ADAMTS13 Inhibitor Research Articles

Thrombotic thrombocytopenic purpura: The role of ADAMTS13.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon, life-threatening disease requiring prompt diagnosis and initiation of therapeutic plasma exchange to improve patient survival. However, diagnosis is often difficult because of atypical presentations and signs and symptoms that resemble other conditions. Measurements of ADAMTS13 activity, ADAMTS13 inhibitor, and ADAMTS13 autoantibody are useful for diagnosing TTP, guiding therapy, and predicting relapse.

Validation of a panel of ADAMTS13 assays for diagnosis of thrombotic thrombocytopenic purpura: activity, functional inhibitor, and autoantibody test.

Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone). A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%. ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study.

Proteasome inhibitor associated thrombotic microangiopathy.

A variety of medications have been implicated in the causation of thrombotic microangiopathy (TMA). Recently, a few case reports have emerged of TMA attributed to the proteasome inhibitors (PI) bortezomib and carfilzomib in patients with multiple myeloma. The aim of this case series was to better characterize the role of PI in the etiology of drug-induced TMA. We describe eleven patients from six medical centers from around the world who developed TMA while being treated with PI. The median time between medication initiation and diagnosis of TMA was 21 days (range 5 days to 17 months). Median laboratory values at diagnosis included hemoglobin-7.5 gdL(-1) , platelet count-20 × 10(9) /L, LDH-698 UL(-1) , creatinine-3.12 mgdL(-1) . No patient had any other cause of TMA, including ADAMTS13 inhibition, other malignancy or use of any other medication previously associated with TMA. Nine patients had resolution of TMA without evidence of hemolysis after withdrawal of PI. Two patients had stabilization of laboratory values but persistent evidence of hemolysis despite medication withdrawal. One patient had recurrence of TMA with rechallenge of PI. There is a strong level of evidence that PI can cause DITMA. In evaluating patients with suspected TMA, PI use should be recognized as a potential etiology, and these medications should be discontinued promptly if thought to be the cause of TMA. Am. J. Hematol. 91:E348-E352, 2016. © 2016 Wiley Periodicals, Inc.

Thrombotic thrombocytopenic purpura associated with Klebsiella pneumonia in the background of alcoholic liver cirrhosis

A 75-year-old male patient with alcoholic liver cirrhosis was emergently admitted due to systemic convulsion and Klebsiella pneumonia. He was referred to us due to severe thrombocytopenia (7,000/μl), and we suspected thrombotic thrombocytopenicpurpura (TTP) considering the coexistence of hemolytic findings and neurological symptoms. We promptly performed plasma exchange and administration of corticosteroids, resulting in full recovery of symptoms and laboratory findings in a week. Thediagnosis of TTP was confirmed by severely decreased ADAMTS13 activity (less than 0.5%) and detection of ADAMTS13 inhibitor. In this case, we speculated that TTP was triggered by Klebsiella pneumonia in the background of advanced alcoholicliver cirrhosis. This is the first report describing the complication of Klebsiella pneumonia and TTP. It is important to be awarethat patients complicated with severe liver disease could be vulnerable to TTP.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model.

Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.

Diagnosis and management of thrombotic thrombocytopenic purpura (TTP) in Australia: findings from the first 5 years of the Australian TTP/thrombotic microangiopathy registry.

