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1.  A shear-based assay for assessing plasma ADAMTS13 activity and inhibitor in patients with thrombotic thrombocytopenic purpura 
Transfusion  2011;51(7):1580-1591.
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.
PMCID: PMC3168518  PMID: 21251003
2.  Mechanisms of microvascular thrombosis in thrombotic thrombocytopenic purpura 
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.
PMCID: PMC3158997  PMID: 19180123
ADAMTS13; shear stress; thrombosis; thrombotic thrombocytopenic purpura; von Willebrand factor
3.  ADAMTS13-binding IgG are present in patients with thrombotic thrombocytopenic purpura 
Thrombosis and haemostasis  2006;95(5):886-892.
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.
PMCID: PMC2603189  PMID: 16676082
ADAMTS13; thrombotic thrombocytopenic purpura; antibody
4.  Long term follow up of congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome) on hemodialysis for 19 years: a case report 
BMC Nephrology  2013;14:156.
Thrombotic thrombocytopenic purpura (TTP) is frequently associated with renal abnormalities, but there have been few reports about renal abnormalities in patients with hereditary TTP. In particular, little is known about the long-term prognosis of patients with childhood-onset congenital TTP.
Case presentation
We report a Japanese patient with congenital TTP (Upshaw–Schulman syndrome) who was followed for 19 years after initiation of hemodialysis when he was 22 years old. At the age of 6 years, the first episode of purpura, thrombocytopenia, and proteinuria occurred without any precipitating cause. He underwent living-related donor kidney transplantation from his mother, but the graft failed after 5 months due to recurrence of TTP. Even after resection of the transplanted kidney and resumption of regular hemodialysis, TTP became refractory to infusion of fresh frozen plasma (FFP). Therefore, splenectomy was performed and his disease remained in remission for 10 years. However, TTP recurred at the age of 39 years. Plasma activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I domain 13) was less than 3%, while ADAMTS13 inhibitor was not detected (< 0.5 Bethesda units/mL). The patient died suddenly after hemodialysis at the age of 41 years. Subsequent genetic analysis of this patient and his parents revealed two different heterozygous mutations of ADAMTS13, including a missense mutation in exon 26 (c.T3650C causing p.I1217T) inherited from his father and a missense mutation in exon 21 (c.G2723A causing p.C908Y) inherited from his mother. The former mutation has not been detected before in Japan, while the latter mutation is common in Japan. A retrospective review showed that serum C3 levels were consistently low while C4 levels were normal during follow-up, and C3 decreased much further during each episode of TTP.
Congenital TTP was diagnosed from the clinical, biochemical, and genetic findings. Infusion of FFP controlled each thrombotic episode, but the effect was limited and of short duration. Review of the complement profile in this patient suggested that a persistently low serum C3 level might be associated with refractory TTP and a worse renal prognosis.
PMCID: PMC3729817  PMID: 23870247
Congenital thrombotic thrombocytopenic purpura; ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I domain 13); Chronic hemodialysis; Complement activation; C3; Alternative pathway
5.  Novel Recombinant Glycosylphosphatidylinositol (GPI)-Anchored ADAMTS13 and Variants for Assessment of Anti-ADAMTS13 Autoantibodies in Patients with Thrombotic Thrombocytopenic Purpura 
Thrombosis and haemostasis  2011;106(5):947-958.
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.
PMCID: PMC3208768  PMID: 21901237
von Willebrand factor cleaving protease; thrombotic microangiopathies; diagnostic test; autoantibody
6.  Rituximab for chronic recurring thrombotic thrombocytopenic purpura: a case report and review of the literature 
British journal of haematology  2004;124(6):787-795.
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.
PMCID: PMC3153075  PMID: 15009067
thrombotic thrombocytopenic purpura; rituximab; plasma exchange
Transfusion  2011;51(10):2237-2243.
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.
PMCID: PMC3151471  PMID: 21470236
8.  Inhibitory autoantibodies against ADAMTS-13 in patients with thrombotic thrombocytopenic purpura bind ADAMTS-13 protease and may accelerate its clearance in vivo 
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.
