Humoral immune response to ADAMTS13 in acquired thrombotic thrombocytopenic purpura

Summary. The apparently spontaneous development of autoantibodies to ADAMTS13 in previously healthy individuals is a major cause of thrombotic thrombocytopenic purpura (TTP). Epitope mapping studies have shown that in most patients antibodies directed towards the spacer domain of ADAMTS13 are present. A single antigenic surface comprising Arg⁶⁶⁰, Tyr⁶⁶¹ and Tyr⁶⁶⁵ that contributes to the productive binding of ADAMTS13 to unfolded von Willebrand factor is targeted by anti‐spacer domain antibodies. Antibodies directed to the carboxyl‐terminal CUB1–2 and TSP2–8 domains have also been observed in the plasma of patients with acquired TTP. As yet it has not been established whether this class of antibodies modulates ADAMTS13 activity. Inspection of the primary sequence of human monoclonal anti‐ADAMTS13 antibodies suggests that the variable heavy chain germline gene segment VH1–69 is frequently incorporated. We suggest a model in which ‘shape complementarity’ between the spacer domain and residues encoded by the VH1–69 gene segment explain the preferential use of this variable heavy chain gene segment. Finally, a model is presented for the development of anti‐ADAMTS13 antibodies in previously healthy individuals that incorporates the recent identification of HLA DRB1*11 as a risk factor for acquired TTP.     

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. Objective: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. Methods and Results: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. Conclusions: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.     

IgG subclass distribution of anti-ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background : ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). Objective : To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. Methodology : An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma. Results : IgG₄ (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG₁ (52%), IgG₂ (50%), and IgG₃ (33%). IgG₄ was found either alone (17/52) or with other IgG subclasses (35/52). IgG₄ was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG₄ and IgG₁ antibodies ( P  < 0.01). Patients with high IgG₄ levels and undetectable IgG₁ are more prone to relapse than patients with low IgG₄ levels and detectable IgG₁. Conclusions : All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG₄, followed by IgG₁, antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG₄ could be useful for the identification of patients at risk of disease recurrence.     

Cardiac involvement in acute thrombotic thrombocytopenic purpura: association with troponin T and IgG antibodies to ADAMTS 13

Summary.  Introduction: Evidence for cardiac involvement in thrombotic thrombocytopenic purpura (TTP) is uncommonly described. Methodology: We retrospectively reviewed 41 patients assessing troponin T as a marker for cardiac involvement in acute TTP with clinical symptoms, electrocardiograms (ECG) and echocardiograms. A histopathological review of five patients who died of acute TTP was also undertaken. Results: In 54% (22/41) of patients, troponin T was ≥ 0.05μg L⁻¹ (normal range 0–0.01 μg L⁻¹). Half (12/22) had cardiac symptoms and 8/22 with a raised troponin T reported chest pain. ECG changes were present in 62% of patients with a raised troponin T. Median anti-ADAMTS 13 IgG antibody was significantly higher ( P  = 0.018) in patients with troponin T ≥ 0.05 μg L⁻¹ (58.5% (range 17–162%), compared with patients with troponin T < 0.05 μg L⁻¹ (35%, range 9–134%). Patients who died had higher troponin T levels (median 0.305 μg L⁻¹) and raised anti-ADAMTS 13 IgG (median 66.5%). On admission, there were no deaths in those with troponin T ≤ 0.04μg L⁻¹. Histology confirmed widespread myocardial microvascular thrombi. Conclusion: Clinical symptoms, ECG changes and echocardiograms are poor predictors of cardiac disease in acute TTP. Troponin T is specific for cardiac muscle and a sensitive marker of myocardial damage. In TTP patients, raised levels (≥ 0.05 μg L⁻¹) signify myocardial necrosis associated with microvascular thrombi. Mortality and acute morbidity was associated with higher admission troponin T and raised IgG antibody (> 67%) to ADAMTS 13.     

An open conformation of ADAMTS‐13 is a hallmark of acute acquired thrombotic thrombocytopenic purpura

Essentials

• Conformational changes in ADAMTS‐13 are part of its mode‐of‐action.
• The murine anti‐ADAMTS‐13 antibody 1C4 discriminates between folded and open ADAMTS‐13.
• ADAMTS‐13 conformation is open in acute acquired thrombotic thrombocytopenic purpura (TTP).
• Our study forms an important basis to fully elucidate the pathophysiology of TTP.