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA). In 2009, the Australian TTP/TMA registry was established to collect data on patients presenting with TTP/TMA throughout Australia. To summarise information on the diagnosis and management of patients with TTP collected in the first 5 years (2009-2014) of the Australian TTP registry. Registry data from June 2009 to October 2014 were reviewed. Fifty-seven patients were identified with TTP (defined as ADAMTS13 activity <10%), accounting for 72 clinical episodes. ADAMTS13 inhibitor testing was performed in nine out of 57 patients (16%), reflecting the limited availability of accredited testing facilities. Sixty-seven out of 72 episodes were treated with therapeutic plasma exchange (PEx) using cryodepleted plasma (40% of episodes), fresh frozen plasma (36%) or a mixture (22%). Median exposure to plasma products was 55.9 L. PEx was commenced ≥2 days from stated diagnosis in 15% of episodes. Adverse reactions to PEx were common with documented allergic reactions (including life threatening) in 21% of episodes. Adjunctive immunosuppression was documented in 76% of episodes (corticosteroid 71% and rituximab 39%). Platelet transfusion was administered in 15% of episodes. Data from the Australian TTP/TMA registry suggest a heterogenous approach to the diagnosis and management of TTP in Australia over the assessed period. These observations highlight areas for improvement and standardisation of practice, including comprehensive diagnostic testing, more immediate access to PEx and a more uniform approach to adjunctive immunosuppression and supportive care.

Thrombotic Thrombocytopenic Purpura (TTP) Management at Foothills Medical Centre: A Retrospective Analysis Between 2005-2010 (Tertiary Centre, Calgary)

TTP is a rare condition of microangiopathic hemolytic anemia and thrombocytopenia with a reported incidence of 4 cases per million people. Untreated mortality is reported to be as high as 90%; plasma exchange (PLEX) has significantly reduced this to 28%. Given the heterogeneity in presentation and severity at diagnosis, there is a wide range of practice with regards to exchanges, management of refractory disease and relapse. The British Committee for Standards in Hematology (BCSH) released clinical guidelines first in 2003, updated in 2012 outlining evidence based approach to practice. Our goal was to examine clinical practices and effect on outcomes at our tertiary centre during 2005-2010, between release of the first and second edition guidelines.Patients were retrospectively identified by searching for the TTP diagnostic code with admission dates between 2005-2010. They were excluded if they were not primarily treated in Calgary, or their thrombotic microangiopathy (TMA) was felt to be due to another diagnosis.Thirty eight patients were identified, with an average age of 48.8 years, and mostly female (men=8 patients, 21%). Ten patients were excluded for alternate diagnoses or main treatment outside Calgary. TTP was classified as primary in 15/45 (39%), and in 33 patients (87%) this was their first episode. Secondary causes included underlying autoimmune disease (24% of patients), malignancy (16% of patients), and pregnancy (4% of patients). 4% of patients were receiving active chemotherapy at the time of diagnosis of TTP. ADAMTS13 level and inhibitor (antibodies) were only examined in 10 cases (26%), and reported in fewer. Measurement of antigen, activity and inhibitors is strongly emphasized in the literature for accurate diagnosis of TTP compared with hemolytic uremic syndrome (HUS), other TMA or congenital TTP, and this was underutilized in this tertiary centre likely due to laboratory availability.Duration in hospital was often long, with an average of 29 days. This is likely due to the frequency of refractory disease, seen in more than half of patients (23/38, 61%). 24% of cases were fatal; this result is consistent with previously reported mortality rates in the era of PLEX. 5 of 9 fatalities were associated with malignancy where there is felt to be no benefit from PLEX, although interestingly 2 patients with malignancy received 5 to 7 days of PLEX, recovered and were discharged from hospital. Two fatalities had a delay of 48 hours or more to initiation of PLEX through misdiagnosis, one developed severe neurologic symptoms during line placement and the other presented atypically with hematuria. The most common complications of TTP included bleeding (16 patients, 42%), neurologic changes without stroke (16 patients, 42%), requirement for hemodialysis (9 patients, 24%), and radiologically identified stroke (6 patients, 16%).35 patients (92%) received PLEX, and the average number of days on PLEX was 16. Twice daily exchange at initiation or exacerbation was common (29%), as were tapers (60%), although literature now suggests tapering does not reduce relapses. Steroids are recommended in most cases for immunosuppression; only 21 of our patients (55%) received these in hospital. Secondary immunosuppression was occasionally instituted in refractory or relapsing cases including cyclophosphamide (13 patients, 34%), rituximab (5 patients, 13%), mycophenolate mofetil and vincristine (1 patient each, 3%). Rituximab is now routinely used in initial treatment to decrease relapses, for refractory disease, and occasionally as prophylaxis with low ADAMTS13 in asymptomatic patients, and other agents generally recommended against given its efficacy and safety, but this was not standardized in our group.Limitations for this project include small patient numbers, the change in definitions of HUS, TMA and TTP over time, and the retrospective nature of patient selection and data collection.In summary, diagnostic and treatment strategies for TTP have become significantly more standardized in the past 10 years. A protocolized strategy for initial investigations, PLEX and immunosuppressive therapies in line with previously published guidelines would be useful in our institution, potentially reduce relapses and improve rapidity of patient response, however our mortality statistics appear similar to those previously published. DisclosuresNo relevant conflicts of interest to declare.

