischemia. The disease is caused by a severe deficiency of the enzyme ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), which can either be acquired, mainly by autoantibodies targeting ADAMTS13, or congenital due to mutations in the ADAMTS13 gene. Thanks to the establishment of national registries worldwide, fundamental and translational research, major advances have been made on the diagnosis, treatment, and fundamental understanding of TTP, since the description of the first TTP case almost 100 years ago. The introduction of therapeutic plasma exchange in the 1970s has significantly improved patient survival, but novel diagnostic assays, targeted treatments (rituximab, caplacizumab, recombinant ADAMTS13), and the unraveling of both ADAMTS13 function and TTP pathophysiology should help to further improve the patients' quality of life. However, differential diagnosis of TTP remains challenging and still a lot of questions remain unanswered to completely understand this rare and devastating disease.

Generation and validation of small ADAMTS13 fragments for epitope mapping of anti-ADAMTS13 autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments. OBJECTIVES: We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients. METHODS: A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples. RESULTS: Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2-T5, T6-T8, and CUB1-2 were sufficient to accurately determine the antibody-binding sites. CONCLUSION: We have developed a tool to profile patients with iTTP according to their anti-ADAMTS13 antibodies for a better insight in their immune response.

Open ADAMTS13, induced by antibodies, is a biomarker for subclinical immune-mediated thrombotic thrombocytopenic purpura.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.

Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function.

Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.

Anti-ADAMTS13 autoantibodies in immune-mediated thrombotic thrombocytopenic purpura do not hamper ELISA-based quantification of ADAMTS13 antigen.

BACKGROUND: The biological diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is based on determination of ADAMTS13 activity (<10%) and anti-ADAMTS13 autoantibodies. ADAMTS13 antigen levels are not routinely measured in iTTP patients, but studies have shown that antigen levels are a valuable prognostic factor. OBJECTIVES: To (a) report the validation of our in-house developed ADAMTS13 antigen enzyme-linked immunosorbent assay (ELISA) and determine ADAMTS13 antigen in a large cohort of healthy donor and iTTP patient plasma samples; and (b) to investigate whether ADAMTS13 antigen determination is not disturbed by the presence of anti-ADAMTS13 autoantibodies. METHODS: Our in-house ADAMTS13 antigen ELISA was validated in terms of sensitivity, repeatability, and reproducibility. ADAMTS13 antigen levels were determined in plasma samples from 423 healthy donors and 112 acute iTTP patients. Purified IgGs from iTTP patients were added to normal human plasma to determine whether anti-ADAMTS13 autoantibodies hampered ADAMTS13 antigen determination. RESULTS: Our in-house ADAMTS13 antigen ELISA has a detection limit of 3% and low intra-assay (coefficient of variation, %CV < 10%) and inter-assay (%CV < 18%) variability. ADAMTS13 antigen levels were significantly reduced (P < .0001) in acute iTTP patients (15 ± 18%) compared to healthy donors (101 ± 18%). The anti-ADAMTS13 autoantibodies in plasma of iTTP patients did not impede ADAMTS13 antigen determinations using our in-house ELISA. CONCLUSIONS: Our in-house ADAMT13 antigen ELISA is a powerful tool to correctly determine ADAMTS13 antigen levels in iTTP patients, which supports routine ADAMTS13 antigen measurements in these patients to have better insight into disease prognosis.

Generation of anti-idiotypic antibodies to detect anti-spacer antibody idiotopes in acute thrombotic thrombocytopenic purpura patients.

In autoantibody-mediated autoimmune diseases, autoantibody profiling allows patients to be stratified and links autoantibodies with disease severity and outcome. However, in immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, stratification according to antibody profiles and their clinical relevance has not been fully explored. We aimed to develop a new type of autoantibody profiling assay for iTTP based on the use of anti-idiotypic antibodies. Anti-idiotypic antibodies against 3 anti-spacer autoantibodies were generated in mice and were used to capture the respective anti-spacer idiotopes from 151 acute iTTP plasma samples. We next deciphered these anti-spacer idiotope profiles in iTTP patients and investigated whether these limited idiotope profiles could be linked with disease severity. We developed 3 anti-idiotypic antibodies that recognized particular idiotopes in the anti-spacer autoantibodies II-1, TTP73 or I-9, that are involved in ADAMTS13 binding; 35%, 24% and 42% of patients were positive for antibodies with the II-1, TTP73 and I-9 idiotopes, respectively. Stratifying patients according to the corresponding 8 anti-spacer idiotope profiles provided a new insight into the anti-spacer II-1, TTP73 and I-9 idiotope profiles in these patients. Finally, these limited idiotope profiles showed no association with disease severity. We successfully developed 3 anti-idiotypic antibodies that allowed us to determine the profiles of the anti-spacer II-1, TTP73 and I-9 idiotopes in iTTP patients. Increasing the number of patients and/or future development of additional anti-idiotypic antibodies against other anti-ADAMTS13 autoantibodies might allow idiotope profiles of clinical, prognostic value to be identified.

