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.     

Relapsing or refractory idiopathic thrombotic thrombocytopenic purpura‐hemolytic uremic syndrome: the role of rituximab

Idiopathic thrombotic thrombocytopenic purpura–hemolytic uremic syndrome (TTP‐HUS) is a rare disease responsive to treatment with plasma exchange (PE) but with a high percentage of relapse or refractory patients. A severe deficiency of ADAMTS‐13 (<5% of normal activity), congenital or caused by an autoantibody, may be specific for TTP and it has been proposed that severe ADAMTS‐13 deficiency now defines TTP. B cells play a key role in both the development and the perpetuation of autoimmunity, suggesting that B‐cell depletion could be a valuable treatment approach for patients with idiopathic TTP‐HUS. This review of the literature focuses on the role of rituximab, a chimeric monoclonal antibody directed against CD20 antigen expressed by B lymphocytes, in patients with relapsing or refractory TTP‐HUS with or without ADAMTS‐13 deficiency, suggesting that rituximab may produce clinical remission in a significant proportion of patients. Rituximab therapy reduces plasma requirement and avoids complications related to salvage‐immunosuppressive therapy. In conclusion, rituximab provides an effective, well‐tolerated, and safe treatment option for patients with idiopathic TTP‐HUS, thus giving an alternative approach to the current treatment based on PE.     

Blood group O and black race are independent risk factors for thrombotic thrombocytopenic purpura associated with severe ADAMTS13 deficiency

BACKGROUND: It has been postulated that blood group O subjects may be partially protected against thrombotic thrombocytopenic purpura (TTP) because they have lower plasma levels of von Willebrand factor. STUDY DESIGN AND METHODS: The Oklahoma TTP Registry enrolled 301 consecutive patients from November 13, 1995 (when systematic ADAMTS13 measurements began), through 2009; 281 (93%) patients had ADAMTS13 measurements. Patients were designated as having severe ADAMTS13 deficiency when the activity measurement by either method was less than 10%. ABO blood group was determined in all 281 patients. The observed frequency of blood group O was compared to the expected frequency. The association between severe ADAMTS13 deficiency and blood group, race, sex, and age were analyzed by logistic regression. RESULTS: The frequency of blood group O was unexpectedly and significantly greater than the race‐ethnicity–adjusted expected frequency in 65 patients with severe ADAMTS13 deficiency (60.0% vs. 47.4%, p = 0.042) but not in 216 patients without severe ADAMTS13 deficiency (44.9% vs. 46.5%, p = 0.639). Blood group O and race‐ethnicity were independently associated with severe ADAMTS13 deficiency among patients with TTP. The probability for severe ADAMTS13 deficiency was 45.8% with O and 32.1% with non‐O blood groups for black patients and 24.1% with O and 15.1% with non‐O blood groups for white patients. CONCLUSION: Among patients with TTP and severe ADAMTS13 deficiency the relative frequency of patients with blood group O was greater than expected, suggesting that blood group O may be a risk factor for TTP associated with severe ADAMTS13 deficiency.     

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.     

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.     

Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a dramatic intravascular platelet-clumping disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal insufficiency and fever. TTP is a rare disease but is almost always fatal if untreated. More than 80% of patients survive with plasma therapy. In healthy individuals, the proteolytic cleavage of ultralarge von Willebrand factor (vWF) multimers prevents spontaneous clumping of platelets in the microcirculation. Patients with TTP have either severe congenital deficiency of von Willebrand factor-cleaving protease (vWF-cp), or have autoantibodies that inhibit the protease. Determination of vWF-cp levels in patient plasma helps to distinguish between TTP and other thrombotic microangiopathies with similar clinical signs and symptoms. vWF-cp is a member of the ADAMTS family of metalloproteases and has been designated ADAMTS13.     

Update on thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, which is classically associated with signs and symptoms of fever, thrombocytopenia, neurologic deficits, hemolytic anemia, and renal failure. It is caused by a deficiency of A Disintegrin-like And Metalloprotease with a ThromboSpondin type1 motif 13 (ADAMTS13), which may be an inherited disorder, but more commonly is an acquired disease due to autoantibodies directed against ADAMTS13. Low ADAMTS13 levels result in increased ultra-large von Willebrand factor multimers, which induce platelet adhesion and thrombosis. Plasma exchange therapy is the standard of care, and has greatly reduced morbidity and mortality. A recent TTP case is reviewed, and treatments for recurrent or refractory TTP are summarized. A scoring system using clinical and laboratory parameters to evaluate which suspected TTP patients will benefit from plasma exchange therapy is also discussed.     

Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a dramatic intravascular platelet-clumping disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal insufficiency and fever. TTP is a rare disease but is almost always fatal if untreated. More than 80% of patients survive with plasma therapy. In healthy individuals, the proteolytic cleavage of ultralarge von Willebrand factor (vWF) multimers prevents spontaneous clumping of platelets in the microcirculation. Patients with TIP have either severe congenital deficiency of von Willebrand factor-cleaving protease (vWF-cp), or have autoantibodies that inhibit the protease. Determination of vWF-cp levels in patient plasma helps to distinguish between TTP and other thrombotic microangiopathies with similar clinical signs and symptoms. vWF-cp is a member of the ADAMTS family of metalloproteases and has been designated ADAMTS13.     

The utility of patient characteristics in predicting severe ADAMTS13 deficiency and response to plasma exchange

BACKGROUND: Idiopathic thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy (TMA) characterized by extreme deficiency of ADAMTS13, an enzyme responsible for cleavage of von Willebrand factor. Plasma exchange therapy is the cornerstone of current treatment and is ineffective for most other forms of TMA. The availability of ADAMTS13 testing has improved diagnostic accuracy and appropriate selection of patients who are most likely to respond to plasma exchange. STUDY DESIGN AND METHODS: We performed a retrospective chart review of 110 cases of clinically suspected TTP with ADAMTS13 test results from 2005 to the present. The primary goal was to identify presenting clinical and/or laboratory features of patients with ADAMTS13 deficiency that would prove useful in increasing the likelihood of, or excluding the possibility of, TTP. In addition, patient outcomes including alternative diagnoses and response to plasma exchange were reviewed. RESULTS: Significant correlations for severe ADAMTS13 deficiency were seen for four of the observed variables: indirect bilirubin, reticulocyte percentage, creatinine, and platelet count; a fifth variable, D‐dimer, just missed significance but performed well in subsequent analysis. Receiver operator characteristics curves for individual variables had area under the curve (AUC) values ranging from 0.75 to 0.85; a combined model had an AUC of 0.98. In addition, we constructed tree models both for clinical use as a diagnostic algorithm and for recursive partitioning to help establish cutoff points for categorical variables in developing an easy‐to‐use clinical prediction score. CONCLUSION: These results may enable the rapid exclusion and accurate diagnosis of TTP using readily available laboratory data.     

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. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

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.     

Use of n-acetylcysteine therapy in patients with relapsed refractory thrombotic thrombocytopenic purpura

There is limited data on the use of NAC in the literature. We would like to present the satisfactory results we obtained in our resistant and relapsed patients as a case series.Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by ADAMTS13 (a disintegrin with thrombospondin type 1 motif and metalloprotease activity, member13) deficiency. Von Willebrand factor (vWF) initiates platelet aggregation and thus thrombus formation. The multimers of vWF are cleaved by ADAMTS13. Because of the decreased activity of ADAMTS13, ultra-large multimers accumulate and end-organ damage occurs. TTP is characterized by microangiopathic hemolytic anemia (MAHA), severe thrombocytopenia, and organ ischemia resulting from vascular occlusion caused by thrombi. Plasma exchange therapy (PEX) remains the mainstay of TTP therapy. Patients who do not respond to PEX and corticosteroids require additional treatments such as rituximab and caplacizumab. NAC reduces disulfide bonds in mucin polymers through its free sulfhydryl group. Thus, the size and viscosity of the mucins are reduced. VWF is structurally similar to mucin. Based on this similarity, Chen and colleagues showed that NAC can reduce the size and reactivity of ultralarge multimers of vWF, such as ADAMTS13. Currently, there is not much information to suggest that NAC has any clinical value in the treatment of TTP. In this case series of 4 refractory patients, we would like to present the responses we obtained with the addition of NAC therapy. NAC can be added to PEX and glucocorticoid therapy as supportive therapy, especially in unresponsive patients.     

Interferon‐α is not elevated in idiopathic thrombotic thrombocytopenic purpura patients

Idiopathic thrombotic thrombocytopenic purpura (TTP) patients have ADAMTS13 deficiency, which is usually caused by ADAMTS13 autoantibodies. However, the triggering factors for the autoantibody production remain unclear. Interferon‐α (IFN‐α) is a cytokine involved with many autoimmune processes such as inducing the activation of peripheral dendritic cells and stimulating T cells and B cells. It also plays an important role in some autoimmune diseases. Elevated IFN‐α levels have been observed in some TTP patients and previous case reports have shown the occurrence of TTP after IFN‐α treatment. Thus, we hypothesized that high levels of IFN‐α would correlate with presence of ADAMTS13 autoantibodies. However, we did not observe elevated IFN‐α levels in 36 TTP patients (mean 5.29 pg/ml, standard deviation (SD) 26.56 pg/ml) compared to healthy controls (mean 0 pg/ml, SD 0 pg/ml), P = 0.59. IFN‐α levels of most patients (94%) were undetectable. Only two patients had increased IFN‐α levels and ADAMTS13 autoantibodies were detected in these two patients. Interestingly, both the patients had an underlying autoimmune disease. Although there have been cases of secondary TTP following IFN‐α treatment, no evidence supports a role of IFN‐α in the development of idiopathic TTP in our patient population. J. Clin. Apheresis 29:336–338 2014. © 2014 Wiley Periodicals, Inc.     

Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the vWF-cleaving protease ADAMTS13. Here we show that ADAMTS13-deficient mice are viable and exhibit normal survival, although vWF-mediated platelet-endothelial interactions are significantly prolonged. Introduction of the genetic background CASA/Rk (a mouse strain with elevated plasma vWF) resulted in the appearance of spontaneous thrombocytopenia in a subset of ADAMTS13-deficient mice and significantly decreased survival. Challenge of these mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP. Surprisingly, no correlation was observed between plasma vWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from vWF. These data suggest that microbe-derived toxins (or possibly other sources of endothelial injury), together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency.     

External validation of the PLASMIC score: a clinical prediction tool for thrombotic thrombocytopenic purpura diagnosis and treatment

Essentials

• Severe ADAMTS‐13 deficiency is key to thrombotic thrombocytopenic purpura (TTP) diagnosis.
• PLASMIC score predicts ADAMTS‐13 deficiency in suspected TTP with high discrimination.
• PLASMIC score is more generalizable with fewer missing data than alternative clinical scores.
• PLASMIC score identifies a subgroup of patients lacking significant response to plasma exchange.

Summary

Background

The PLASMIC score was recently published to distinguish patients with severe ADAMTS‐13 deficiency from those without for early identification of thrombotic thrombocytopenia purpura (TTP).

Objective

We performed an independent external validation of the PLASMIC score for clinical prediction of severe ADAMTS‐13 deficiency.

Patients/Methods

We studied an independent cohort of 112 consecutive hospitalized patients with suspected thrombotic microangiopathy and appropriate ADAMTS‐13 testing (including 21 patients with TTP diagnosis).

Results

The PLASMIC score model predicted severe ADAMTS‐13 deficiency with a c statistic of 0.94 (0.88–0.98). When dichotomized at high (score 6–7) vs. low‐intermediate risk (score 0–5), the model predicted severe ADAMTS‐13 deficiency with positive predictive value of 72%, negative predictive value of 98%, sensitivity of 90% and specificity of 92%. In the low‐intermediate risk group (score 0–5) there was no significant improvement in overall survival associated with plasma exchange.

Conclusions

The PLASMIC score model had excellent applicability, discrimination and calibration for predicting severe ADAMTS‐13 deficiency. The clinical algorithm allowed identification of a subgroup of patients who lacked a significant response to empiric treatment.     

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw–Schulman syndrome)

Summary.  ADAMTS13, the specific von Willebrand factor (VWF)-cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw–Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations. Six probands with congenital TTP and their family were tested for ADAMTS13 activity in plasma using a two-site immunoradiometric assay and for ADAMTS13 gene mutations using polymerase chain reaction and sequencing. ADAMTS13 activity was severely deficient (< 5%) in the six probands and one mildly symptomatic sibling but normal (> 50%) in all the parents and the asymptomatic siblings. Ten novel candidate ADAMTS13 mutations were identified in all families, showing either a compound heterozygous or a homozygous status in all probands plus the previous sibling and a heterozygous status in the parents. The mutations were spread all over the gene, involving the metalloprotease domain (I79M, S203P, R268P), the disintegrin domain (29 bp deletion in intron/exon 8), the cystein-rich domain (acceptor splice exon 12, R507Q), the spacer domain (A596V), the 3rd TSP1 repeat (C758R), the 5th TSP1 repeat (C908S) and the 8th TSP1 repeat (R1096stop). This study emphasizes the role of ADAMTS13 mutations in the pathogenesis of congenital TTP and suggests that several structural domains of this metalloprotease are involved in both its biogenesis and its substrate recognition process.     

Lessons learned from the Oklahoma thrombotic thrombocytopenic purpura-hemolytic uremic syndrome registry

The Oklahoma TTP-HUS Registry provides a complete community perspective of thrombotic thrombocytopenic purpura (TTP). This is possible because plasma exchange is the essential treatment for TTP and the Oklahoma Blood Institute provides all plasma exchange procedures for a region encompassing most of the State, including 58 of Oklahoma's 77 counties. The Registry is an inception cohort of consecutive patients for whom plasma exchange treatment was requested for a diagnosis of either TTP or hemolytic uremic syndrome (HUS). All 382 patients identified from January 1, 1989 to December 31, 2007 have consented to be enrolled. Complete follow-up is available for 380 of 382 patients. Patients are described both by clinical categories, related to their associated conditions and clinically apparent etiologies, and by the presence of severe ADAMTS13 deficiency. ADAMTS13 activity has been measured on 235 (93%) of 254 patients since 1995. Registry data have provided new perspectives on the definition and diagnoses of these syndromes as well as their outcomes. Long-term follow-up has documented that relapse is common among patients with ADAMTS13 deficiency but rarely occurs in patients without ADAMTS13 deficiency. Long-term follow-up has also documented persistent abnormalities of health-related quality-of-life and cognitive function. In addition to providing new perspectives on the natural history of these syndromes, The Oklahoma TTP-HUS Registry provides a support group for our patients, information about evaluation and management for community physicians, and a resource for research and educational programs.