Plasma Levels of Human Neutrophil Peptides and Complement Activation Markers in Patients with Acquired Autoimmune Thrombotic Thrombocytopenic Purpura

Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome, resulting from autoantibodies against the metalloprotease ADAMTS13. Autoantibodies bind and inhibit plasma ADAMTS13 activity, leading to exaggerated platelet agglutination and thrombus formation in small arteries and capillaries. However, plasma ADAMTS13 deficiency or the presence of anti-ADAMTS13 autoantibodies is not sufficient to cause the disease. Infection or inflammation often precedes the acute onset of TTP. We hypothesize that neutrophil activation or complement activation vial alternative pathway may be the trigger for TTP. In this study, plasma samples were collected prior to plasma exchange from 58 adult patients with acute TTP between 2006 and 2015 at the University of Alabama at Birmingham Medical Center. All patients were diagnosed having TTP on the basis of severe thrombocytopenia, microangiopathic hemolytic anemia, and severe deficiency of plasma ADAMTS13 activity (less than 10%) and the presence of ADAMTS13 inhibitors. All patients were treated by plasma exchange. Thirty plasma samples from healthy individuals were collected for controls. Plasma levels of human neutrophil peptides (HNP-1, -2, and -3) released from activated neutrophils and alternative pathway complement activation markers (iC3b, C4d, C5b-9, and Bb) were determined by enzyme-linked immunosorbent assays (ELISAs). We showed that plasma levels of HNP-1, -2, and -3 in TTP patients were elevated on average by 10-fold when compared with those in the healthy controls (44.12 ± 39.53 ng/ml vs. 3.54 ± 2.97 ng/ml, means ± SD) (p <0.0001). Whereas plasma levels of Bb and iC3b were only modestly increased in TTP patients (2.94 ± 1.82 µg/ml vs. 1.39 ± 0.34 µg/ml for Bb, p <0.0001, and 16.21 ± 9.71 µg/ml vs. 12.13 ± 3.07 µg/ml for iC3b, p =0.028). No significant difference was detected in the plasma levels of C4d and C5b-9 between TTP patients and healthy controls (p >0.05). To differentiate HNP-1 from HNP-2 or HNP-3, we developed a LC/MS mass spectrometric assay and showed that all subtypes of HNPs in TTP patients were significantly elevated when compared with those in the healthy controls (p<0.001). In general, there was good concordance between ELISA and LC/MS MS in the measurement of all three HNPs. These results demonstrate that innate immunity (i.e. activation of neutrophils) and to a lesser degree the activation of complements via alternative pathway may play a role in pathogenesis of TTP in light of severe ADAMTS13 deficiency. Our findings provide rationales for developing novel targeted therapies for acquired TTP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Investigator-Initiated Phase 2 Trial of Rituximab in Adult Japanese Patients with Treatment-Resistant Acquired Thrombotic Thrombocytopenic Purpura

