Background From 20 to 50% of patients who survive an acute episode of the acquired form of thrombotic thrombocytopenic purpura relapse but clinical and laboratory markers of recurrence are not well established. Design and MethodsIn 109 patients enrolled in an international registry we evaluated, in the frame of a retrospective cohort study, the predictive role of the metalloprotease ADAMTS13 as measured in plasma during remission. Anti-ADAMTS13 antibodies and von Willebrand factor were also evaluated in a smaller number of the same patients. ResultsMedian values of ADAMTS13 activity and antigen were significantly lower in patients with recurrent thrombotic thrombocytopenic purpura than in those with no recurrence (activity: 12% vs. 41%; p=0.007; antigen: 36% vs. 58%; p=0.003). A severe deficiency of ADAMTS13 activity (10% or less) was associated with a higher likelihood of recurrence (odds ratio 2.9; 95% confidence interval 1.3 to 6.8; p=0.01). Anti-ADAMTS13 antibodies were also more prevalent in patients with recurrent thrombotic thrombocytopenic purpura (odds ratio 3.1; 95% confidence interval 1.4 to 7.3; p=0.006). The presence during remission of both severe ADAMTS13 deficiency and anti-ADAMTS13 antibodies increased the likelihood of recurrence 3.6 times (95% confidence interval 1.4 to 9.0; p=0.006). The presence of ultralarge von Willebrand factor multimers and of associated diseases or conditions did not increase recurrence. ConclusionsSurvivors of an acute episode of acquired thrombotic thrombocytopenic purpura with severely reduced levels of ADAMTS13 and/or with anti-ADAMTS13 antibodies during remission have an approximately three-fold greater likelihood of developing another episode of thrombotic thrombocytopenic purpura than patients with higher protease activity and no antibody.Key words: thrombotic thrombocytopenic purpura, ADAMTS13, von Willebrand factor, risk factors, recurrence.Citation: Peyvandi F, Lavoretano S, Palla R, Feys H B., Vanhoorelbeke K, Battaglioli T, Valsecchi C, Canciani MT, Fabris F, Zver S, Réti M, Mikovic D, Karimi M, Giuffrida G, Laurenti L, antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission. Haematologica 2008 Feb; 93(2):232-239. DOI: 10.3324/haematol.11739 ©2008 Ferrata Storti Foundation. This is an open-access paper. ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission IntroductionAcquired thrombotic thrombocytopenic purpura (TTP) is a rare but severe disease characterized by microangiopathic hemolytic anemia and consumptive thrombocytopenia leading to disseminated microvascular thrombosis that causes variable signs and symptoms of organ ischemia and damage.1 The pathophysiology of TTP has become clearer after it was demonstrated that the plasma metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin 1 repeats) cleaves the platelet-adhesive plasma protein von Willebrand factor (VWF) at the peptide bond Tyr1605-Met1...

SummaryThe congenital or acquired deficiency of the von Willebrand factor (VWF) cleaving protease, ADAMTS-13 has been specifically associated with a diagnosis of thrombotic thrombocytopenic purpura (TTP), a microangiopathy characterized by the formation of occlusive platelet thrombi. The mechanisms of TTP were investigated in 100 patients diagnosed on the basis of the presence of at least three of the following: thrombocytopenia, haemolytic anaemia, elevated serum levels of lactate dehydrogenase and neurological symptoms. Plasma levels of ADAMTS-13 were severely reduced (<10% of normal) in 48%, moderately reduced (between 10% and 46%) in 24% and normal (>46%) in 28%. A neutralizing antibody was the cause of the deficiency in 38% of the cases, with a higher prevalence of this mechanism (87%) in the 48 patients with severely reduced ADAMTS-13. Double heterozygosity for a 29 base pair (bp) deletion and a nucleotide insertion and homozygosity for a 6 bp deletion in the ADAMTS13 gene were identified only in two patients born from consanguineous marriages. In conclusion, this study indicated that ADAMTS-13 was normal in nearly one-third of patients with TTP and that ADAMTS-13 deficiency was not associated with the presence of neutralizing antibodies in more than half of the patients.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr 1605 -Met 1606 peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 M urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37°C, with an apparent k cat /K m Х 3.4 ؋ 10 4 M ؊1 s ؊1 , but this value decreased by ϳ10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO 4 ؊ > Cl ؊ > F ؊ ). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIb␣ binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

Summary Thrombotic microangiopathies (TMAs) are rare but serious complications of bone marrow transplantation (BMT). Clinical manifestations are similar to those of thrombotic thrombocytopenic purpura (TTP), but prognosis is generally poorer despite plasma exchange. The enzymatic activity of the plasma metalloprotease ADAMTS13, which cleaves ultralarge thrombogenic multimers of von Willebrand factor (VWF) derived from activated endothelial cells, is very low or undetectable in patients with classic TTP, and protease deficiency is thought to play a mechanistic role in the formation of platelet thrombi in the microcirculation. This is the first prospective study to evaluate the incidence of TMA in 46 consecutively recruited patients undergoing autologous or allogeneic BMT and explore in parallel the behaviour of ADAMTS13, VWF antigen and VWF multimer size. The incidence of post‐BMT TMA was 6% (three of 46); all cases occurred after allogeneic BMT. Compared with baseline values plasma ADAMTS13 activity was significantly reduced in patients undergoing BMT, particularly after the conditioning regimen (mean values: 50 ± 22 vs. 77 ± 32%; P < 0·0001). In the three patients who developed TMA, ADAMTS13 decreased after conditioning, but was very low in one case only (8%). VWF antigen levels progressively increased after the conditioning regimen (228 ± 75 vs. 178 ± 76% at baseline, P = 0·002). The mean proportion of high‐molecular weight VWF multimers did not change in the various stages of BMT, even though ultralarge multimers were transiently found in same cases with and without TMA. Hence, the measurements evaluated in this study are not clinically useful to predict the occurrence of post‐BMT TMA.