Summary

Background

Acquired thrombotic thrombocytopenic purpura (aTTP) is an autoimmune disorder characterized by absent ADAMTS‐13 activity and the presence of anti‐ADAMTS‐13 autoantibodies. Recently, it was shown that ADAMTS‐13 adopts a folded or an open conformation.

Objectives

As conformational changes in self‐antigens play a role in the pathophysiology of different autoimmune diseases, we hypothesized that the conformation of ADAMTS‐13 changes during acute aTTP.

Methods

Antibodies recognizing cryptic epitopes in the spacer domain were generated. Next, the conformation of ADAMTS‐13 in 40 healthy donors (HDs), 99 aTTP patients (63 in the acute phase versus 36 in remission), 12 hemolytic–uremic syndrome (HUS) patients and 63 sepsis patients was determined with ELISA.

Results

The antibody 1C4 recognizes a cryptic epitope in ADAMTS‐13. Therefore, we were able to discriminate between a folded and an open ADAMTS‐13 conformation. We showed that ADAMTS‐13 in HDs does not bind to 1C4, indicating that ADAMTS‐13 circulates in a folded conformation. Similar results were obtained for HUS and sepsis patients. In contrast, ADAMTS‐13 of acute aTTP patients bound to 1C4 in 92% of the cases, whereas, in most cases, this binding was abolished during remission, showing that the conformation of ADAMTS‐13 is open during an acute aTTP episode.

Conclusions

Our study shows that, besides absent ADAMTS‐13 activity and the presence of anti‐ADAMTS‐13 autoantibodies, an open ADAMTS‐13 conformation is also a hallmark of acute aTTP. Demonstrating this altered ADAMTS‐13 conformation in acute aTTP will help to further unravel the pathophysiology of aTTP and lead to improved therapy and diagnosis.     

ADAMTS‐13 activity and autoantibodies classes and subclasses as prognostic predictors in acquired thrombotic thrombocytopenic purpura

Summary. Background: Thrombotic thrombocytopenic purpura (TTP) is a rare life‐threatening disease. Of surviving patients, 45% develops an exacerbation or a late recurrence. Severe ADAMTS‐13 deficiency, both during the acute episode and remission, is a well‐established predictor of recurrence. The predictive value of anti‐ADAMTS‐13 antibodies, their inhibitory activity and Ig class subtype for disease recurrence is still to be established. Objectives: To analyze ADAMTS‐13‐related biomarkers (ADAMTS‐13 and anti‐ADAMTS‐13 immunoglobulins, classes and subclasses) and their potential relationship with prognosis. Patients/Methods: In 115 patients with TTP, we assessed the association between levels of these biomarkers and the severity of acute episodes; we analysed also the hazard ratio (HR) and 95% confidence interval (CI) of recurrence in association with biomarkers levels retrieved at the previous acute episode or during remission, using Cox regression models. Results: During the acute phase, higher IgA, IgG1 and IgG3 titers showed the strongest association with acute episode severity. In the survival analyzes, the only biomarker significantly associated with a high hazard of recurrence after an acute episode was the presence of IgG. Conversly, low ADAMTS‐13 activity or antigen levels (< 10%), the presence of ADAMTS‐13 inhibitor or IgG during remission were all significantly associated with a higher hazard of recurrence. Conclusions: Both the Ig class and subclass are of predictive value for acute episode severity in patients with TTP. Although markers that could predict the risk of recurrence in the acute phase are limited, a thorough assessment of ADAMTS‐13‐related parameters during remission is warranted.     

Complete ADAMTS13 remission in a patient with refractory autoimmune-mediated thrombotic thrombocytopenic purpura after infliximab

Autoimmune thrombotic thrombocytopenic purpura (aTTP) is caused by autoantibodies to the von Willebrand Factor cleaving enzyme, ADAMTS13. Despite recent advances in the treatment of acute aTTP, relapse rates remain high. Guidance for the treatment of patients in clinical remission but with persistent severe ADAMTS13 deficiency who fail to respond to rituximab remains unclear. We report a case of a 29-year-old man diagnosed with aTTP at the age of 11. Over a period of 18 years, he had five clinical relapses with persistent severe ADAMTS13 deficiency (<10%) and presence of autoantibodies during clinical remissions despite immunosuppressive therapy with rituximab, bortezomib and azathioprine. While in a clinical remission, he was diagnosed with Crohn’s disease and initially treated with adalimumab. When he subsequently developed antibodies to adalimumab, he was transitioned to infliximab. ADAMTS13 activity increased to 24% by 2 months of infliximab induction, and four months later the ADAMTS13 activity improved to 42%. This case demonstrates the importance of managing concurrent inflammatory disorders and suggests that TNF may play a role in autoantibody development and ADAMTS13 depletion.     

