Silvia Pontiggia

Measurement and prevalence of circulating ADAMTS13-specific immune complexes in autoimmune thrombotic thrombocytopenic purpura

The formation of ADAMTS13‐specific circulating immune complexes (CICs) may be a pathophysiologic mechanism in autoimmune thrombotic thrombocytopenic purpura (TTP), but has not been systematically investigated.

ADAMTS13 content in plasma-derived factor VIII/von Willebrand factor concentrates

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy syndrome caused by a congenital or acquired deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF) and thus prevents the formation of platelet‐rich thrombi in the microcirculation. TTP can be fatal if not appropriately and timely treated with the infusion of fresh frozen plasma (FFP) or exchange plasmapheresis, that reverse the process of microangiopathy by removing anti‐ADAMTS13 autoantibodies and replacing functional ADAMTS13. The treatment of TTP with FFP is not free from risks and must be administered in hospitals or clinics, owing to the substantial amount of plasma volume infused or exchanged and the frequent need of catheter application. Moreover, most FFPs are not subjected to treatments to remove or inactivate blood‐borne infectious agents. A number of recent reports indicate that certain plasma‐derived VWF‐factor VIII (FVIII) concentrates are clinically effective in the treatment of congenital TTP. In this study, we measured ADAMTS13 levels in various plasma‐derived VWF‐FVIII concentrates, showing that Koate®‐DVI (Grifols), contained relatively high amounts of ADAMTS13 and that Alphanate® (Grifols) was the closest other product in terms of protease content. Koate®‐DVI contains, on average (five lots tested), 0.091 ± 0.007 Units of ADAMTS13 activity per IU of FVIII. On the basis of this analysis and other reports of VWF‐FVIII concentrate utilization in congenital TTP, potential dosing, and future clinical developments are discussed. Am. J. Hematol. 88:895–898, 2013.

FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

Pregnancy complications in acquired thrombotic thrombocytopenic purpura: a case-control study

BackgroundPregnant women with a history of acquired thrombotic thrombocytopenic purpura (TTP) are considered at risk for disease recurrence and might be at risk for miscarriage, similar to other autoimmune disorders. However, the exact entity of these risks and their causes are unknown. The aim of this study was to evaluate risk factors associated with adverse pregnancy outcome, in terms of both gravidic TTP and miscarriage, in women affected by previous acquired TTP.MethodsWe conducted a nested case–control study in women with a history of acquired TTP enrolled in the Milan TTP registry from 1994 to October 2012, with strict inclusion criteria to reduce referral and selection bias.ResultsFifteen out of 254 women with acquired TTP were included, namely four cases with gravidic TTP, five with miscarriage, and six controls with uncomplicated pregnancy. In the cases, ADAMTS13 activity levels in the first trimester were moderately-to-severely reduced (median levels <3% in gravidic TTP and median levels 20% [range 14-40%] in the women with miscarriage) and anti-ADAMTS13 antibodies were invariably present, while in the control group ADAMTS13 activity levels were normal (median 90%, range 40-129%), with absence of detectable anti-ADAMTS13 antibodies. Reduced levels of ADAMTS13 activity (<25%) in the first trimester were associated with an over 2.9-fold increased risk for gravidic TTP and with an over 1.2-fold increased risk for miscarriage (lower boundary of the confidence interval of the odds ratio). In addition, the presence of anti-ADAMTS13 antibodies during pregnancy was associated with an over 6.6-fold increased risk for gravidic TTP and with an over 4.1-fold increased risk for miscarriage.ConclusionsADAMTS13 activity evaluation and detection of anti-ADAMTS13 antibody could help to predict the risk of complications in pregnant women with a history of acquired TTP.

von Willebrand factor propeptide to antigen ratio in acquired thrombotic thrombocytopenic purpura