The inherited deficiency of the von Willebrand factor-cleaving protease ADAMTS13 is associated with rare forms of thrombotic thrombocytopenic purpura (TTP). We investigated a woman with a family history of chronic recurrent TTP and undetectable plasma levels of ADAMTS13 activity. Genetic analysis revealed two missense mutations in the heterozygous state: p.Val88Met substitution in the metalloprotease domain and p.Gly1239Val substitution in the first CUB domain of ADAMTS13. To explore the mechanism of ADAMTS13 deficiency in this patient, the wild type (WT; ADAMT13(WT)) and each mutant construct (ADAMTS13(Val88Met), ADAMTS13(Gly1239Val)) were transiently expressed in HEK 293 and COS-7 cells. To recapitulate the compound heterozygous state of the patient, both mutant ADAMTS13 proteins were also expressed together. The p.Val88Met mutation led to a defect of secretion of the protease associated with a reduction of enzymatic activity, the p.Gly1239Val mutation led to a secretion defect causing intracellular accumulation of the protease. The mechanistic effects of the mutations were further explored by means of differential immunofluorescence, that demonstrated an homogeneous distribution of ADAMTS13(WT) in the Cis-Golgi and endoplasmic reticulum (ER) compartments, a reduction of ADAMTS13(Val88Met) in both compartments, while ADAMTS13(Gly1239Val) failed to reach the Cis-Golgi compartment and remained in the ER.

In a patient who presented with a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous Arg67His mutation was identified in the prothrombin gene.

analysis identified a homozygous deletion of nucleotides 2930-2935 (GTGCCC) in exon 23 of ADAMTS13 in the 2 probands, but also in one asymptomatic sibling. 13 In order to explain the patients' phenotype, we then studied the mechanistic effect of the deletion by means of expression studies in mammalian cells. Design and Methods PatientsTwo South Iranian patients (2 brothers), off-spring of first cousins, were affected by chronic recurrent TTP that first developed during adulthood (Figure 1). Patient II:2, a 26 year old male, had his first episode of TTP at the age of 23 years and 6 subsequent recurrent episodes with no precipitating event or triggering agent. Bleeding symptoms such as purpura and petechiae were present at each episode, accompanied by fever and vomiting, whereas mild neurological symptoms (visual disorders and drowsiness) were observed only during the first episode. The clinical diagnosis was established at the time of the first episode by the presence of thrombocytopenia (platelet count no higher tha 20×10 9 /L), Coombs negative hemolytic anemia (Hb 10.3 g/L), fragmented erythrocytes and high serum level of lactate dehydrogenase (LDH 1055 UI/L). The patient was successfully treated during each acute episode with plasma exchange and high dose of corticosteroids, before the molecular diagnosis. After his sixth episode of TTP, to prevent further relapses, the patient started a prophylactic treatment with fresh frozen plasma (FFP) (30 ml/kg) every three weeks. Patient II:3, a 31 year old male, developed his first episode of TTP at the age of 29 years, in association with an episode of pneumonia. He had purpura and petechiae on his legs, a platelet count of 29×10 9 /L and Coombs negative hemolytic anemia (Hb 8.5 g/L, LDH 954 UI/L). Daily FFP infusions (30 mL/kg) were effective as reflected by a progressive increase in the platelet count. Since the first disease episode, the patient receives an FFP infusion when his platelet count falls below 100×10 9 /L. According to the family history, another male sibling (II:1) had a TTP episode at the age of 23 years and died because of multiorgan failure. Another 4 brothers and one sister are all healthy and have never had any signs or symptoms of TTP. This study was carried out with the approval of the local ethics committee Ex-vivo analysisADAMTS13 activity and antigen were measured in plasma samples and in the conditioned media of cells transfected by wild type (ADAMTS13WT) and mutant (ADAMTS13del6bp) expression vectors using respectively a collagen binding assay (CBA) and an immunoassay, both modified as previously described.14 The presence of neutralizing (inhibitors) and non-neutralizing anti-ADAMTS13 autoantibodies in patients' plasma was also evaluated.14 The coding region and intron-exon boundaries of the ADAMTS13 gene (NT_017539) were amplified and sequenced as previously reported. 5 In vitro expression studiesThe complete ADAMTS13 wild-type cDNA was inserted into the mammalian expression vector pcDNA TM 3.1/V5-His TOPO ® TA (Invitrogen, Carlsbad, CA, U...