PMCID: PMC2577225  PMID: 16879212
a disintegrin and metalloprotease with thrombospondin type 1 repeats; autoimmune disorder; thrombotic microangiopathy; von Willebrand factor
9.  Pregnancy complications in acquired thrombotic thrombocytopenic purpura: a case–control study 
Pregnant women with a history of acquired thrombotic thrombocytopenic purpura (TTP) are considered at risk for disease recurrence and might be at risk for miscarriage, similar to other autoimmune disorders. However, the exact entity of these risks and their causes are unknown. The aim of this study was to evaluate risk factors associated with adverse pregnancy outcome, in terms of both gravidic TTP and miscarriage, in women affected by previous acquired TTP.
We conducted a nested case–control study in women with a history of acquired TTP enrolled in the Milan TTP registry from 1994 to October 2012, with strict inclusion criteria to reduce referral and selection bias.
Fifteen out of 254 women with acquired TTP were included, namely four cases with gravidic TTP, five with miscarriage, and six controls with uncomplicated pregnancy. In the cases, ADAMTS13 activity levels in the first trimester were moderately-to-severely reduced (median levels <3% in gravidic TTP and median levels 20% [range 14-40%] in the women with miscarriage) and anti-ADAMTS13 antibodies were invariably present, while in the control group ADAMTS13 activity levels were normal (median 90%, range 40-129%), with absence of detectable anti-ADAMTS13 antibodies. Reduced levels of ADAMTS13 activity (<25%) in the first trimester were associated with an over 2.9-fold increased risk for gravidic TTP and with an over 1.2-fold increased risk for miscarriage (lower boundary of the confidence interval of the odds ratio). In addition, the presence of anti-ADAMTS13 antibodies during pregnancy was associated with an over 6.6-fold increased risk for gravidic TTP and with an over 4.1-fold increased risk for miscarriage.
ADAMTS13 activity evaluation and detection of anti-ADAMTS13 antibody could help to predict the risk of complications in pregnant women with a history of acquired TTP.
PMCID: PMC4279798  PMID: 25431165
ADAMTS13; Anti-ADAMTS13 antibodies; Miscarriage; Pregnancy; Thrombotic thrombocytopenic purpura
10.  Two Mechanistic Pathways for Thienopyridine-Associated Thrombotic Thrombocytopenic Purpura 
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.
PMCID: PMC3167088  PMID: 17868804
11.  Pathophysiology of thrombotic thrombocytopenic purpura 
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.
PMCID: PMC3159000  PMID: 20058209
TTP; von Willebrand factor; ADAMTS13; Shear stress; Microvascular thrombosis
12.  Management of thrombotic thrombocytopenic purpura: current perspectives 
Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy which causes significant morbidity and mortality unless promptly recognized and treated. The underlying pathogenesis of TTP is a severe deficiency in ADAMTS13 activity, a metalloprotease that cleaves ultralarge von Willebrand factor multimers. This deficiency is either autoantibody mediated (acquired TTP) or due to deleterious mutations in the gene encoding ADAMTS13 (congenital TTP). The elucidation of this disease mechanism has reinforced the rationale and place of current therapies (eg, plasma exchange) as well as providing a basis for the prospective evaluation of immunotherapy with rituximab in addition to classic immunosuppression (eg, corticosteroid) in autoantibody-mediated TTP. This review discusses the current evidence base for therapeutic interventions in acquired and congenital TTP as well as providing a practical approach to the other aspects of investigation and management for which a firm evidence base is lacking. Novel agents that are currently being evaluated in prospective trials and future directions of therapy are also discussed which are expected to make an important contribution to improving outcomes in patients with TTP.
PMCID: PMC3921093  PMID: 24523598
TTP; rituximab; ADAMTS13
13.  The Kidney in Thrombotic Thrombocytopenic Purpura 
Minerva medica  2007;98(6):731-747.
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.
PMCID: PMC2430013  PMID: 18299685
Thrombotic thrombocytopenic purpura; ADAMTS13; von Willebrand factor; Thrombosis; Renal failure; Shear stress
14.  Enzymatically Active ADAMTS13 Variants Are Not Inhibited by Anti-ADAMTS13 Autoantibodies 
The Journal of biological chemistry  2005;280(48):39934-39941.
