Elien Roose

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13

Recently, conformational activation of ADAMTS‐13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2‐CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF).

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.

ADAMTS13 deficiency promotes microthrombosis in a murine model of diet-induced liver steatosis.

ADAMTS13 cleaves ultralarge multimeric von Willebrand Factor (VWF), thereby preventing formation of platelet-rich microthrombi. ADAMTS13 is mainly produced by hepatic stellate cells, and numerous studies have suggested a functional role of ADAMTS13 in the pathogenesis of liver diseases. The aim of our study was to investigate a potential role of ADAMTS13 in formation of hepatic microthrombi and development of non-alcoholic steatohepatitis (NASH), and furthermore to evaluate whether plasmin can compensate for the absence of ADAMTS13 in removal of thrombi. Therefore, we used a model of high-fat diet-induced steatosis in Adamts13 deficient (Adamts13-/-) and wild-type (WT) control mice. Microthrombi were more abundant in the liver of obese Adamts13-/- as compared to obese WT or to lean Adamts13-/- mice. Obese Adamts13-/- mice displayed lower platelet counts and higher prevalence of ultra-large VWF multimers. Hepatic plasmin-α2-antiplasmin complex levels were comparable for obese WT and Adamts13-/- mice and were lower for lean Adamts13-/- than WT mice, not supporting marked activation of the fibrinolytic system. High fat diet feeding, as compared to normal chow, resulted in enhanced liver triglyceride levels for both genotypes (p < 0.0001) and steatosis (p < 0.0001 for WT mice, p = 0.002 for Adamts13-/- mice) without differences between the genotypes. Expression of markers of inflammation, oxidative stress, steatosis and fibrosis was affected by diet, but not by genotype. Thus, our data confirm that obesity promotes NASH, but do not support a detrimental role of ADAMTS13 in its development. However, Adamts13 deficiency in obese mice promotes hepatic microthrombosis, whereas a compensatory role of plasmin in removal of microthrombi in the absence of ADAMTS13 could not be demonstrated.

ADAMTS13 and anti-ADAMTS13 autoantibodies in thrombotic thrombocytopenic purpura – current perspectives and new treatment strategies

ABSTRACT A deficiency in ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type-1 repeats, member 13) is associated with thrombotic thrombocytopenic purpura (TTP). Congenital TTP is caused by a defect in the ADAMTS13 gene resulting in decreased or absent enzyme activity; acquired TTP results from autoantibodies that either inhibit the activity or increase the clearance of ADAMTS13. Despite major progress in recent years in our understanding of the disease, many aspects around the pathophysiology of TTP are still unclear. Newer studies expanded the TTP field from ADAMTS13 and inhibitory antibodies to immune complexes, cloned autoantibodies, and a possible involvement of other proteases. Additionally, several new treatment strategies supplementing plasma-exchange and infusion are under investigation for a better and more specific treatment of TTP patients. In this review, we discuss the recent insights in TTP pathophysiology and describe upcoming therapeutic opportunities.

Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy linked to a deficiency in the metalloprotease ADAMTS13. In the current study, a novel mouse model for acquired TTP was generated to facilitate development and validation of new therapies for this disease. Therefore, a large panel (n = 19) of novel anti-mouse ADAMTS13 (mADAMTS13) monoclonal antibodies (mAbs) of mouse origin was generated. Inhibitory anti-mADAMTS13 mAbs were identified using the FRETS-VWF73 assay. Four mAbs strongly inhibited mADAMTS13 activity in vitro (∼68–90% inhibition). Injecting a combination of 2 inhibitory mAbs (13B4 and 14H7, 1.25 mg/kg each) in Adamts13+/+ mice resulted in full inhibition of plasma ADAMTS13 activity (96 ± 4% inhibition, day 1 post injection), leading to the appearance of ultra-large von Willebrand factor (UL-VWF) multimers. Interestingly, the inhibitory anti-mADAMTS13 mAbs 13B4 and 14H7 were ideally suited to induce long-term ADAMTS13 deficiency in Adamts13+/+ mice. A single bolus injection resulted in full ex vivo inhibition for more than 7 days. As expected, the mice with the acquired ADAMTS13 deficiency did not spontaneously develop TTP, despite the accumulation of UL-VWF multimers. In line with the Adamts13-/- mice, TTP-like symptoms could only be induced when an additional trigger (rVWF) was administered. On the other hand, the availability of our panel of anti-mADAMTS13 mAbs allowed us to further develop a sensitive ELISA to detect ADAMTS13 in mouse plasma. In conclusion, a novel acquired TTP mouse model was generated through the development of inhibitory anti-mADAMTS13 mAbs. Consequently, this model provides new opportunities for the development and validation of novel treatments for patients with TTP. In addition, these newly developed inhibitory anti-mADAMTS13 mAbs are of great value to specifically study the role of ADAMTS13 in mouse models of thrombo-inflammatory disease.