ADAMTS13 Gene Mutations Influence ADAMTS13 Conformation and Disease Age-Onset in the French Cohort of Upshaw-Schulman Syndrome.

BACKGROUND: Congenital thrombotic thrombocytopaenic purpura (TTP) or Upshaw-Schulman syndrome (USS) is a rare, life-threatening, inherited thrombotic microangiopathy (TMA). USS is mostly due to bi-allelic recessive sequence variations of the a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13) gene inducing a severe ADAMTS13 deficiency (activity < 10 IU/dL). In healthy individuals, ADAMTS13 circulates in a folded conformation where CUB domains interact with the spacer domain. The spacer-CUB interaction is abrogated when ADAMTS13 is conformationally activated. OBJECTIVE: This article evaluates the influence of ADAMTS13 sequence variations on both clinical/biological phenotype and ADAMTS13 conformation in USS. PATIENTS AND METHODS: All USS patients from the French registry for TMAs (1 January 2000 to 1 June 2017) were investigated for ADAMTS13 genotype, phenotype (activity, antigen and autoantibodies) and conformation. Clinical records were analysed (inaugural acute TTP and follow-up). Child-onset USS was compared with adult-onset USS. RESULTS: Fifty-six USS patients from 51 families (34 child-onset and 22 adult-onset cases) were enrolled. Child-onset USS was characterized by a large panel of ADAMTS13 sequence variations (n = 43), spread all over ADAMTS13 gene and not correlated with either clinical features or plasmatic ADAMTS13 parameters. In contrast, adult-onset USS, consisting exclusively in pregnancy-induced TTP, included a smaller and distinct panel of ADAMTS13 sequence variations (n = 20) because of one mutation (p.Arg1060Trp) present in 82% of patients. ADAMTS13 conformation was studied in 16 USS patients (5 child-onset and 11 adult-onset USS, encompassing 16 distinct ADAMTS13 sequence variations) whose ADAMTS13 antigen levels were detectable: 14 of 16 patients (87.5%) exhibited abnormalities of ADAMTS13 conformation. CONCLUSION: In USS, age-onset defines two entities and ADAMTS13 sequence variations modify ADAMTS13 conformation.

Preemptive rituximab prevents long-term relapses in immune-mediated thrombotic thrombocytopenic purpura.

Preemptive rituximab infusions prevent relapses in immune thrombotic thrombocytopenic purpura (iTTP) by maintaining normal ADAMTS13 activity. However, the long-term outcome of these patients and the potential adverse events of this strategy need to be determined. We report the long-term outcome of 92 patients with iTTP in clinical remission who received preemptive rituximab after identification of severe ADAMTS13 deficiency (activity <10%) during the follow-up. Thirty-seven patients had >1 iTTP episode, and the median cumulative relapse incidence before preemptive rituximab was 0.33 episode per year (interquartile range [IQR], 0.23-0.66). After preemptive rituximab, the median cumulative relapse incidence in the whole population decreased to 0 episodes per year (IQR, 0-1.32; P < .001). After preemptive rituximab, ADAMTS13 activity recovery was sustained in 34 patients (37%) during a follow-up of 31.5 months (IQR, 18-65), and severe ADAMTS13 deficiency recurred in 45 patients (49%) after the initial improvement. ADAMTS13 activity usually improved with additional courses of preemptive rituximab. In 13 patients (14%), ADAMTS13 activity remained undetectable after the first rituximab course, but retreatment was efficient in 6 of 10 cases. In total, 14 patients (15%) clinically relapsed, and 19 patients (20.7%) experienced benign adverse effects. Preemptive rituximab treatment was associated with a change in ADAMTS13 conformation in respondent patients. Finally, in the group of 23 historical patients with iTTP and persistently undetectable ADAMTS13 activity, 74% clinically relapsed after a 7-year follow-up (IQR, 5-11). In conclusion, persistently undetectable ADAMTS13 activity in iTTP during remission is associated with a higher relapse rate. Preemptive rituximab reduces clinical relapses by maintaining a detectable ADAMTS13 activity with an advantageous risk-benefit balance.