About 500 persons in Japan are estimated to have thrombotic thrombocytopenic purpura (TTP), which is a life-threatening disorder. In contrast with the USA and EU, off-label use of rituximab for TTP is not allowed by the Japanese public health insurance system. We therefore have limited experience of the effects of rituximab in Japanese patients with adult TTP and have not investigated possible ethnicity-related differences in its effects. To evaluate the safety and efficacy of rituximab in Japanese adult patients with TTP, we conducted an investigator-initiated phase 2 clinical trial in Japan between January and December of 2014. This study was sponsored by the Japanese government and the study design was based instructions from the regulatory agency, PMDA (Phamaceuticals and Medical Devices Agency). Adult Japanese patients with acquired TTP that had proven refractory to at least five plasma exchanges (PEX) or had serum concentrations of more than 2 BU/mL of ADAMTS13 inhibitor or both were eligible for this study. Rituximab was infused at 375 mg/m2 weekly for 4 weeks. The study protocol allowed use of both plasma exchange and corticosteroids with the investigational drug. Thirteen patients were registered and seven found to be eligible and treated with rituximab. All seven patients were evaluable for safety and six for efficacy. Three of these six (50%) were women; the median age was 41.5 years. Median platelet counts at baseline were 22 × 109/L. All of these six patients were refractory to previous PEX and ADMTS13 inhibitor concentrations were higher than 2 BU/mL in two (33%) of them. All six patients no longer required PEX after receiving rituximab treatment. Five (83%) and two (33%) patients achieved platelet counts of 100 × 109/L and 150 × 109/L 4 weeks after initiating rituximab, respectively. Median platelet counts at week 4 after commencing rituximab were 136 × 109/L (range: 64-201 × 109/L). The median time from initiating PEX to achieving platelet counts over 150 × 109/L was 22 days. By the study endpoint (4 weeks after initiating rituximab treatment), ADAMTS13 activity had normalized to 77.3% from 3.4%, accompanied by disappearance of ADAMTS13 inhibitor. Both anemia and neurological impairment also improved in all patients. The median percentages of peripheral blood CD20-positive B cells were 22.9% and 0.2% before and at the completion of treatment with rituximab, respectively. Serum IgG decreased slightly from 1,116 mg/dL at baseline to 921 mg/dL 4 weeks after rituximab treatment. Five of seven patients experienced adverse events, two of which were severe; namely septic shock and cytomegalovirus infection; no patients died. No unpublished severe adverse events were noted. No recurrence of TTP occurred during a median follow-up of 7 months. These data suggest that rituximab is safe and effective for acute refractory/high risk acquired TTP in Japanese patients and that there are no ethnic differences in effectiveness or safety of rituximab for adult TTP. However, there were too few patients to draw any firm conclusions. Further investigation is necessary to optimize the treatment schedule of rituximab. We are preparing to apply to the Japanese regulatory agency for approval of new indications for rituximab for TTP. DisclosuresOff Label Use: Investigational drug, rituximab, was provided by Zenyaku Kogyo Co. Ltd.. Matsumoto:Baxter Bioscience: Consultancy.

N-Acetylcysteine Treatment in Two Patients with Relapsed Thrombotic Thrombocytopenic Purpura Increased ADAMTS13 Activity, Free Thiol Concentration in Plasma, and Inhibited Platelet Activation

