Roberta Palla

ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission

Acquired thrombotic thrombocytopenic purpura (TTP) is often due to anti-ADAMTS13 antibodies that inhibit the proteolytic activity of the plasma metallo-protease and/or accelerate its clearance. Survivors of an acute episode of TTP with severely reduced levels of ADAMTS13 and /or with anti-ADAMTS13 antibodies during remission are at high risk of developing another epidode of TTP. 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 Methods In 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. Results Median 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. Conclusions Survivors 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.

Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders

Summary.  Background:  The European Network of Rare Bleeding Disorders (EN‐RBD) was established to bridge the gap between knowledge and practise in the care of patients with RBDs.

ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura

Congenital thrombotic thrombocytopenic purpura (TTP) (also known as Upshaw‐Schulman syndrome, USS) is a rare, life‐threatening disease characterized by thrombocytopenia and microangiopathic hemolytic anemia, associated with the deficiency of the von Willebrand factor‐cleaving protease (ADAMTS13) due to mutations in the corresponding gene. The spectrum of clinical phenotype in congenital TTP is wide, encompassing neonatal‐onset disease and adult‐onset disease, forms with a single disease episode and chronic‐relapsing forms. We review ADAMTS13 gene variants associated with inherited ADAMTS13 deficiency and congenital TTP. To date, 76 mutations of ADAMTS13 are reported in the literature. Missense mutations, which constitute nearly 60% of ADAMTS13 mutations, preferentially localize in the 5′‐half of the gene encoding the N‐terminal half of the protein, where the domains that are indispensable for ADAMTS13 catalytic function are situated. In vitro expression studies in cell cultures have shown that defects in protein secretion and catalytic activity are the main mechanisms responsible for the deficiency of ADAMTS13 in congenital TTP patients. Even if data from the literature suggest the existence of genotype–phenotype correlations, a clear relationship between the type and the effect of ADAMTS13 genetic defects with disease manifestations remains to be established. Hum Mutat 30:1–9, 2009.

Rare bleeding disorders

Summary.  Deficiencies of coagulation factors other than factor VIII and factor IX (afibrinogenemia, FII, FV, FV+FVIII, FVII, FX, FXI, FXIII) that cause bleeding disorders (RBDs) are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500.000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. The study of the genetic basis of these disorders could represent an important tool for prevention through prenatal diagnosis. Treatment of patients with RBDs during bleeding episodes or surgery is a challenge because of the lack of experience and the paucity of data. For some deficiency factor concentrates are still non available and severe complications can occur. These complications can be minimized by assessment of risks of bleeding and thrombosis, use of haemostatic means other than blood components or no therapy at all. The RBDs pose a problem for guideline writers because there are no suitable clinical trials to supply good evidence for how these people are best treated. The lack of adequate information on clinical manifestations, treatment and genetic basis of RBDs could be improved by the collection of data in an International Database (http://www.rbdd.org), linkable to others previously published. This could be a useful tool to fill the gap between clinical data and clinical practice. This article reviews the genetic basis of RBDs, problems and complications of treatment, problems in the preparation of suitable guidelines for treatment and the future perspectives of the International Registry on RBDs.

Genetic diagnosis of haemophilia and other inherited bleeding disorders

Summary.  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 Willebrands 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 Willebrands disease and of rare bleeding disorders.

Introduction. Rare bleeding disorders: general aspects of clinical features, diagnosis, and management.

Rare bleeding disorders (RBDs) are autosomal recessive diseases including the inherited deficiencies of coagulation factors such as fibrinogen, factor (F) II, FV, FV + FVIII, FVII, FX, FXI, FXIII, and multiple deficiency of vitamin K-dependent factors, with clinical manifestations ranging from mild to severe. They represent 3 to 5% of all the inherited coagulation deficiencies with a prevalence in the general population varying between 1 in 500,000 and 1 in 2 million, being higher in areas where consanguineous marriages are diffuse. Despite the progress made in past years, as a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, the actual management of bleeding episodes and particularly the prophylactic treatment in patients affected with RBDs are not well established. In this introductory article, the main features, diagnosis, available treatment options, and treatment complications of RBDs will be discussed.

Rare bleeding disorders: diagnosis and treatment.

Despite the worldwide prevalence of rare bleeding disorders (RBDs), knowledge of these conditions and their management is suboptimal; health care professionals often have little diagnostic and treatment experience with variable access to diagnostic modalities required for accurate identification. Therefore, patients often experience morbidity and mortality due to delayed diagnosis. As RBDs represent a small potential commercial market, few, if any, specific therapies exist for these conditions. As a result, affected individuals commonly face delayed diagnosis, incomplete laboratory evaluation, and limited treatment options. Standardization and customization of coagulation assays, full genome sequencing, and global clotting assays will significantly improve diagnosis of patients with RBDs. In addition, new therapeutic modalities, both recombinant and plasma derived, are emerging, at least in developed countries. Registries and clinical trials have demonstrated decreased bleeding and improved outcomes when patients are appropriately diagnosed and properly treated. Expansion and harmonization of international registries has been initiated to correlate genotype, laboratory, and clinical phenotypes including bleeding severity to improve the diagnosis and therapeutic approach. This review focuses on the latest advances in our understanding, diagnosis, and treatment of RBDs.

ADAMTS-13 assays in thrombotic thrombocytopenic purpura

Summary.  ADAMTS‐13, the thirteenth member of the ADAMTS (A Disintegrin And Metalloprotease with Thrombo‐Spondin 1 repeats) family, is the plasma metalloprotease responsible for regulating the multimeric structure of VWF. In congenital or acquired deficiency it is actively involved in the pathophysiology of thrombotic thrombocytopenic purpura (TTP), a rare but life threatening disease characterized by microangiopathic haemolytic anaemia and consumptive thrombocytopenia leading to disseminated microvascular thrombosis and variable signs and symptoms of organ ischemia and damage. In the last few years, a number of in house and commercial laboratory assays for ADAMTS‐13 and its autoantibodies have been developed. The features and clinical utility of ADAMTS‐13 assays are summarized in this review.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

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 Tyr1605-Met1606 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 kcat/Km ≅ 3.4 × 104 m-1 s-1, but this value decreased by ∼10-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 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{ClO}_{4}^{-}{>}\mathrm{Cl}^{-}{>}\mathrm{F}^{-}\) \end{document}). 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 GpIbα 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.

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.