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.
PMCID: PMC2582217  PMID: 16203734
15.  A case of thrombotic thrombocytopenic purpura induced by acute pancreatitis 
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.
PMCID: PMC3333833  PMID: 22536085
thrombotic thrombocytopenic purpura; ADAMTS13; acute pancreatitis; plasma exchange
16.  Structure-function and regulation of ADAMTS13 protease 
ADAMTS13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS13 activity are emerging risk factors for developing myocardial and cerebral infarction, preeclampsia, and malignant malaria. Moreover, Adamts13−/− mice develop more severe inflammatory responses, leading to increased ischaemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS13 (TSP1 2–8 repeats and CUB domains) may function as a disulphide bond reductase to prevent an elongation of ultra large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.
PMCID: PMC3713533  PMID: 23809107
17.  ADAMTS-13 in the Diagnosis and Management of Thrombotic Microangiopathies 
Thrombotic microangiopathies (TMAs) comprise a group of distinct disorders characterized by microangiopathic hemolytic anemia, thrombocytopenia, and microvascular thrombosis. For many years distinction between these TMAs, especially between thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), remained purely clinical and hard to make. Recent discoveries shed light on different pathogenesis of TTP and HUS. Ultra-large von Willebrand factor (UL-VWF) platelet thrombi, resulting from the deficiency of cleavage protease which is now known as ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), were found to cause TTP pathology, while Shiga toxins or abnormalities in regulation of the complement system cause microangiopathy and thrombosis in HUS. TMAs may appear in various conditions such as pregnancy, inflammation, malignancy, or exposure to drugs. These conditions might cause acquired TTP, HUS, or other TMAs, or might be a trigger in individuals with genetic predisposition to ADAMTS-13 or complement factor H deficiency. Differentiation between these TMAs is highly important for urgent initiation of appropriate therapy. Measurement of ADAMTS-13 activity and anti-ADAMTS-13 antibody levels may advance this differentiation resulting in accurate diagnosis. Additionally, assessment of ADAMTS-13 levels can be a tool for monitoring treatment efficacy and relapse risk, allowing consideration of therapy addition or change. In the past few years, great improvements in ADAMTS-13 assays have been made, and tests with increased sensitivity, specificity, reproducibility, and shorter turnaround time are now available. These new assays enable ADAMTS-13 measurement in routine clinical diagnostic laboratories, which may ultimately result in improvement of TMA management.
PMCID: PMC4222415  PMID: 25386342
ADAMTS-13; aHUS; HUS; thrombotic microangiopathies; TTP; UL-VWF; Von Willebrand factor
18.  Complement activation associated with ADAMTS13 deficiency in human and murine thrombotic microangiopathy 
This study addressed the contribution of ADAMTS13-deficiency to complement activation in thrombotic thrombocytopenic purpura (TTP). Renal tissue and blood samples were available from 12 TTP patients. C3 and C5b-9 deposition were demonstrated in the renal cortex of two TTP patients, by immunofluorescence and immunohistochemistry, respectively. C3 was also demonstrated in the glomeruli of Shiga toxin-2 treated Adamts13−/− mice (n=6 of 7) but less in mice that were not Shiga toxin-2-treated (n=1 of 8, p<0.05) or wild-type mice (n=0 of 7). TTP patient plasma (n=9) contained significantly higher levels of complement-coated endothelial microparticles than control plasma (n=13), as detected by flow cytometry. Exposure of histamine-stimulated primary glomerular endothelial cells to platelet-rich-plasma from patients, or patient platelet-poor-plasma combined with normal platelets, in a perfusion system, under shear, induced C3 deposition on von Willebrand factor (VWF)-platelet strings (on both VWF and platelets) and on endothelial cells. Complement activation occurred via the alternative pathway. No C3 was detected when cells were exposed to TTP plasma that was pre-incubated with EDTA or heat-inactivated, or to control plasma. In the perfusion system patient plasma induced more release of C3- and C9-coated endothelial microparticles compared to control plasma. The results indicate that the microvascular process induced by ADAMTS13 deficiency triggers complement activation on platelets and the endothelium, which may contribute to formation of thrombotic microangiopathy.
PMCID: PMC3750088  PMID: 23878316
19.  Ribosomal and Immune Transcripts Associate with Relapse in Acquired ADAMTS13-Deficient Thrombotic Thrombocytopenic Purpura 
PLoS ONE  2015;10(2):e0117614.
Approximately 40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe acquired ADAMTS13 deficiency experience one or more relapses. Risk factors for relapse other than severe ADAMTS13 deficiency and ADAMTS13 autoantibodies are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation. This cross-sectional study asked whether autoantibodies against RNA-binding proteins or peripheral blood gene expression profiles measured during remission are associated with history of prior relapse in acquired ADAMTS13-deficient TTP. Peripheral blood from 38 well-characterized patients with autoimmune ADAMTS13-deficient TTP in remission was examined for autoantibodies and global gene expression. A subset of TTP patients (9 patients, 24%) exhibited a peripheral blood gene signature composed of elevated ribosomal transcripts that associated with prior relapse. A non-overlapping subset of TTP patients (9 patients, 24%) displayed a peripheral blood type I interferon gene signature that associated with autoantibodies to RNA-binding proteins but not with history of relapse. Patients who had relapsed bimodally expressed higher HLA transcript levels independently of ribosomal transcripts. Presence of any one potential risk factor (ribosomal gene signature, elevated HLA-DRB1, elevated HLA-DRB5) associated with relapse (OR = 38.4; p = 0.0002) more closely than any factor alone or all factors together. Levels of immune transcripts typical of natural killer (NK) and T lymphocytes positively correlated with ribosomal gene expression and number of prior episodes but not with time since the most recent episode. Flow cytometry confirmed elevated expression of cell surface markers encoded by these transcripts on T and/or NK cell subsets of patients who had relapsed. These data associate elevated ribosomal and immune transcripts with relapse history in acquired, ADAMTS13-deficient TTP.
PMCID: PMC4324966  PMID: 25671313
20.  A case of congenital TTP presenting with microganiopathy in adulthood 
BMC Hematology  2014;14(1):16.
Congenital thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman Syndrome is a rare inherited deficiency of ADAMTS13. Unlike the more common acquired TTP which is characterized by an acquired inhibitor of ADAMTS13, patients with congenital TTP have an absolute deficiency of ADAMTS13 without an inhibitor. Congenital TTP generally presents in infancy with repeat episodes of acute hemolysis and evidence of microangiopathy, these episodes are usually triggered by illness or physiological stress. Congenital TTP can be effectively treated with plasma infusion either during acute episodes or on a prophylactic schedule to prevent episodes.
Case presentation
We present a case of a 25 year old Caucasian woman with no know family history of hematological disorders with congenital TTP. She presented with episodes of hemolysis since infancy, but without clear evidence of microangiopathy until the age of 25. At presentation to our center the patient was documented to have thrombocytopenia, elevated creatinine, and schistocytes. She was initially treated with plasma infusion at a rate of 60 ml/hr continuously for a 24 hr period with resolution of her thrombocytopenia and hemolysis. At the time of writing this article she is maintained on a prophylactic schedule of biweekly plasma infusions at 10 mg/kg and is maintaining a normal platelet count with no evidence of hemolysis.
Congenital TTP is a rare condition, and the above case is atypical as the patient did not present with clear evidence of microangiopathy until adulthood. Although this a rare condition it is important for physicians to be aware of as it, especially the possibility of atypical presentations, as the condition is potentially fatal and effective treatment exists.
PMCID: PMC4168056  PMID: 25243071
TTP; ADAMTS13; Hemolysis; Upshaw-Schulman; Microangiopathy
21.  Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes* 
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.
PMCID: PMC2613173  PMID: 18957966
22.  Inhibition of platelet-aggregating activity in thrombotic thrombocytopenic purpura plasma by normal adult immunoglobulin G. 
Journal of Clinical Investigation  1984;73(2):548-555.
Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of autologous and homologous platelets, and effect which was inhibited by normal plasma. IgG purified from seven normal adults at a concentration of 0.7 mg/ml completely inhibited the platelet aggregation induced by plasma obtained from two TTP patients with active disease. The inhibition of platelet aggregation by human adult IgG was concentration dependent, and the inhibitory activity of human IgG was neutralized by rabbit antihuman IgG. Fab fragments inhibited the TTP plasma-induced platelet aggregation as well as intact IgG, whereas Fc fragments had no effect. Platelet aggregation caused by ADP, collagen, epinephrine, or thrombin was not affected by purified human IgG. The prior incubation of IgG with TTP plasma caused a significantly greater reduction of platelet aggregation by TTP plasma than that of IgG and platelet suspension, suggesting that the IgG inhibits TTP plasma-induced platelet aggregation through direct interaction with platelet aggregating factor in TTP plasma. IgG obtained initially from five infants and young children under the age of 4 yr did not possess any inhibitory activity. When one of the children reached 3 yr of age, his IgG inhibited the aggregation induced by one TTP plasma, but not that caused by another plasma. The IgG procured from the same boy at 4 yr of age inhibited the aggregation induced by both TTP plasmas. The IgG purified from the TTP plasma during active disease failed to inhibit the aggregation caused by the same plasma. After recovery, however, the IgG effectively inhibited aggregation. These observations suggest that platelet-aggregating factors present in the TTP plasma are heterogeneous in nature and that the IgG present in the normal adult plasma, which inhibits the TTP plasma-induced platelet aggregation, may be partially responsible for the success of plasma infusion therapy in TTP.
PMCID: PMC425047  PMID: 6538207
23.  Acquired Idiopathic ADAMTS13 Activity Deficient Thrombotic Thrombocytopenic Purpura in a Population from Japan 
PLoS ONE  2012;7(3):e33029.
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.
PMCID: PMC3299727  PMID: 22427934
American journal of hematology  2011;86(9):743-751.
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.
PMCID: PMC3420338  PMID: 21850657
infection; thrombotic thrombocytopenic purpura; TTP; hemolytic uremic syndrome; HUS; ADAMTS13
25.  Antibody Inhibitors to von Willebrand Factor Metalloproteinase and Increased Binding of von Willebrand Factor to Platelets in Ticlopidine-Associated Thrombotic Thrombocytopenic Purpura 
Annals of internal medicine  2000;132(10):794-799.
Thrombotic thrombocytopenic purpura (TTP) affects 1 in 1600 to 1 in 5000 patients who receive ticlopidine, but little is known about the pathogenesis of this complication.
To investigate whether von Willebrand factor (vWF), which has been associated with idiopathic TTP, is involved in the pathogenesis of ticlopidine-associated TTP.
Case series.
Three tertiary care, university-affiliated medical centers.
Seven patients who developed TTP 2 to 7 weeks after initiation of ticlopidine therapy. Controls were 7 consecutive patients without thrombocytopenia who had been receiving ticlopidine for 3 to 5 weeks and 10 randomly selected hospitalized patients.
Platelet-bound vWF in patients’ EDTA-anticoagulated whole blood samples; vWF proteinase activity in patients’ plasma samples; inhibitory activity of IgG isolated from patients’ plasma samples against the proteinase from the controls’ plasma samples; and vWF multimeric patterns in patients’ EDTA-anticoagulated plasma samples.
Binding of vWF to single platelets was increased in the three patients tested during the most thrombocytopenic phase of TTP episodes. Initial plasma samples from all seven patients lacked the largest vWF multimers and were severely deficient in vWF metalloproteinase. IgG molecules, isolated from plasma samples of five patients, inhibited metalloproteinase in plasma samples from the controls. In patients examined, these abnormalities resolved upon the remission that accompanied plasma exchange and discontinuation of ticlopidine therapy.
In the patients who developed ticlopidine-associated TTP, autoantibodies to the vWF metalloproteinase were formed; this led to the same type of vWF abnormalities observed in patients with idiopathic acute TTP. The findings suggest that failure to process large and unusually large vWF multimers in vivo caused binding of vWF to platelets, systemic platelet thrombosis, and TTP.
PMCID: PMC3152977  PMID: 10819702

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