▪Introduction: Thrombotic thrombocytopenic purpura (TTP) is a catastrophic and potentially fatal disorder caused by systemic microvascular thrombosis due to von Willebrand factor (VWF)-platelet thrombi. TTP is caused by congenital or acquired deficiency of the plasma metalloprotease ADAMTS13. Based on an earlier study (Chen J et al., J Clin Invest 2011, 121:593-603), we proposed N-acetylcysteine (NAC) as an adjunct treatment for TTP. This study showed that NAC reduced the size and activity of VWF in vitro in human plasma and in vivo in a TTP mouse model. In 2013 and 2014, two case reports described treatment of refractory TTP patients with NAC, one receiving a low dose of NAC [300 mg/kg (total 15 g) for the 1st 24 hrs, followed by 2.5 g/day for two weeks concurrently with plasma exchange] (Shortt J et al., N Engl J Med 2013, 368: 90-92; Shortt J et al., Transfusion 2014, 54:2362-2363) and the other receiving high-dose NAC [300 mg/kg/day (11 g/day) for 10 days between plasma exchanges] (Li GW et al. Transfusion 2014, 54: 1221-1224). The patient treated with high-dose NAC improved rapidly (the patient woke up from coma 18 hr after NAC treatment was initiated), but the patient treated with lower dose NAC did not appear to respond. Thus, it is as yet unclear whether NAC is an effective treatment for TTP. Therefore, more clinical studies and detailed analyses are required to examine the effects of NAC in TTP patients. Here we report the results of clinical and biochemical studies on two patients with relapsed TTP treated with NAC. Before, during, and after NAC treatment, we determined the concentrations of NAC, cysteine, and glutathione in plasma; VWF concentration, multimer structure, and functions; ADAMTS13 concentration and activity; and platelet counts and activation status (P-selectin expression and phosphatidylserine exposure).Methods: Two females with a history of prior episodes of TTP presented with acute TTP [ADAMTS13 < 10%, positive for ADAMTS13 inhibitors, platelet count ≤ 10,000/uL, lactate dehydrogenase (LDH) > 600 IU/L] and both were treated with NAC per IRB-approved protocol [150 mg/kg bolus over 1 hr and 150 mg/kg as continuous infusion until the next therapeutic plasma exchange (TPE)]. They received daily TPE until their platelet counts normalized, and intravenous NAC during days 2-5. Blood was collected daily for 8 days for research assays. ADAMTS13 concentrations in patient plasma were measured by ELISA. ADAMTS13 activity was measured using HRP-conjugated A2 peptide substrate (Wu J-J et al. J Thromb Haemost 2006, 4:129-136). Concentrations of NAC, total cysteine, and total free thiols (free thiol cysteine and free thiol NAC) in plasma were determined by mass spectrometry. Plasma VWF multimer patterns were analyzed by 1.5% agarose gel electrophoresis followed by western blotting with an HRP-conjugated polyclonal VWF antibody. Platelets in whole blood were labeled for platelet markers (CD41a or CD42b) together with one of the activation markers, P-selectin or phosphatidylserine (lactadherin). The labeled platelets were analyzed by flow cytometry.Results: Platelet counts in both patients started to increase 1 day after NAC infusion and continued to increase after discontinuation of NAC and TPE. After NAC infusion, the free thiol concentration (NAC and cysteine) in plasma increased 4 and 59 fold in patients 1 and 2, respectively. This was accompanied by increasing ADAMTS13 specific activity (ADAMTS13 activity/ADAMTS13 antigen). In patient 1, the specific activity increased from 127% (prior to NAC infusion but after TPE) to 270% during NAC infusion; in patient 2, the specific activity increased from 56% to 86%. In patient 1, the VWF multimer size decreased during NAC treatment and the VWF multimers migrated slightly faster. NAC also appeared to inhibit platelet activation. Before NAC infusion, the platelets in both patients were positive for phosphatidylserine (PS, > 30%) and P-selectin (> 15%), compared to 2% and 5%, respectively, in a normal control. The percentages of PS- and P-selectin-positive platelets decreased to less than 18% and 10% respectively, during NAC treatment.Summary: NAC treatment of two patients with TTP in conjunction with TPE was well tolerated and associated with recovery of platelet count and LDH, increased ADAMTS13 specific activity and total free thiol concentration in plasma, reduced platelet activation, and decreased VWF multimer size in one patient. DisclosuresKonkle:CSL Behring: Consultancy; Pfizer: Consultancy; Baxalta: Consultancy, Research Funding; Biogen: Consultancy, Research Funding; Octapharma: Research Funding; Novo Nordisk: Consultancy.

Neutralization of inhibitory antibodies and restoration of therapeutic ADAMTS‐13 activity levels in inhibitor‐treated rats by the use of defined doses of recombinant ADAMTS‐13

Acquired thrombotic thrombocytopenic purpura (TTP) is caused by an autoantibody-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS-13. Acute episodes of the disease are treated with a combination of immunosuppression and repeated cycles of plasma exchange to remove anti-ADAMTS-13 autoantibodies and, at the same time, replenish functional ADAMTS-13. Although this is often effective, the mortality rate has remained between 10% and 20%, highlighting the need for safer treatment options. We previously showed that, in vitro, human recombinant ADAMTS-13 (rADAMTS-13) is able to override neutralizing antibodies and restore ADAMTS-13 activity in plasma from patients with acquired TTP. In the present study, we assessed the in vivo feasibility of this strategy by using a rat model. Wild-type rats were adjusted to an ADAMTS-13 inhibitor (inhibitor) titer of ~ 10 BU mL(-1) with goat anti-ADAMTS-13 IgG, and treated with increasing doses of rADAMTS-13. Blood samples were drawn and analyzed for ADAMTS-13-specific parameters, including FRETS-VWF73 activity, inhibitor, and ADAMTS-13-specific immune complexes (ICs). The pharmacokinetics of ADAMTS-13 activity and inhibitors were evaluated. Administration of inhibitor titer-adjusted doses of rADAMTS-13 to inhibitor-treated rats predictably restored activity. Inhibitors were readily neutralized through formation of ADAMTS-13-specific ICs, which were cleared at a higher rate than the free inhibitor. Surplus protease was enzymatically active in plasma, and showed similar pharmacokinetics to ADAMTS-13 in not inhibitor-treated rats. Defined doses of rADAMTS-13 neutralized circulating anti-ADAMTS-13 antibodies and enabled reconstitution of ADAMTS-13 activity in plasma in our model, indicating that the protease may be a promising candidate for further exploration in treating acute episodes of acquired TTP.

Inhibition of ADAMTS-13 by Doxycycline Reduces von Willebrand Factor Degradation During Supraphysiological Shear Stress: Therapeutic Implications for Left Ventricular Assist Device-Associated Bleeding

ObjectivesThe aim of this study was to investigate a potential therapy for left ventricular assist device (LVAD)–associated bleeding. BackgroundNonsurgical bleeding is the most frequent complication of LVAD support. Recent evidence has demonstrated that supraphysiological shear stress from continuous-flow LVADs accelerates von Willebrand factor (vWF) metabolism by the action of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) (the vWF protease). An acquired vWF deficiency causes bleeding. This suggests that ADAMTS-13 is a clinical target to reduce vWF degradation. We tested the hypothesis that inhibition of ADAMTS-13 with doxycycline, an inexpensive, clinically approved drug, reduces vWF degradation during shear stress. MethodsWhole blood was collected from human donors (n = 15), and purified, recombinant ADAMTS-13 protein was obtained. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the dose relationship between doxycycline and ADAMTS-13 activity prior to shear stress (n = 10). To determine the effect of shear stress, plasma and recombinant ADAMTS-13 were exposed to LVAD-like supraphysiological shear stress (approximately 175 dyne/cm2). vWF multimers and degradation fragments were characterized with electrophoresis and immunoblotting (n = 10). Förster resonance energy transfer was used to quantify plasma ADAMTS-13 activity (n = 10). An ELISA was used to quantify vWF:collagen binding activity. Platelet aggregometry was performed with adenosine 5′-diphosphate, collagen, and ristocetin (vWF-platelet pathway) agonism (n = 10). ResultsDoxycycline significantly decreased plasma ADAMTS-13 activity (p = 0.01) and the activity of recombinant human ADAMTS-13 protein by 21%. After plasma was exposed to shear stress, the same pattern of vWF degradation was observed as previously reported for LVAD patients, and vWF:collagen binding activity decreased significantly (p = 0.002). Doxycycline significantly decreased ADAMTS-13 activity (p = 0.04) and the activity of recombinant ADAMTS-13 by 18%, protected large vWF multimers from degradation, and significantly decreased the levels of the 5 smallest vWF fragments by 12 ± 2% (p < 0.05). As a result, vWF:collagen binding activity was significantly restored (p = 0.004). ADAMTS-13 inhibition with doxycycline did not hyperactivate platelets. ConclusionsInhibition of ADAMTS-13 by doxycycline decreased vWF degradation and improved vWF function during supraphysiological shear stress without hyperactivating platelets. ADAMTS-13 is a clinical target to reduce vWF degradation, improve vWF function, and potentially reduce bleeding during LVAD support.

Prise en charge du purpura thrombotique thrombopénique dans deux centres français : à partir de 27 patients

IntroductionThrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy due to platelet microthrombosis involved in multiple systems associated with multiple organ dysfunctions and often severe disease course. MethodsIn order to enhance the understanding of TTP, the clinical features, laboratory characteristics, treatment and outcome of 27 patients with TTP were retrospectively analyzed and investigated in two centres (Annecy and Grenoble). ResultsAll the 27 patients presented with haemolytic anemia and decreased platelet counts. Eight patients had fever. Thirteen patients had kidney involvement including proteinuria and renal function abnormalities. Eighteen patients had neurological manifestations. Association of the 5 characteristic features of TTP was rarely found (11%) which could lead to diagnosis delay. The von Willebrand factor-cleaving protease (ADAMTS13) activity was less than 10% in all patients. At the same time, plasma ADAMTS13 inhibitors were detected in 20 patients out of the 27. After treatment with plasma exchange, glucocorticoid and rituximab, 23 patients (85%) achieved complete remission. Four patients died, in which two cases were secondary to late diagnosis. ConclusionTTP is a thrombotic microangiopathy often associated with multiple organ dysfunctions. Salvage therapy, based on some studies experiences and discussed with the Reference Center, could help getting answers in most severe cases. Awareness of physicians is an important step to further limit any delay in medical care, and to further improve the prognosis of patients with TTP.

Poor responder to plasma exchange therapy in acquired thrombotic thrombocytopenic purpura is associated with ADAMTS13 inhibitor boosting: visualization of an ADAMTS13 inhibitor complex and its proteolytic clearance from plasma.

Plasma exchange (PE) is the first-line treatment for primary acquired thrombotic thrombocytopenic purpura (aTTP) with severe deficiency of ADAMTS13 activity (ADAMTS13:AC). Some patients are poor responders to PE, raising concern over multiple pathogenetic pathways. Based on 52 aTTP patients in our national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor responder to PE, we examined an ADAMTS13 inhibitor (ADAMTS13:INH) complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis. Of 52 aTTP patients, 20 were good responders and 32 were poor responders. In the latter group, plasma ADAMTS13:AC levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (±SD) plasma ADAMTS13:INH titers (Bethesda unit/mL) were 5.7 (±4.5) before PE, but decreased to 1.4 (±0.8) on the fourth PE day and then remarkably increased to 14.8 (±10.0) on the 10th PE day, termed "inhibitor boosting," and then slowly decreased to undetectable level over 1 month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13:INH were correlated with a poor response. We visualized an ADAMTS13:INH (immunoglobulin G) complex in a patient plasma by an IEF analysis and found proteolytic fragment of ADAMTS13 antigen by a two-dimensional IEF and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Findings from this cohort of aTTP patients demonstrated that inhibitor boosting often occurs in aTTP patients in Japan. Poor responders could be predicted by elevated pre-PE ADAMTS13:INH levels on admission, but not by routinely collected clinical or laboratory data.

