Functional assays are commonly used to measure the antibodies of ADAMTS13 found in patients of thrombotic thrombocytopenic purpura (TTP). In this study we used an enzyme-linked immunoassay to analyze the ADAMTS13-binding IgG levels in six groups of individuals: normal, random hospitalized patients, acute TTP,TTP after receiving plasma therapy, TTP in remission, and other types of thrombotic microangiopathy (TMA). The results showed thatADAMTS13-binding IgG levels were elevated in 100% of the acute TTP group, 75% of the TTP group after receiving plasma therapy, and 40% of the remission group. Overall, the ADAMTS13-binding IgG levels correlated with the inhibitory activity levels against ADAMTS13 (r=−0.69,P<0.0001). The assay also detected elevated IgG binding levels in 5% – 15% of the normal, random, and other TMA control groups. Addition of purified ADAMTS13 protein to the plasma samples suppressed the IgG binding in each of the acute TTP patients, but in none of the non-TTP groups. Serial measurement in a patient that had two exacerbations of TTP within the first three weeks revealed that the ADAMTS13 activity levels remained < 0.1 U/ml during this period, and the ADAMTS13-binding IgG remained elevated, suggesting thatADAMTS13 analysis may provide valuable insight to the disease status during the course of therapy. Analysis of ADAMTS13-binding IgG is helpful for the diagnosis and management of TTP.
ADAMTS13; thrombotic thrombocytopenic purpura; antibody
Thrombotic thrombocytopenic purpura (TTP) is a multisystemic microvascular disorder that may be caused by an imbalance between unusually large von Willebrand factor multimers and the cleaving protease ADAMTS13. In acquired TTP, especially in secondary TTP with various underlying diseases, the diagnosis is difficult because there are many cases that do not exhibit severe deficiency of ADAMTS13 or raised levels of ADAMST13 inhibitors. It is well known that collagen disease, malignancy, and hematopoietic stem cell transplantation can be underlying conditions that induce TTP. However, TTP induced by acute pancreatitis, as experienced by our patient, has rarely been reported. Our patient completely recovered with treatments using steroids and plasma exchange (PE) only. In cases where patients develop acute pancreatitis with no apparent causes for hemolytic anemia and thrombocytopenia, the possibility of TTP should be considered. Treatments for TTP including PE should be evaluated as soon as a diagnosis is made.
thrombotic thrombocytopenic purpura; ADAMTS13; acute pancreatitis; plasma exchange
Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either pre-denatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiological relevance of the assay results is uncertain.
We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitor. We also compared this assay with a fluorogenic peptide assay.
We found that an incubation of purified plasma VWF with 0.5-1.0 μl of citrated plasma under constant vortexing at 2,500 rpm for 60 minutes in the presence of 5 mM CaCl2, 1.7 μM ZnCl2 and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity.
Our fluid shear-based assay may be useful for investigating basic biological function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.
Thrombotic thrombocytopenic purpura (TTP) is a disorder with characteristic von Willebrand factor (VWF)-rich microthrombi affecting the arterioles and capillaries of multiple organs. The disorder frequently leads to early death unless the patients are treated with plasma exchange or infusion. Studies in the last decade have provided ample evidence to support that TTP is caused by deficiency of a plasma metalloprotease, ADAMTS13. When exposed to high shear stress in the microcirculation, VWF and platelets are prone to form aggregates. This propensity of VWF and platelet to form microvascular thrombosis is mitigated by ADAMTS13, which cleaves VWF before it is activated by shear stress to cause platelet aggregation in the circulation. Deficiency of ADAMTS13, due to autoimmune inhibitors in patients with acquired TTP and mutations of the ADAMTS13 gene in hereditary cases, leads to VWF–platelet aggregation and microvascular thrombosis of TTP. In this review, we discuss the current knowledge on the pathogenesis, diagnosis and management of TTP, address the ongoing controversies, and indicate the directions of future investigations.
