Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura

BACKGROUND: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. METHODS: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. RESULTS: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. CONCLUSION: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.     

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.     

The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission

Summary.  Background:  Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. Objective:  To study ADAMTS13 and VWF-related features in TTP patients in remission. Methods:  ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibα-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. Results:  ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. Conclusion : ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.     

Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity

BACKGROUND: ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. ADAMTS13 activity is deficient in patients with thrombotic thrombocytopenic purpura (TTP). The determination of plasma levels of ADAMTS13 activity is a prerequisite for a differential diagnosis of thrombotic microangiopathies. Here, a unique and highly sensitive enzyme immunoassay (EIA) of ADAMTS13 activity is described. STUDY DESIGN AND METHODS: ADAMTS13 hydrolyzes the peptide bond between Y1605 and M1606 of VWF. In this assay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. A panel of mouse monoclonal antibodies (MoAbs) that specifically recognizes Y1605, which is the C-terminal edge residue of the VWF-A2 domain and is generated by the enzymatic cleavage, has been produced. These antibodies were prepared with a synthetic decapeptide, termed N-10 (1596-DREQAPNLVY-1605), as the immunogen. Twenty-six clones specific to N10 were obtained, and one anti-N10 MoAb was used in this study. RESULTS: With horseradish peroxidase-conjugated anti-N10 MoAb, a standard enzyme assay was established. This assay was highly sensitive, and the detection limit was 0.5 percent of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda units per mL. ADAMTS13 activity was measured in 20 patients with Upshaw-Schulman syndrome, a congenital TTP, and 61 acquired TTP patients. The activity measured by this assay and by the classic VWF multimer assay showed high correlation. CONCLUSION: A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine.     

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.     

影响获得性血栓性血小板减少性紫癜首次缓解期内复发的相关指标研究

目的 探讨血管性血友病因子裂解蛋白酶(ADAMTS) 13活性和抗ADAMTS13抗体表达水平,与获得性血栓性血小板减少性紫癜(TTP)于首次缓解期内复发的关系.方法 选择2008年3月至2014年6月于陕西延安大学附属医院和陕西省渭南市富平县医院诊治的37例获得性TTP患者为研究对象,按照其在首次缓解随访期内是否复发,分为研究组(n=15)和对照组(n=22).分别采用残余胶原结合试验、ELISA、免疫印迹等方法,检测两组患者的ADAMTS13活性,ADAMTS13抗原水平,抗ADAMTS13抗体,ADAMTS13抑制物,血管性血友病因子(vWF)抗原和超大分子量vWF(ULVWF)多聚体等指标,并且进行统计学分析;采用多因素非条件logistic回归分析法,评估获得性TTP患者于首次缓解期内复发的独立影响因素.本研究遵循的程序符合病例收集医院人体试验委员会所制定的伦理学标准,得到该伦理会批准,分组征得受试对象本人的知情同意,并与之签订临床研究知情同意书.结果 ①研究组患者的中位ADAMTS13活性为11％(7％～124％),低于对照组的53％(7％～151％),差异有统计学意义(u=4.018,P＜0.05).研究组与对照组患者的ADAMTS13活性显著降低率分别为53.3％(8/15)和22.7％(5/22),二者比较,差异有统计学意义(P=0.049).②37例获得性TTP患者的血浆ADAMTS13活性和ADAMTS13抗原水平呈正相关关系(rs=0.810,P=0.001).研究组患者的中位ADAMTS13抗原水平为33％(3％～99％),低于对照组的59％(3％～128％),差异有统计学意义(u=4.121,P＜0.05).研究组与对照组ADAMTS13抗原水平显著降低率分别为13.3％(2/15)和9.1％(2/22),二者比较,差异有统计学意义(P=0.008).③研究组患者的抗ADAMTS13抗体检出率为66.7％(10/15),高于对照组的36.4％(8/22),差异有统计学意义(P=0.007).④研究组患者中抗ADAMTS13抑制物检出率为46.7％ (7/15),高于对照组患者的18.2％ (4/22),差异有统计学意义(P=0.011).⑤研究组与对照组患者ULVWF多聚体检出率分别为20.0％(3/15)和13.6％(3/22),二者比较,差异有统计学意义(P=0.042).⑥多因素非条件logistic回归分析结果显示,获得性TTP患者于首次缓解期内复发的独立危险因素包括ADAMTS13活性显著降低(OR=2.95,95％ CI:1.13～6.96,P＜0.05),检出抗ADAMTS13抗体(OR=3.31,95％CI:1.08～8.19,P＜0.05),检出抗ADAMTS13抑制物(OR=3.24,95 ％CI:1.24～9.03,P＜0.05).结论 获得性TTP患者在首次缓解期内,ADAMTS13活性水平显著降低,存在抗ADAMTS13抗体及抗ADAMTS13抑制物,会显著增加疾病复发风险,可以考虑将其作为预测获得性TTP患者于首次缓解期内复发的重要指标.     

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.     

Von Willebrand factor and ADAMTS13 balancing primary haemostasis

Von Willebrand factor (VWF) is an adhesive, multi-functional huge multimerized protein with multiple domains harboring binding sites for collagen, platelet glycoprotein receptors and coagulation factor VIII (FVIII). The functional domains enable VWF to bind to the injured vessel wall, to recruit platelets to the site of injury by adhesion and aggregation and to bind and protect FVIII, an important cofactor of the coagulation cascade. VWF function in primary haemostasis is located in particular in the arterial and micro-circulation. This environment is exposed to high shear forces with hydrodynamic shear rates ranging over several orders of magnitude from 10?1 to 10? s-1 and requires particular mechanisms to enable platelet adhesion and aggregation under these variable conditions. The respective VWF function is strictly correlating with its multimer size. Lack or reduction of large VWF multimers is seen in patients with von Willebrand disease (VWD) type 2A which correlates with reduction of both VWF:platelet GPIb-binding and VWF:collagen binding and a bleeding phenotype. To prevent unlimited platelet adhesion and aggregation which is the cause of the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), VWF function is regulated by its specific protease ADAMTS13. Whereas a particular susceptibility of VWF to ADAMTS13 proteolysis is the cause of a frequent VWD type 2A phenotype, lack or dysfunction of ADAMTS13, either acquired by ADAMTS13 antibodies or by inherited ADAMTS13 deficiency (Upshaw-Schulman Syndrome), causes TTP. Therefore VWD and TTP represent the opposite manifestations of VWF related disorders, tightly linked to each other.     

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.     

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

BACKGROUND: Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either predenatured von Willebrand factor (VWF) or small peptides derived from the VWF‐A2 domain. The physiologic relevance of the assay results is uncertain. STUDY DESIGN AND METHODS: We sought to develop a novel shear‐based assay to assess plasma ADAMTS13 activity and inhibitors. We also compared this assay with a fluorogenic peptide assay. RESULTS: We found that an incubation of purified plasma VWF with 0.5 to 1.0 µL of citrated plasma under constant vortexing at 2500 rpm for 60 minutes in the presence of 5 mmol/L CaCl₂ and 1.7 µmol/L ZnCl₂ and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear‐based assay appeared to be more sensitive than the guanidine‐denaturization assay for determining plasma ADAMTS13 activity. CONCLUSIONS: Our fluid shear‐based assay may be useful for investigating basic biologic function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.     

ADAMTS13 Deficiency and Thrombotic Thrombocytopenic Purpura Associated with Trimethoprim-Sulfamethoxazole

Thrombotic thrombocytopenic purpura (TTP) is a hematological disease characterized by microangiopathic hemolytic anemia and thrombocytopenia. Although the link between ADAMTS13 deficiency and idiopathic TTP has been well-established, the role of trimethoprim-sulfamethoxazole (TMP-SMX) in the pathogenesis of TTP is not yet well elucidated. To the best of our knowledge, there have been only two previous reports linking this medication with the development of TTP. We present the case of a healthy woman, age 26 years, who developed TTP during TMP-SMX therapy for urinary tract infection. She was found to have ADAMTS13 deficiency with anti-ADAMTS13 antibodies. Her condition responded to discontinuation of the TMP-SMX, plasmapheresis, and rituximab therapy. We speculate that the acquired ADAMTS13 deficiency might have been triggered by the TMP-SMX therapy.     

ADAMTS13 activity and the presence of acquired inhibitors in human immunodeficiency virus-related thrombotic thrombocytopenic purpura

BACKGROUND: Little is known about the pathophysiology of human immunodeficiency virus (HIV)-related thrombotic thrombocytopenic purpura (TTP). It is generally assumed that acquired ADAMTS13 deficiency is due to the presence of autoantibody inhibitors, but limited data are available regarding ADAMTS13 activity and inhibitors in such patients. STUDY DESIGN AND METHODS: By use of a collagen-binding assay, ADAMTS13 activity was analyzed at presentation in 20 patients with HIV-related TTP. The presence of inhibitors in patients with reduced ADAMTS13 activity was assessed with mixing studies. The correlation between ADAMTS13 activity and inhibitors and other laboratory and clinical parameters was assessed. RESULTS: The patients fell clearly into two groups with regard to ADAMTS13 activity. Six patients (30%) had activity within the normal range, whereas the remaining 14 patients had severely reduced levels. Of the patients with reduced activity, only 5 patients had a detectable inhibitor whereas 8 showed no evidence of an inhibitor. There was significant correlation between normal ADAMTS13 activity and lower CD4 counts (p = 0.049). von Willebrand factor (VWF) antigen levels were significantly higher in patients with reduced ADAMTS13 activity (p = 0.03). Low activity in the absence of a detectable inhibitor was associated with significantly higher D-dimer levels (p = 0.01) and worse clinical outcome. CONCLUSION: The heterogeneity with regard to ADAMTS13 activity and the absence of inhibitors in the majority of patients indicate that other factors are important in the pathogenesis of HIV-related TTP. VWF release and localized coagulation activation due to direct viral or cytokine-mediated endothelial cell injury is likely to be playing a major role.     

ADAMTS13: origins,applications, and prospects

ADAMTS13 is an enzyme that acts by cleaving prothrombotic von Willebrand factor (VWF) multimers from the vasculature in a highly regulated manner. In pathologic states such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs), VWF can bind to the endothelium and form large multimers. As the anchored VWF chains grow, they provide a greater surface area to bind circulating platelets (PLTs), generating unique thrombi that characterize TTP. This results in microvasculature thrombosis, obstruction of blood flow, and ultimately end‐organ damage. Initial presentations of TTP usually occur in an acute manner, typically developing due to an autoimmune response toward, or less commonly a congenital deficiency of, ADAMTS13. Triggers for TMAs that can be associated with ADAMTS13 deficiency, including TTP, have been linked to events that place a burden on hemostatic regulation, such as major trauma and pregnancy. The treatment plan for cases of suspected TTP consists of emergent therapeutic plasma exchange that is continued on a daily basis until normalization of PLT counts. However, a subset of these patients does not respond favorably to standard therapies. These patients necessitate a better understanding of their diseases for the advancement of future therapeutic options. Given ADAMTS13’s key role in the cleavage of VWF and the prevention of PLT‐rich thrombi within the microvasculature, future treatments may include anti‐VWF therapeutics, recombinant ADAMTS13 infusions, and ADAMTS13 expression via gene therapy.     

Rituximab maintenance for relapsed refractory thrombotic thrombocytopenic purpura

BACKGROUND: Rituximab, an anti‐CD20 chimeric monoclonal antibody, has been used successfully to treat patients with relapsed or refractory thrombotic thrombocytopenic purpura (TTP); however, the optimal dose and frequency and the role of rituximab maintenance remain uncertain. STUDY DESIGN AND METHODS: We describe a 45‐year‐old woman with chronic relapsing immune thrombocytopenia who responded to rituximab retreatment administered in four doses over the course of 12 months. Previously, she had received four doses of rituximab and sustained a remission for 19 months. During her latest TTP relapse, multiple treatments were administered including rituximab retreatment. After the first dose (375 mg/m²), she developed serum sickness requiring further doses to be deferred. Three subsequent doses were administered at 4‐month intervals over the course of 12 months. ADAMTS13 activity was measured by von Willebrand factor (VWF) digestion. ADAMTS13 inhibition was measured by a modification of the VWF digestion assay and anti‐ADAMTS13 antibodies were measured by enzyme‐linked immunoassay (enzyme‐linked immunosorbent assay, American Diagnostica). RESULTS: Clinical and laboratory remission were achieved after one dose of rituximab, with normalization of ADAMTS13 activity and disappearance of ADAMTS13 inhibitor. Three subsequent doses of rituximab were given without incident and the patient remained in remission after 3.5 years of follow‐up (2.5 years since her last dose of rituximab). CONCLUSION: Maintenance dosing of rituximab should be considered in some patients with relapsing TTP.     

ADAMTS-13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences

BACKGROUND: The recently discovered plasma enzyme ADAMTS-13 cleaves the A2-domain of von Willebrand factor (VWF). A defective cleaving protease results in unusually large VWF multimers, which cause thrombotic thrombocytopenic purpura (TTP). AIM: Analysis of the ADAMTS-13 antigen levels in TTP patients compared with normal donors. METHODS: An antigen ELISA test was built, based on high affinity anti-ADAMTS-13 monoclonal antibodies, which were generated using genetic immunization. RESULTS: Specificity of the ADAMTS-13 antigen test was confirmed, as (i) plasma from a patient with acquired TTP but presenting without inhibitor did not contain antigen and (ii) the binding of recombinant ADAMTS-13 was inhibited by increasing amounts of normal plasma. The assay is sensitive as it can detect antigen levels as low as 1.6% of normal. The concentration in normal pooled human plasma was determined (1.03 +/- 0.15 microg mL(-1)) and arbitrarily set to 1 U mL(-1). The antigen levels in congenital TTP samples (34 +/- 21 mU mL(-1), n = 2), as well as in samples from patients with acquired TTP (231 +/- 287 mU mL(-1), n = 11), were clearly reduced when compared with normal Caucasian donors (951 +/- 206 mU mL(-1), n = 16). Remarkably, normal Chinese donors have a significantly lower antigen titer (601 +/- 129 mU mL(-1), n = 15), when compared with normal Caucasians. CONCLUSIONS: Our results show that acquired TTP patients suffer mainly from ADAMTS-13 antigen depletion, thereby indicating the importance of ADAMTS-13 antigen determination in diagnosis and patient follow-up.     

The role of ADAMTS13 in the pathogenesis of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome

The identification, characterization, and clinical observation of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin-1-like domains) have provided important insights into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a plasma enzyme essential for postsecretion proteolytic processing of von Willebrand factor (VWF). Absence of ADAMTS13 is associated with the occurrence of abnormally large multimers of VWF and is also associated with the occurrence of TTP. Initial assumptions that absent ADAMTS13 was itself the etiology of TTP have been tempered by subsequent observations that ADAMTS13 activity can be severely deficient without clinical abnormalities and that patients can have characteristic clinical features of TTP without severe ADAMTS13 deficiency. A current interpretation of these observations is that ADAMTS13 deficiency is a major risk factor for the development of TTP, but it is neither always necessary nor sufficient to cause TTP. This interpretation is consistent with other vascular and thrombotic disorders in which multiple risk factors and associated conditions contribute to the etiology of acute events.     

Diagnosis of thrombotic thrombocytopenic purpura

As hallmark of TTP, generalized hyaline thrombi in the patient's microcirculation is known. These thrombi are composed of platelets and VWF. A severe defect of the VWF cleaving protease (VWF-CP) was found in all known patients with the inherited form of TTP. In contrary, although a severe deficiency of VWF-CP is specific for the acquired form, too, only a fraction of these patients is characterized by a severe deficiency. In most patients with a severe deficiency autoantibodies directed against VWF-CP is detectable in plasma. However, many patients with acquired TTP do not show any severe deficiency. Because treatment differs in inherited and acquired forms and as persistance of autoantibodies during clinical remission is of prognostic value, the determination of the activity of VWF-CP and of antibodies against VWF-CP are important parts in the workup of patients with TTP. In all methods for the determination of the activity of VWF-CP the first step is proteolysis of a specific substrate for the protease. In the second step the activity of the protease is measured by analysis of the residual VWF multimers, by the generation of specific fragments, by using the residual VWF:CB or VWF:RCo as marker of the loss of multimers or with help of specific monoclonal antibodies. In less than 30 min the cone and plate(let) aggregometer helps to distinguish between different forms of thrombotic microangiopathies. While adhesion and aggregation of platelets from a healthy person are clearly enhanced after addition of a small amount of plasma from a TTP patient, both characteristics are weakened by plasma from patients with other forms of thrombotic microangiopathy (dilution effect). Molecular genetics are established methods in the differentiation between inherited and acquired forms of TTP in those cases without autoantibodies against VWF-CP.     

Thrombotic thrombocytopenic purpura in childhood

Thrombotic thrombocytopenic purpura (TTP) is a micro-angiopathic disease due to deficiency of the specific VWF cleaving protease (VWF-CP) ADAMTS13. The acquired form is caused by autoantibodies against VWF-CP, whereas mutations of the ADAMTS13 gene are responsible for inherited TTP. In childhood both forms exist with predominance of inherited TTP. The phenotype of TTP in childhood can be rather variable. Besides the classical clinical picture, abortive forms may occur that can delay the identification of patients at risk. The patients are frequently assumed to suffer from idiopathic thrombocytopenia (ITP) or Evans syndrome. Further efforts are necessary to accelerate correct diagnosis and to establish a risk-adapted prophylactic therapy.     

Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura

Summary. Background: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross‐link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti‐ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. Objectives: To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. Methods: Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. Results: The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute‐phase but not remission‐phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute‐phase but not remission‐phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose‐dependently inhibited by addition of exogenous α₂‐antiplasmin. Examination of the endogenous α₂‐antiplasmin antigen and activity confirmed deficiency of α₂‐antiplasmin function in acute‐phase but not remission‐phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase‐type plasminogen activator was added to an (unrelated) congenital α₂‐antiplasmin‐deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. Conclusion: We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.     

Multi‐step binding of ADAMTS‐13 to von Willebrand factor

Summary. Background: ADAMTS‐13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non‐catalytic domains in ADAMTS‐13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS‐13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS‐13. Methods: Immunoprecipiation of VWF–ADAMTS‐13 complexes using anti‐VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co‐immunoprecipitation demonstrated that ADAMTS‐13 binds to native multimeric VWF (K_d of 79 ± 11 nmol L^?1) with no measurable proteolysis. Upon shear‐induced unfolding of VWF, binding increased 3‐fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS‐13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS‐13 spacer, probably thrombospondin‐1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS‐13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS‐13 binds to circulating VWF and may therefore be incorporated into a platelet‐rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.