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw–Schulman syndrome)

Summary.  ADAMTS13, the specific von Willebrand factor (VWF)-cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw–Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations. Six probands with congenital TTP and their family were tested for ADAMTS13 activity in plasma using a two-site immunoradiometric assay and for ADAMTS13 gene mutations using polymerase chain reaction and sequencing. ADAMTS13 activity was severely deficient (< 5%) in the six probands and one mildly symptomatic sibling but normal (> 50%) in all the parents and the asymptomatic siblings. Ten novel candidate ADAMTS13 mutations were identified in all families, showing either a compound heterozygous or a homozygous status in all probands plus the previous sibling and a heterozygous status in the parents. The mutations were spread all over the gene, involving the metalloprotease domain (I79M, S203P, R268P), the disintegrin domain (29 bp deletion in intron/exon 8), the cystein-rich domain (acceptor splice exon 12, R507Q), the spacer domain (A596V), the 3rd TSP1 repeat (C758R), the 5th TSP1 repeat (C908S) and the 8th TSP1 repeat (R1096stop). This study emphasizes the role of ADAMTS13 mutations in the pathogenesis of congenital TTP and suggests that several structural domains of this metalloprotease are involved in both its biogenesis and its substrate recognition process.     

Congenital and acquired ADAMTS13 deficiency: Two mechanisms,one patient

Thrombotic thrombocytopenic purpura (TTP) is a life‐threatening microangiopathy with a heterogeneous and largely unpredictable course. It is caused by ADAMTS13 deficiency, that can be either congenital or due to anti‐ADAMTS13 autoantibodies development. ADAMTS13 deficiency is necessary but not always sufficient to cause acute clinical manifestations and trigger factors may be needed. We report the case of a woman diagnosed with congenital TTP in her adulthood, presenting with anti‐ADAMTS13 autoantibodies in acute phase during ticlopidine consumption. Noteworthy, the two ADAMTS13 mutations identified in this patient are novel: one is a splice‐site mutation located in intron 11 (c.1308+2_5delTAGG) and the other is a point missense mutation in exon 29 (c.4184T>C leading to p.Leu1395Pro substitution). Since congenital TTP is an extremely rare disease and drug‐induced TTP is an uncommon side effect of treatment with ticlopidine, the simultaneous occurrence of both mechanisms of disease in one patient is exceptional. This case represents TTP as a multifactorial disease, with ADAMTS13 genetic abnormality and environmental exposures acting together in determining individual clinical phenotype. J. Clin. Apheresis 30:252–256, 2015. © 2014 Wiley Periodicals, Inc.     

Recombinant ADAMTS13 normalizes von Willebrand factor‐cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Summary. Background: Severe deficiency of the von Willebrand factor (VWF)‐cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non‐inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme. Objectives: To explore the use of recombinant ADAMTS13 (rADAMTS13) as a potential therapeutic agent in acquired TTP, we investigated its efficacy in normalizing VWF‐cleaving activity in the presence of ADAMTS13 inhibitors. Methods: Thirty‐six plasma samples from TTP patients were adjusted to predefined inhibitor titers, and recovery of ADAMTS13 activity was analyzed following supplementation with rADAMTS13. Results: We showed a linear relation between the inhibitor titer measured and effective rADAMTS13 concentration necessary for reconstitution of VWF‐cleaving activity in the presence of neutralizing autoantibodies. Conclusions: Our results support the further investigation of the potential therapeutic applicability of rADAMTS13 as an adjunctive therapy in acquired TTP.     

一例两种新的ADAMTS13基因突变导致的遗传性血栓性血小板减少性紫癜

目的 对1例高度可疑的遗传性血栓性血小板减少性紫癜（TTP）患者的vWF裂解蛋白酶（ADAMTS13，vWF-cp）基因突变进行检测，以明确诊断并制定相应的治疗方案。方法 用残余胶原结合实验（residual-collagen binding assay）动态检测1例TTP患者ADAMTS13的活性和抑制物水平，用PCR方法扩增ADAMTS13的29个外显子及外显子邻近处的内含子，并直接测序检测突变，对其亲属的基因组DNA在患者突变区域扩增并测序。结果 患者ADAMTS13活性降低，未发现抑制物存在测序显示患者的ADAMTS13的凝血酶敏感蛋白1（TSP1）基序重复区第21外显子碱基2708处和25号外显子碱基3283处出现C→T的错义突变（均为纯合子），分别导致该碱基参与编码的丝氨酸密码子（TCG）变为亮氨酸密码子（TTG）（S903L），精氨酸密码子（CGG）变为色氨酸密码子（TGG）（R1095W）患者父母上述突变位置处的基因均呈杂合状态，其兄的该段碱基序列正常，并且在采集的25位家族成员巾共发现11名上述突变位点的正常携带者。结论 该患者是由于ADAMTS13基因纯合子错义突变导致翻译发生错误所致的遗传性TTP。     

Coexistence of thrombotic thrombocytopenic purpura and immune thrombocytopenic purpura in an Asian woman: a case report

A 33-year-old Chinese woman with a history of immune thrombocytopenic purpura presented with heavy menstrual bleeding. She was found to have thrombocytopenia, plasma ADAMTS13 activity of 0%, and positivity for the plasma ADAMTS13 inhibitor. She was diagnosed with the coexistence of thrombotic thrombocytopenic purpura and immune thrombocytopenic purpura. The patient was treated by plasmapheresis, a glucocorticoid, and rituximab. Her platelet level returned to normal, and she was discharged 28 days after admission. The number of plasmapheresis sessions and the timing of rituximab administration may be the key aspects of management of patients with thrombotic thrombocytopenic purpura who have underlying immune dysfunction caused by diseases such as immune thrombocytopenic purpura.     

Caplacizumab: a change in the paradigm of thrombotic thrombocytopenic purpura treatment

ABSTRACT

Introduction: Immune thrombotic thrombocytopenic purpura (iTTP) is an immune-mediated deficiency in von Willebrand factor-cleaving protease ADAMTS13 allowing unrestrained adhesion of von Willebrand factor multimers to platelets and microthrombosis. Caplacizumab, an anti-von Willebrand factor humanized, bivalent single-domain nanobody preventing its binding to the platelet has been investigated and approved for use in the treatment of iTTP.

Areas covered: The purpose of this article is to summarize the available clinical data on the efficacy and safety of caplacizumab in iTTP and to provide our opinion on the place of caplacizumab in current treatment regimens.

Expert opinion: Caplacizumab is a new drug with a complementary mechanism of action with respect to the standard available therapeutics. It demonstrated efficacy in clinical trials through a faster platelet count normalization and protection of patients from exacerbations and refractoriness. Caplacizumab is well tolerated with minor bleeds as the most important side effect. The efficacy of caplacizumab now needs to be assessed in real-life but definitely, this drug opens hope for a significant improvement in iTTP prognosis at the very early, critical step of the disease.

Trial registration: ClinicalTrials.gov identifier: NCT03922308.

Trial registration: ClinicalTrials.gov identifier: NCT02553317.     

A phenotype–genotype correlation of ADAMTS13 mutations in congenital thrombotic thrombocytopenic purpura patients treated in the United Kingdom

Summary. Background: ADAMTS13 mutations play a role in thrombotic thrombocytopenic purpura (TTP) pathogenesis. Objectives: To establish a phenotype–genotype correlation in a cohort of congenital TTP patients. Patients/Methods: Clinical history and ADAMTS13 activity, antigen and anti‐ADAMTS13 antibody assays were used to diagnose congenital TTP, and DNA sequencing and in vitro expression were performed to identify the functional effects of the ADAMTS13 mutations responsible. Results: Seventeen (11 novel) ADAMTS13 mutations were identified in 17 congenital TTP patients. All had severely reduced ADAMTS13 activity and antigen levels at presentation. Six patients with pregnancy‐associated TTP and six patients with childhood TTP were homozygous or compound heterozygous for ADAMTS13 mutations located in the metalloprotease (MP), cysteine‐rich, spacer and/or distal thrombospondin type 1 domains. The adults had TTP precipitated by pregnancy, and had overall higher antigen levels (median, 30 ng mL^?1; range, < 10–57 ng mL^?1) than the children (median, 14 ng mL^?1; range, < 10–40 ng mL^?1). Presentation in the neonatal period was associated with more intensive treatment requirements. The two neonates with the most severe phenotype had mutations in the first thrombospondin type 1 motif of ADAMTS13 (p.R398C, p.R409W, and p.Q436H). Using transfected HEK293T cells, we have shown that p.R398C and p.R409W block ADAMTS13 secretion, whereas p.Q436H allows secretion at reduced levels. Conclusions: This study confirms the heterogeneity of ADAMTS13 defects and an association between ADAMTS13 genotypes and TTP phenotype.