TTP; von Willebrand factor; ADAMTS13; Shear stress; Microvascular thrombosis
Deficiency of von Willebrand factor (VWF) cleaving protease ADAMTS13 has been demonstrated to be the proximate cause of a subset of thrombotic microangiopathic haemolytic anaemias (MAHA) typical for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 gene mutations cause the hereditary form; acquired deficiency has been attributed to presence of an autoantibody, which may represent a specific subset of MAHA best termed ‘autoimmune thrombotic thrombocytopenic purpura’. We describe a patient with relapsing TTP because of ADAMTS13 inhibitors, who failed to achieve sustained remission despite therapies with plasma exchange, steroids, vincristine, staphylococcal protein A and splenectomy. The ADAMTS13 inhibitor titre remained elevated and clinical stability was only maintained by plasma exchange every 2–3 d over a period of 268 d. The patient then received rituximab therapy (eight doses of 375 mg/m2 weekly), during which she required five plasma exchanges in the first 10 d, two exchanges in the next 3 weeks, and none thereafter for 450 d and ongoing. The ADAMTS13 inhibitor titre decreased and enzyme activity increased. We compared this case with that of seven previously reported TTP cases also treated with rituximab; experience suggests that rituximab therapy deserves further investigation for patients with either refractory or relapsing TTP caused by ADAMTS13 inhibitors.
thrombotic thrombocytopenic purpura; rituximab; plasma exchange
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 (VWF).
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 <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, gender, and age were analyzed by logistic regression.
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.
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 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing von Wille-brand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP.
Recent studies have demonstrated that thrombotic thrombocytopenic purpura (TTP), a serious thrombotic disorder affecting the arterioles and capillaries of multiple organs, is caused by a profound deficiency in the von Willebrand factor cleaving metalloprotease, ADAMTS13. ADAMTS13, a 190-kD plasma protease originating primarily in hepatic stellate cells, prevents microvascular thrombosis by cleaving von Willebrand factor when the substrate is conformationally unfolded by high levels of shear stress in the circulation. Deficiency of ADAMTS13, due to genetic mutations or inhibitory autoantibodies, leads to accumulation of superactive forms of vWF, resulting in vWF-platelet aggregation and microvascular thrombosis. Analysis of ADAMTS13 has led to the recognition of subclinical TTP and atypical TTP presenting with thrombocytopenia or acute focal neurological deficits without concurrent microangiopathic hemolysis. Infusion of plasma replenishes the missing ADAMTS13 and ameliorates the complications of hereditary TTP. The patients are at risk of both acute and chronic renal failure if they receive inadequate plasma therapy. The more frequent, autoimmune type of TTP requires plasma exchange therapy and perhaps immunomodulatory measures. Current studies focus on the factors affecting the phenotypic severity of TTP and newer approaches to improving the therapies for the patients.
ADAMTS13; shear stress; thrombosis; thrombotic thrombocytopenic purpura; von Willebrand factor
Many patients with acquired thrombotic thrombocytopenic purpura (TTP) harbor autoantibodies that may bind and/or inhibit ADAMTS-13 proteolytic activity and accelerate its clearance in vivo.
To test this hypothesis, we determined ADAMTS-13 activity and antigen levels in parallel plasma samples from patients clinically diagnosed with TTP. Collagen binding, GST-VWF73 and FRETS-VWF73 assays were used to determine ADAMTS-13 activity and to detect inhibitory autoantibodies. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation plus Western blotting (IP/WB) were used to detect total anti-ADAMTS-13 IgG (inhibitory and non-inhibitory).
Among 40 patients with TTP (21 idiopathic and 19 non-idiopathic), inhibitory autoantibodies were detected (by FRETS-VWF73) in 52% of idiopathic and 0% of non-idiopathic TTP patients. In contrast, non-inhibitory IgG autoantibodies were detected in 29% of idiopathic and 50% of non-idiopathic TTP patients. The concentration of inhibitory IgG autoantibody in idiopathic TTP patients was significantly higher than that of non-inhibitory IgG in either idiopathic or non-idiopathic TTP patients. Idiopathic TTP patients demonstrated significantly reduced ADAMTS-13 activity compared with non-idiopathic patients, but only slightly lower ADAMTS-13 antigen levels. Interestingly, patients with inhibitory autoantibodies exhibited significantly lower ADAMTS-13 antigen levels than those with only non-inhibitory IgG autoantibodies or no autoantibody. Serial plasma exchanges increased levels of ADAMTS-13 activity and antigen concurrently in patients with inhibitory autoantibodies.