ADAMTS13 deficiency in mice does not affect adipose tissue development

BACKGROUND BMI and ADAMTS13 levels are positively correlated in man. Development of obesity is associated with angiogenesis and inflammation, and increased ADAMTS13 synthesis in the liver. METHODS Male wild-type (WT) and ADAMTS13 deficient (Adamts13-/-) mice were kept on normal chow (SFD) or high fat diet (HFD) for 15 weeks. RESULTS HFD feeding of WT mice resulted in significantly enhanced levels of ADAMTS13 antigen and activity as compared to SFD feeding. ADAMTS13 deficiency had no significant effect on body weight gain, subcutaneous (SC) or gonadal (GN) adipose tissue mass, or on adipocyte size. In GN fat of obese (HFD) Adamts13-/- mice, adipocyte density was higher and blood vessel density lower as compared to obese WT mice. No marked effects of genotype were observed on mRNA expression of adipogenic, endothelial, inflammatory or oxidative stress markers in adipose tissue. Analysis of metabolic parameters and of glucose and insulin tolerance did not reveal significant differences between both obese genotypes, except for higher adiponectin and cholesterol levels in obese Adamts13-/- as compared to WT mice. CONCLUSION Our data do not support a functional role of ADAMTS13 in adiposity nor in associated angiogenesis or inflammation in mice. GENERAL SIGNIFICANCE ADAMTS13 deficiency may cause thrombotic thrombocytopenic purpura (TTP). Obesity, which is associated with enhanced ADAMTS13 levels is nevertheless considered to be an independent risk factor for TTP. To resolve this apparent contradiction, we show that ADAMTS13 does not directly promote development of adipose tissue in a mouse model.

Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73-1, ELH2-1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73-1 and TR8C11 bind to the cysteine-spacer domains, while the antibody ELH2-1 recognizes the T2-T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.

Anti-ADAMTS13 Antibodies and a Novel Heterozygous p.R1177Q Mutation in a Case of Pregnancy-Onset Immune-Mediated Thrombotic Thrombocytopenic Purpura

In this study, we investigated a case of pregnancy-onset thrombotic thrombocytopenic purpura (TTP). The patient had severely decreased ADAMTS13 ( a d isintegrin a nd m etalloprotease with t hrombo s pondin type 1 motif, member 13) activity levels during acute phase and the presence of inhibitory anti-ADAMTS13 autoantibodies was demonstrated, which led to the diagnosis of immune-mediated TTP. However, ADAMTS13 activity was only mildly restored during remission, although inhibitory anti-ADAMTS13 antibodies were no longer detected. We hypothesized that genetic abnormalities could account for this discrepancy between ADAMTS13 activity and antigen. Genetic analysis revealed the presence of two heterozygous substitutions on the same allele: a single nucleotide polymorphism (SNP) c.2699C > T (p.A900V), located in the beginning of the T5 domain, and a mutation c.3530G > A (p.R1177Q) located in the third linker region of ADAMTS13. In vitro testing of those substitutions by expression of recombinant proteins revealed a normal secretion but a reduced ADAMTS13 activity by the novel p.R1177Q mutation, which could partially explain the subnormal activity levels found during remission. Although changes in the linker region might induce conformational changes in ADAMTS13, the p.R1177Q mutation in the third linker region of ADAMTS13 did not expose a cryptic epitope in the metalloprotease domain. In conclusion, we report on an immune-mediated pregnancy-onset TTP patient who had inhibitory anti-ADAMTS13 autoantibodies during acute phase, but not during remission. Genetic analysis confirmed the diagnosis of immune-mediated TTP and revealed the novel p.R1177Q mutation which mildly impaired ADAMTS13 activity.

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Essentials Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP). Obese ADAMTS‐13‐deficient mice were triggered with von Willebrand factor (VWF). Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model. VWF enhances phagocytosis of platelets by macrophages, dose‐dependently.

Child-onset thrombotic thrombocytopenic purpura caused by p.R498C and p.G259PfsX133 mutations in ADAMTS13

Patients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene.