J. A. Kremer Hovinga

Thrombotic thrombocytopenic purpura

Summary.  This overview summarizes the history of thrombotic thrombocytopenic purpura (TTP) from its initial recognition in 1924 as a most often fatal disease to the discovery in 1997 of ADAMTS‐13 deficiency as a major risk factor for acute disease manifestation. The cloning of the metalloprotease, ADAMTS‐13, an essential regulator of the extremely adhesive unusually large von Willebrand factor (VWF) multimers secreted by endothelial cells, as well as ADAMTS‐13 structure and function are reviewed. The complex, initially devised assays for ADAMTS‐13 activity and the possible limitations of static in vitro assays are described. A new, simple assay using a recombinant 73‐amino acid VWF peptide as substrate will hopefully be useful. Hereditary TTP caused by homozygous or double heterozygous ADAMTS‐13 mutations and the nature of the mutations so far identified are discussed. Recognition of this condition by clinicians is of utmost importance, because it can be easily treated and – if untreated – frequently results in death. Acquired TTP is often but not always associated with severe, autoantibody‐mediated ADAMTS‐13 deficiency. The pathogenesis of cases without severe deficiency of the VWF‐cleaving protease remains unknown, affected patients cannot be distinguished clinically from those with severely decreased ADAMTS‐13 activity. Survivors of acute TTP, especially those with autoantibody‐induced ADAMTS‐13 deficiency, are at a high risk for relapse, as are patients with hereditary TTP. Patients with thrombotic microangiopathies (TMA) associated with hematopoietic stem cell transplantation, neo‐plasia and several drugs, usually have normal or only moderately reduced ADAMTS‐13 activity, with the exception of ticlopidine‐induced TMA. Diarrhea‐positive‐hemolytic uremic syndrome (D+ HUS), mainly occurring in children is due to enterohemorrhagic Escherichia coli infection, and cases with atypical, D− HUS may be associated with factor H abnormalities. Treatment of acquired idiopathic TTP involves plasma exchange with fresh frozen plasma (FFP), and probably immunosuppression with corticosteroids is indicated. We believe that, at present, patients without severe acquired ADAMTS‐13 deficiency should be treated with plasma exchange as well, until better strategies become available. Constitutional TTP can be treated by simple FFP infusion that rapidly reverses acute disease and – given prophylactically every 2–3 weeks – prevents relapses. There remains a large research agenda to improve diagnosis of TMA, gain further insight into the pathophysiology of the various TMA and to improve and possibly tailor the management of affected patients.

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.

ADAMTS-13, von Willebrand factor and related parameters in severe sepsis and septic shock

Background: Insufficient control of von Willebrand factor (VWF) multimer size as a result of severely deficient ADAMTS‐13 activity results in thrombotic thrombocytopenic purpura associated with microvascluar thrombosis and platelet consumption, features not seldom seen in severe sepsis and septic shock. Methods: ADAMTS‐13 activity and VWF parameters of 40 patients with severe sepsis or septic shock were compared with those of 40 healthy controls of the same age and gender and correlated with clinical findings and sepsis outcome. Results: ADAMTS‐13 activity was significantly lower in patients than in healthy controls [median 60% (range 27–160%) vs. 110% (range 63–200%); P < 0.001]. VWF parameters behaved reciprocally and both VWF ristocetin cofactor activity (RCo) and VWF antigen (VWF:Ag) were significantly (P < 0.001) higher in patients compared with controls. Neither ADAMTS‐13 activity nor VWF parameters correlated with disease severity, organ dysfunction or outcome. However, a contribution of acute endothelial dysfunction to renal impairment in sepsis is suggested by the significantly higher VWF propeptide and soluble thrombomodulin levels in patients with increased creatinine values as well as by their strong positive correlations (creatinine and VWF propeptide rs = 0.484, P < 0.001; creatinine and soluble thrombomodulin rs = 0.596, P < 0.001). Conclusions: VWF parameters are reciprocally correlated with ADAMTS‐13 activity in severe sepsis and septic shock but have no prognostic value regarding outcome.

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.

Treatment of Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP), characterized by thrombocytopenia and microangiopathic haemolytic anaemia, was almost universally fatal until the introduction of plasma exchange (PE) therapy in the 1970s. Based on clinical studies, daily PE has become the first‐choice therapy since 1991. Recent findings may explain its effectiveness, which may include, in particular, the removal of anti‐ADAMTS13 autoantibodies and unusually large von Willebrand factor multimers and/or supply of ADAMTS13 in acquired idiopathic or congenital TTP. Based on currently available data, the favoured PE regimen is daily PE [involving replacement of 1–1·5 times the patients plasma volume with fresh‐frozen plasma (FFP)] until remission. Adverse events of treatment are mainly related to central venous catheters. The potential reduction of plasma related side‐effects, such as transfusion‐related acute lung injury (TRALI) or febrile transfusion reactions by use of solvent–detergent treated (S/D) plasma instead of FFP is not established by controlled clinical studies. Uncontrolled clinical observations and the hypothesis of an autoimmune process in a significant part of the patients with acquired idiopathic TTP suggest a beneficial effect of adjunctive therapy with corticosteroids. Other immunosuppressive treatments are not tested in controlled trials and should be reserved for refractory or relapsing disease. There is no convincing evidence for the use of antiplatelet agents. Supportive treatment with transfusion of red blood cells or platelets has to be evaluated on a clinical basis, but the transfusion trigger for platelets should be very restrictive. Further controlled, prospective studies should consider the different pathophysiological features of thrombotic microangiopathies, address the prognostic significance of ADAMTS13 and explore alternative exchange fluids to FFP, the role of immunosuppressive therapies and of new plasma saving approaches as recombinant ADAMTS13 and protein A immunoadsorption.

Measurement of ADAMTS-13 activity in plasma by the FRETS-VWF73 assay: comparison with other assay methods.

Thrombocytopenia and microangiopathic hemolytic anemia together with a severely deficient ADAMTS-13 activity (<5%) confirm the diagnosis of acute thrombotic thrombocytopenic purpura (TTP) [1], even though not all patients with a clinical diagnosis of TTP have a severe ADAMTS-13 deficiency [2]. Since the initial reports by Furlan et al. [3] and Tsai and Lian [4], numerous assays for the determination of ADAMTS-13 activity have been developed. The majority of these assays comprise two steps [5]. First, a von Willebrand factor (VWF) substrate is subjected to proteolysis by plasma ADAMTS-13. This is followed by a second step of either quantifying VWF digestion products [3,4,6] or determination of residual VWF function [7,8]. These assays are time-consuming, often difficult to perform and hence restricted to specialized laboratories. Recently, Kokame et al. [9] reported a new fluorescence resonance energy transfer assay using a truncated, synthetic 73amino-acid VWF peptide as a substrate for the determination of ADAMTS-13 activity (FRETS-VWF73 assay). While the performances of several of the older assays have been evaluated and compared in two multicenter studies [10,11], this new method has not yet been formally evaluated in this way. Therefore, we compared the results obtained with the FRETSVWF73 assay with those obtained with other ADAMTS-13 activity assays in a series of 30 plasma samples. The FRETS-VWF73 assay was performed essentially as described [9] with the following modifications. Pefabloc SC (Boehringer, Mannheim, Germany) was added to the assay buffer (5 mmol L Bis-Tris, 25 mmol L CaCl2, 0.005% Tween-20, pH 6.0) at a final concentration of 1 mmol L. Assay calibration was obtained by using a normal human plasma pool (NHP; Swiss Red Cross Blood Services, Bern, Switzerland) diluted 1:25 (100%) in assay buffer. Further calibration samples were obtained by serial predilutions of NHP of 3:4 (75%), 1:2 (50%), 1:4 (25%), 1:10 (10%), 1:20 (5%) in heat-inactivated fresh frozen plasma (FFP, incubated for 30 min at 56 C followed by 15 min of centrifugation at 15 000 · g) to correct for a plasma matrix effect in the lower activity range of the standard curve. All of these standard samples as well as heat-inactivated FFP (0% ADAMTS-13 activity) and all test samples were subsequently diluted 1:25 in assay buffer. Next, 100 lL of each diluted standard or patient sample was incubated at 37 C in a 96-well white plate (NUNC, Roskilde, Denmark). After 10 min, 100 lmol L of 4 lmol L FRETS-VWF73 peptide substrate dissolved in assay buffer was added to each well and evolution of fluorescence recorded at 37 C in a TECAN Spectrafluor microplate reader (Tecan, Zürich, Switzerland) equipped with a 340 nm excitation filter (band width 35 nm) and a 450 nm emission filter (band width 25 nm). Fluorescence was measured every 5 min and the reaction rate was calculated by linear regression analysis of fluorescence over time from 5 to 60 min. ADAMTS-13 activity was determined in the same 30 plasma samples of patients with acquired or hereditary TTP and other conditions that had been previously analyzed forADAMTS-13 activity in the context of the first multicenter study by quantitative immunoblotting assay, residual collagen binding activity assay using two different VWF substrates, residual ristocetin cofactor activity assay, and immune radiometric assay [10]. For the FRETS-VWF73 assay, aliquots of the same samples, which had been stored at )70 C, were analyzed three times with different FRETS-VWF73 peptide substrate lots (550 116, 550 703 and 550 827). Patient samples with an ADAMTS-13 activity<20%were screened for the presence of inhibitory autoantibodies for which the patients’ plasma was heat-inactivated as described above, mixed 1:1 (v:v) with NHP and incubated for 2 h at 37 C. This mixture was then diluted 1:25 in assay buffer. The key to the plasma samples and former results remained confidential until completion of all analyses. FRETS-VWF73 inter-assay reproducibility expressed by the mean coefficient of variation, calculated as the ratio between the standard deviation and the mean of the triplicate measurements of all samples multiplied by 100, was 6% and thus excellent. Predilution of calibration curve samples in heatinactivated FFP was superior to dilution of NHP in assay buffer alone as judged from the standard curve correlation coefficient as well as reproducibility, especially in the lower ADAMTS-13 activity range (0–25%). This lower range is not formally covered by the original method described by Kokame et al., as their standard curve point next to 0% corresponds to an ADAMTS-13 activity of 25% [9]. Correspondence: J. A. Kremer Hovinga, Department of Hematology and Central Hematology Laboratory, Inselspital, University Hospital, CH-3010 Bern, Switzerland. Tel.: + 41 31 632 90 22; fax: + 41 31 632 34 06; e-mail: johanna.kremer@insel.ch

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

Summary.  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.

Hyperbilirubinemia interferes with ADAMTS-13 activity measurement by FRETS-VWF73 assay: diagnostic relevance in patients suffering from acute thrombotic microangiopathies

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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.

Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS-13).

Summary.  Background: Over the last 4 years ADAMTS‐13 measurement underwent dramatic progress with newer and simpler methods. Aims: Blind evaluation of newer methods for their performance characteristics. Design: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS‐13‐deficient plasma (arbitrarily set at 0%) into one normal‐pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested ‘blind’ 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. Results: There were eight functional and three antigen assays. Linearity of observed‐vs.‐expected ADAMTS‐13 levels assessed as r2 ranged from 0.931 to 0.998. Between‐run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10–15% for five methods and up to 20% for the remaining three. F‐values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS‐13 levels (the higher the F‐value, the better the capacity) ranged from 3965 to 137. Between‐method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS‐13 offer the best performance characteristics. Conclusions: New assays for ADAMTS‐13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.