The identification of severe ADAMTS-13 deficiency and autoantibodies or inhibitors appears to be assay-dependent; the inhibitory IgG autoantibodies, in addition to binding and inhibiting ADAMTS-13 proteolytic activity, may accelerate ADAMTS-13 clearance in vivo.
a disintegrin and metalloprotease with thrombospondin type 1 repeats; autoimmune disorder; thrombotic microangiopathy; von Willebrand factor
ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers and maintains proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura (TTP). At present, ADAMTS13 expression levels are determined by immunoblotting.
We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. Results were validated using confocal microscopy, immunoblotting and an activity assay (FRETS-VWF73).
We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable to previously established methods for ADAMTS13 detection. Specifically, we compared the endogenous expression levels of ADAMTS13 in various liver cell lines using flow cytometry and obtained results that parallel immunoblot analysis. Knock-down of ADAMTS13 expression via targeted siRNA resulted in significantly reduced median signal, displaying the sensitivity of this detection method. A further analysis of reliability and specificity was achieved through plasmid DNA and transfection reagent dose response studies.
The flow cytometry method described here is useful in determining the expression of both endogenous and recombinant forms of intracellular ADAMTS13. Flow cytometry is a convenient, efficient and cost effective way to measure the expression levels of ADAMTS13.
ADAMTS13; flow cytometry; intracellular protein expression
Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the microtiter-plate assay and the cell-based assay may detect differential antigenic epitopes. Moreover, antigens clustered on cell membrane may enhance antibody binding affinity, thereby increasing analytical sensitivity. Finally, our assay was able to determine kinetic changes of plasma levels of anti-ADAMTS13 IgGs in TTP patients during plasma therapy. Together, our findings suggest that the novel cell-based assay may be applicable for rapid identification and mapping of anti-ADAMTS13 autoantibodies in patients with acquired TTP.
von Willebrand factor cleaving protease; thrombotic microangiopathies; diagnostic test; autoantibody
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.
Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome is a life-threatening condition with various etiopathogeneses. Without therapy approximately 90% of all patients die from the disease.
We report the case of a 17-year-old Caucasian woman with widespread hematomas and headache. Due to hemolytic anemia, thrombocytopenia, and schistocytosis, thrombotic thrombocytopenic purpura-hemolytic uremic syndrome was suspected and plasma exchange therapy was initiated immediately. Since her thrombocyte level did not increase during the first week of therapy, plasma treatment had to be intensified to a twice-daily schedule. Further diagnostics showed markedly reduced activities of both ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 - also known as von Willebrand factor-cleaving protease) and factor H. Test results for antibodies against both proteins were positive. While plasma exchange therapy was continued, rituximab was given once weekly for four consecutive weeks. After the last dose, thrombocytes and activities of ADAMTS-13 and factor H increased into the normal range. Our patient improved and was discharged from the hospital.
Since no clinical symptoms/laboratory findings indicated a malignant or specific autoimmune-mediated disorder, the diagnosis made was thrombotic thrombocytopenic purpura-hemolytic uremic syndrome due to idiopathic combined, autoantibody-mediated ADAMTS-13/factor H deficiency.
The kidney is commonly affected in thrombotic thrombocytopenic purpura (TTP), a multi-system disorder with microvascular thrombosis of the capillaries and arterioles. Nevertheless, due to difference in its diagnostic criteria, the frequency and severity of renal dysfunction in TTP remains controversial. With the recent studies indicating that severe deficiency of a VWF cleaving protease, ADAMTS13, is the main cause of platelet thrombosis in TTP, it is now possible to define TTP at the molecular level. Among patients with acquired TTP due to inhibitory antibodies of ADAMTS13, renal dysfunction is usually mild; oliguria, fluid overload, hypertension, and need of dialysis support are infrequent. When any of these complications occur, one must re-examine the diagnosis of TTP and consider the possibility that the patient has another cause for these complications. In a patient with hereditary TTP, acute renal failure may ensue. However, the renal dysfunction is mostly reversible if the patients are promptly treated with plasma to replenish ADAMTS13. Patients with TTP, particularly of the hereditary type, may develop chronic renal failure. This complication may be a consequence of repeated insults by overt or subclinical microvascular thrombosis to the kidney, or it may have a separate cause. Therapy of hereditary TTP should aim not only to prevent acute exacerbations but also to minimize the risk of irreversible renal injury.
Thrombotic thrombocytopenic purpura; ADAMTS13; von Willebrand factor; Thrombosis; Renal failure; Shear stress
ADAMTS13, a reprolysin-like metalloprotease, limits platelet-rich thrombus formation in the small arteries by cleaving von Willebrand factor (vWF) at the Tyr1605-Met1606 peptide bond. Deficiency of plasma ADAMTS13 activity, due to either an inherited or an acquired etiology, may lead to a potentially lethal syndrome, thrombotic thrombocytopenic purpura (TTP). Molecular cloning and characterization of the ADAMTS13 gene have provided further insight into the structure-function relationships, biosynthesis, and regulation of the ADAMTS13 protease, in addition to understanding the pathogenesis of TTP and perhaps other thrombotic disorders. ADAMTS13 consists of a short propeptide, a typical reprolysin-like metalloprotease domain, followed by a disinte-grin-like domain, first thrombospondin type 1 (TSP1) repeat, Cys-rich domain, and spacer domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two CUB domains. ADAMTS13 is synthesized mainly in hepatic stellate cells, but also in vascular endothelial cells. Recognition and cleavage of vWF require the proximal carboxyl terminal domains, but not the middle and distal carboxyl terminal domains. Cleavage of vWF appears to be modulated by shear force, binding to platelet or platelet glycoprotein-1bα, heparin, inflammatory cytokine (interleukin-6), and chloride ion. At the site of thrombus formation, the ADAMTS13 may be inactivated by thrombin, plasmin, and factor Xa. Having a sensitive and specific assay for ADAMTS13 activity is not only critical to understand the basic biology of ADAMTS13 protease, but also to facilitate a more timely and accurate clinical diagnosis of TTP, and to initiate potentially life-saving plasma exchange therapy. Although many assays have been developed and tested for clinical applications, the fluorescent resonance energy transfer-vWF73 assay appears to be the simplest and most promising assay to date.
Thrombotic thrombocytopenic purpura (TTP); von Willebrand factor; microvascular thrombosis; ADAMTS13; metalloprotease; assays; clinical application
Thrombotic thrombocytopenic purpura is a rare, life-threatening disease characterised by microangiopathic haemolytic anaemia, thrombocytopenia and symptoms related to organ ischaemia, mainly involving the brain and the kidney. It is associated with a deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. The congenital form (Upshaw-Schulman syndrome) is rare and is associated with mutations of the ADAMTS13 gene on chromosome 9q34. The clinical symptoms of congenital thrombotic thrombocytopenic purpura are variable, with some patients developing their first episode during the neonatal period or childhood and others becoming symptomatic in adulthood.
Materials and methods
We describe a case of thrombotic thrombocytopenic purpura, who presented to our attention with a relapsing form of the disease: the first episode occurred at the age of 13 months. Phenotype and genotype tests were performed in the patient and his family.
The undetectable level of ADAMTS13 in the patient was caused by two novel heterozygote missense mutations on the ADAMTS13 gene: one mutation is c.788C > T (p.Ser263Phe) on exon 7 and the second is c.3251G > A (p.Cys1084Tyr) on exon 25 of the ADAMTS13 gene. All the relatives who have been investigated were found to carry one of these missense mutations in a heterozygous state.
Although Upshaw-Schulman syndrome is a rare disease, it should be considered in all children with thrombocytopenia and jaundice in the neonatal period. In fact, once a child is confirmed to carry mutations of the ADAMTS13 gene causing early thrombotic thrombocytopenic purpura, prophylactic treatment should be started to avoid recurrence of symptoms. Genotype tests of relatives would also be important for those women in the family who could be carriers of ADAMTS13 mutations, particularly during pregnancy.
ADAMTS13; thrombotic thrombocytopenic purpura; mutation; Upshaw-Schulman syndrome; TTP
Thrombotic thrombocytopenic purpura (TTP) is a type of thrombotic microangiopathy (TMA). Studies report that the majority of TTP patients present with a deficiency of ADAMTS13 activity. In a database of TMA patients in Japan identified between 1998 and 2008, 186 patients with first onset of acquired idiopathic (ai) ADAMTS13-deficient TTP (ADAMTS13 activity <5%) were diagnosed. The median age of onset of TTP in this group of patients was 54 years, 54.8% were female, 75.8% had renal involvement, 79.0% had neurologic symptoms, and 97.8% had detectable inhibitors to ADAMTS13 activity. Younger patients were less likely to present with renal or neurologic dysfunction (p<0.01), while older patients were more likely to die during the TTP hospitalization (p<0.05). Findings from this cohort in Japan differ from those reported previously from the United States, Europe, and Korea with respect to age at onset (two decades younger in the other cohort) and gender composition (60% to 100% female in the other cohort). We conclude that in one of the largest cohorts of ai-TTP with severe deficiency of ADAMTS13 activity reported to date, demographic characteristics differ in Japanese patients relative to those reported from a large Caucasian registry from Western societies. Additional studies exploring these findings are needed.
The absence of specific diagnostic criteria, the urgency to begin plasma exchange treatment, and the risk for complications from plasma exchange make the initial evaluation of patients with suspected thrombotic thrombocytopenic purpura (TTP) difficult. Systemic infections may mimic the presenting clinical features of TTP. In the Oklahoma TTP-HUS (hemolytic-uremic syndrome) Registry, 1989–2010, 415 consecutive patients have been clinically diagnosed with their first episode of TTP; in 31 (7%) the presenting clinical features were subsequently attributed to a systemic infection. All 31 patients had diagnostic criteria for TTP; 16 (52%) had the complete “pentad” of microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal failure and fever. Four (16%) of 25 patients who had ADAMTS13 measurements had <10% activity; three patients had a demonstrable ADAMTS13 inhibitor. Compared to 62 patients with severe ADAMTS13 deficiency (<10%) who had no recognized alternative disorders, patients with systemic infections had more frequent fever, coma, renal failure, and the complete “pentad” of clinical features. Seventeen different infectious etiologies were documented. A systematic literature review identified 67 additional patients with a diagnosis of TTP or HUS and also a systemic infection. Among all 98 patients, infections with 41 different bacteria, viruses, and fungi were documented, suggesting that many different systemic infections may mimic the presenting clinical features of TTP. Initial plasma exchange treatment is appropriate in critically ill patients with diagnostic features of TTP, even if a systemic infection is suspected. Continuing evaluation to document a systemic infection is essential to determine the appropriateness of continued plasma exchange.
infection; thrombotic thrombocytopenic purpura; TTP; hemolytic uremic syndrome; HUS; ADAMTS13
Thrombotic thrombocytopenic purpura (TTP) is a rare clinical disorder which was associated with poor prognosis for a long time. The outcome has been improved by the consistent introduction of thera-peutic plasma exchange (TPE) as standard treatment of TTP.
Patients and Methods
We describe our experience in the use of solvent/detergent-treated plasma (SDP) for TPE in TTP. We retrospectively analyzed acute TTP epi-sodes in 8 patients (mean age = 27 years, range 18–44 years) treated with TPE using SDP with regard to tolerability and efficacy.
All 8 patients were positive for anti-ADAMTS-13 antibody. Seven out of 8 had a se-vere ADAMTS-13 deficiency. All patients responded rapidly to SDP TPE with an increase in platelet count to above 150 × 109/l. Hemolytic anemia disappeared over the treatment period. Approximately 2,000 l SDP were used for more than 500 treatments. Treatment with SDP was well tolerated; none of the patients experienced an adverse drug reaction after exposure to SDP. No major complications occurred even after multiple TPE.
Our investigations suggest that TPE using SDP as replacement fluid is an effective treatment for TTP. The data described also indicate that SDP might offer the benefit of reducing adverse drug reactions.
Thrombotic thrombocytopenic purpura; TTP; Therapeutic plasma exchange; TPE; Solvent/detergent-treated plasma; SDP; ADAMTS-13
The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13−/− mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in αIIbβ3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.
Thrombotic thrombocytopenia purpura (TTP) caused by a deficiency in ADAMTS-13 activity is considered to involve a subset of thrombotic microangiopathy (TMA). Although concept of TTP is included under the umbrella of TMA, discrimination of TTP from TMA is occasionally difficult in an autoimmune disorder. Herein, we report a case with TTP associated with systemic lupus erythematosus (SLE). In this case, it was difficult to discriminate TTP from TMA and the measurement of ADAMTS-13 activity was useful for obtaining an accurate diagnosis. SLE patients having thrombocytopenia in complication with anemia should be considered a monitoring of ADAMTS-13 activity even though the patients lacked symptoms of TTP related to the microvascular coagulation.
Recent advances have demonstrated that thrombotic thrombocytopenic purpura (TTP), characterized by widespread thrombosis in the arterioles and capillaries, is caused by deficiency of a circulating zinc metalloprotease ADAMTS13. Two types of TTP are recognized: Autoimmune TTP caused by inhibitory antibodies of ADAMTS13 and hereditary TTP in association with genetic mutations of the ADAMTS13 gene. This article reviews the characteristic and function of ADAMTS13, the mechanism by which ADAMTS13 deficiency may cause thrombosis, and the causes of ADAMTS13 deficiency. It also discusses how the new knowledge may improve the diagnosis and treatment of this previously mysterious disorder.
Thrombotic thrombocytopenic purpura; von Willebrand factor; ADAMTS13; Shear stress; Hemolytic uremic syndrome
We sought to describe clinical and laboratory findings for a large cohort of patients with thienopyridine-associated thrombotic thrombocytopenic purpura (TTP).
The thienopyridine derivatives, ticlopidine and clopidogrel, are the 2 most common drugs associated with TTP in databases maintained by the U.S. Food and Drug Administration (FDA).
Clinical reports of TTP associated with clopidogrel and ticlopidine were identified from medical records, published case reports, and FDA case reports (n = 128). Duration of thienopyridine exposure, clinical and laboratory findings, and survival were recorded. ADAMTS13 activity (n = 39) and inhibitor (n = 30) were measured for a subset of individuals.
Compared with clopidogrel-associated TTP cases (n = 35), ticlopidine-associated TTP cases (n = 93) were more likely to have received more than 2 weeks of drug (90% vs. 26%), to be severely thrombocytopenic (84% vs. 60%), and to have normal renal function (72% vs. 45%) (p < 0.01 for each). Compared with TTP patients with ADAMTS13 activity >15% (n = 13), TTP patients with severely deficient ADAMTS13 activity (n = 26) were more likely to have received ticlopidine (92.3% vs. 46.2%, p < 0.003). Among patients who developed TTP >2 weeks after thienopyridine, therapeutic plasma exchange (TPE) increased likelihood of survival (84% vs. 38%, p < 0.05). Among patients who developed TTP within 2 weeks of starting thienopyridines, survival was 77% with TPE and 78% without.
Thrombotic thrombocytopenic purpura is a rare complication of thienopyridine treatment. This drug toxicity appears to occur by 2 different mechanistic pathways, characterized primarily by time of onset before versus after 2 weeks of thienopyridine administration. If TTP occurs after 2 weeks of ticlopidine or clopidogrel therapy, therapeutic plasma exchange must be promptly instituted to enhance likelihood of survival.
The zinc metalloprotease ADAMTS13 is a multidomain protein that cleaves von Willebrand Factor (VWF) and is implicated in Thrombotic Thrombocytopenic Purpura (TTP) pathogenesis. Understanding the mechanism of this protein is an important goal. Conformation sensitive antibodies have been used to monitor protein conformation and to decipher the molecular mechanism of proteins as well as to distinguish functional and non-functional mutants.
We have characterized several antibodies against ADAMTS13, both monoclonal and polyclonal. We have used flow cytometry to estimate the binding of these antibodies to ADAMTS13 and demonstrate that antibodies raised against the TSP and disintegrin domains detect conformation changes in the ADAMTS13. Thus for example, increased binding of these antibodies was detected in the presence of the substrate (VWF), mainly at 37°C and not at 4°C. These antibodies could also detect differences between wild-type ADAMTS13 and the catalytically deficient mutant (P475S). The flow cytometry approach also allows us to estimate the reactivity of the antibody as well as its apparent affinity.
Our results suggest that these antibodies may serve as useful reagents to distinguish functional and non-functional ADAMTS13 and analyze conformational transitions to understand the catalytic mechanism.
Deficiency of ADAMTS13 results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in the present study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (~5–70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor compared to the Adamts13−/− mice expressing eGFP. Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13−/− expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13−/− mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.