von Willebrand factor (VWF) is a large, adhesive, multimeric glycoprotein that plays amajor role in hemostasis bymediating platelet adhesion and aggregation at the sites of vessel wall injury and serving as the carrier of factor VIII [1]. The pre-proVWF, which is synthesized in endothelial cells and megakaryocytes, undergoes intracellular modifications, including signal peptide cleavage, C-terminal dimerization, glycosylation, sulfation, and N-terminal multimerization [2]. Proteolysis then occurs in the trans-Golgi, where the VWF propeptide (VWFpp) is cleaved, but remains stored together with mature VWF in a-granules (megakaryocytes) and Weibel–Palade bodies (endothelial cells). After the secretion of VWFpp and VWF into plasma from endothelial cells as a result of physiologic or pathologic stimuli, VWFpp dissociates from VWF [3,4]. It has been demonstrated that the molar ratio of the propeptide to mature VWF could serve as a tool with which to assess the extent of endothelial cell activation under physiologic and clinical conditions [3,5,6] and assess modified VWF clearance [4]. An increased VWFpp/VWF antigen (VWF:Ag) ratio, which is a marker for enhanced clearance of VWF, has been found in some von Willebrand disease and disseminated intravascular coagulation patients [7–9]. Thrombotic thrombocytopenic purpura (TTP) is a rare, lifethreatening disease, characterized by acute episodes of thrombocytopenia and microangiopathic hemolytic anemia [10]. It is believed that endothelial cell injury may be a triggering mechanism for the onset of thrombosis in TTP. In this regard, we herein evaluate the behavior of the VWFpp/VWF:Ag ratio in patients with acquired TTP at different stages of the disease. The study was conducted on 95 samples collected from 69 patients with acquired TTP (52 females and 17 males, median age 46 years, range 12–73 years), selected from a larger cohort of 136 patients from the Milan TTP registry (http://www. ttpdatabase.org) according to criteria described elsewhere [11]. Samples were also collected from 61 healthy controls (38 males and 23 females, median age 43 years, range 21–64 years). Patient samples were categorized as follows: 29 from patients with severe ADAMTS13 deficiency (< 10%) during their first episodeof acuteTTP; 19 frompatientswith severeADAMTS13 deficiency (< 10%) during their recurrent episode of acute TTP; 20 from patients with severe deficiency of ADAMTS13 (< 10%) during remission after an acute episode of TTP; and 27 from patients with normal plasma levels of ADAMTS13 (> 46%) during remission after an acute episode of TTP. Blood was collected in 3.2% buffered citrate solution (1 : 9, v/v) and EDTA in 5 mM disodium salt (1 : 9, v/v), centrifuged at 3000 · g for 20 min at 4 C, and aliquoted and stored at ) 80 C until assessment. All blood samples were obtained after patients had given informed consent. VWF levels (VWF:Ag) were measured with an immunoturbidometric assay (HemosIL von Willebrand factor antigen) on an ACL Top (IL Instrumentation Laboratory Company, Bedford, TX, USA) [12]. VWFpp was measured with an ELISA, using the MW1939 antibody pair anti-human VWFpp (Sanquin, Amsterdam, The Netherlands). ADAMTS13 activity was measured with the collagen-binding assay, as described previously [13]. VWF multimeric analysis was performed as described by Budde et al. [14], with some modifications. According to Budde et al. [14], the reference plasma lane was divided into small, intermediate and largermultimers of regular size, and the proportion of larger multimers in the sample was calculated by dividing the area corresponding to larger multimers by the total area of the lane. As described by Lotta et al., the ultralarge VWF (ULVWF) multimer ratio was calculated as the ratio of the proportion of large multimers in the sample to the corresponding proportion in the reference plasma within the same gel. A normal range was established by calculating the ULVWF ratio in 36 healthy subjects [15]. Laboratory results for the study groups are summarized in Table 1. There was an equal distribution ofO and non-O blood groups in acute TTP cases (36.6% vs. 36.4%), remission cases Correspondence: Flora Peyvandi, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Via Pace 9, Milan 20122, Italy. Tel.: +39 255035414; fax: +39 254100125. E-mail: flora.peyvandi@unimi.it

ADAMTS13-specific circulating immune complexes as potential predictors of relapse in patients with acquired thrombotic thrombocytopenic purpura.

BACKGROUND Acquired thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy due to the development of autoantibodies against the VWF-cleaving protease ADAMTS13. ADAMTS13-specific circulating immune complexes (CICs) have been described in patients with acquired TTP, but their clinical relevance remained to be established. The aim of this study was to assess the association between ADAMTS13-specific CICs and ADAMTS13-related measurements, clinical and laboratory markers of disease severity, and occurrence of TTP relapse, in autoimmune TTP patients. MATERIAL AND METHODS We measured ADAMTS13-specific CICs in 51 patients with severe ADAMTS13 deficiency and anti-ADAMTS13 autoantibodies, at the first episode of acquired TTP. The associations between ADAMTS13-specific CICs and the variables of interest were assessed by linear, logistic and Cox proportional hazard regression models, where appropriate. RESULTS The prevalence of ADAMTS13-specific CICs in patients experiencing the first TTP episode was 39% (95% confidence intervals [CI]: 26-52%). ADAMTS13-specific CICs were not associated neither with laboratory markers of disease severity, nor with patterns of clinical presentation. Conversely, among 45 survivors, a positive association was found between the presence of ADAMTS13-specific CICs and the risk of recurrence within 2years after the first TTP episode (adjusted hazard ratio, 3.4 [95% CI: 0.9 to 13.5]). CONCLUSIONS ADAMTS13-specific CICs seem to be able to predict the recurrence of acute TTP episodes in the first 2years after disease onset. Therefore, their measurement might be used as a tool to stratify the risk of disease relapse, with potential influence on surveillance and therapeutic choices during remission phase.

Thrombotic microangiopathy without renal involvement: two novel mutations in complement-regulator genes.

Essentials The differential diagnosis among thrombotic microangiopathies (TMAs) is challenging. We studied a case of TMA with neurologic symptoms, no renal impairment and normal ADAMTS‐13 levels. Two novel mutations in complement factor I and thrombomodulin genes were identified. Complement‐regulator genes can be involved in TMAs with normal ADAMTS‐13 regardless of renal damage.

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.

Next-generation sequencing and in vitro expression study of ADAMTS13 single nucleotide variants in deep vein thrombosis

Background Deep vein thrombosis (DVT) genetic predisposition is partially known. Objectives This study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies. Methods Wild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays. Results In vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile [15%; 95% confidence interval [CI] 14–16], p.Asp187His [19%; 95%[CI] 17–21], p.Arg421Cys [24%; 95%[CI] 22–26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio [OR] 2, 95%[CI] 0.18–22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His ([OR] 1.14, 95%[CI] 0.07–18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys ([OR] 0.76, 95%[CI] 0.21–2.68). Conclusions Three SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT.

A novel CD46 mutation in a patient with microangiopathy clinically resembling thrombotic thrombocytopenic purpura and normal ADAMTS13 activity

The differential diagnosis and etiological classification of the two main forms of thrombotic microangiopathy (TMA) [thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS)] remain challenging.1 The terms TTP and HUS are used to describe the clinical presentation of these diseases. HUS has prominent renal involvement, whereas neurological manifestations are common in TTP. The distinction is not always reliable; neurological complications can be present in patients with aHUS and renal failure not requiring dialysis can be present in patients affected by TTP.