Thrombotic thrombocytopenic purpura in a patient with lenalidomide-responsive multiple myeloma

Dear Editor, We report a case of myeloma-associated thrombotic thrombocytopenic purpura (TTP). An 81-year-old female with multiple myeloma initially diagnosed 4 years prior was treated with 4 cycles of lenalidomide, bortezomib and dexamethasone, high-dose melphalan conditioned autologous stem cell transplant, followed by bortezomib maintenance. Subsequent biochemical progression was treated with lenalidomide 25 mg daily (days 1–21 in a 28-day cycle) and dexamethasone 20mg weekly, complicated by venous thromboembolism, treated with enoxaparin for 6 months, then switched to aspirin. Lenalidomide was discontinued. She again experienced biochemical disease progression and was treated with lenalidomide and dexamethasone (LD), with prophylactic enoxaparin. Comorbidities included hypertension, hyperlipidemia, and well-controlled rheumatoid arthritis. She in addition took amlodipine, hydrochlorothiazide, valacyclovir, potassium, and vitamin D/calcium supplements. On cycle 5, day 15, she presented with dizziness, nausea, fatigue, petechiae and hematuria. Examination revealed widespread petechiae, normal neurologic examination, and mild dehydration. Laboratory studies revealed a platelet count of 2×10/μL, hemoglobin 9.1 g/dL, white blood cell count 2.8× 10/μL, reticulocyte 2.8 %, creatinine 1.77 mg/dL, total bilirubin 4.3 mg/dL, indirect bilirubin 3.2 mg/dL, prothrombin time 15.2 s, activated partial thromboplastin time 27.6 s, fibrinogen 422 mg/dL and serum lactate dehydrogenase of 3861 IU/L, haptoglobin <3 mg/dL, and negative DAT and elevated D-dimer 2.55 mcg/mL. The troponin I was elevated at 5.36 ng/mL. Electrocardiogram showed no ischemic changes. Enoxaparin was discontinued, and platelets were transfused without achieving platelet increment. Blood smear revealed schistocytes and thrombocytopenia (Fig. 1). The ADAMTS13 activity was 7 % but ADAMTS13 inhibitor was not detected. The platelet count and serum lactate dehydrogenase over time are depicted in Fig. 2. Intravenous methylprednisolone 1 g was given for 3 days, with plasma exchange (1.5 times plasma volume) performed daily for 4 days. Myeloma restaging investigations were consistent with a partial

標準治療抵抗性の血栓性血小板減少性紫斑病に対するリツキシマブ投与のタイミングについての考察(Optimal timing of rituximab administration for refractory thrombotic thrombocytopenic purpura: a retrospective case series)

【目的】血漿交換とステロイド投与による標準治療に反応しない，難治性の血栓性血小板減少性紫斑病（TTP）にはリツキシマブの投与が有効であるが，その必要性や投与時期の判断には，ADAMTS13活性やADAMTS13に対するinhibitorの測定が必要となる。しかし，これらマーカーのリアルタイムな測定は限られた施設でしか行われていない。本研究では，当施設で経験したTTPを後ろ向きに検討し，簡便な血液データの推移から，難治性の判断およびリツキシマブ投与の判断が可能かどうかについて検討する。【対象】2008年から2013年の期間に経験したTTP 8例。【結果】標準治療に加えて，リツキシマブ投与が必要であったのは5例であった。これら難治性TTP群では，標準治療を続けるうちにinhibitorが再上昇する，inhibitor boostingが生じていた。血漿交換を続けるも，inhibitorが上昇し続けたため，リツキシマブ投与を行い，4例を救命し得た。この5例においては，7日以内のinhibitor boostingが起こった日に一致して，血小板数が再度低下する傾向が認められた。【結語】ADAMTS13活性やinhibitorの迅速な測定ができない場合でも，簡便な血小板数測定によってinhibitor boostingを察知し，早期にリツキシマブ投与の必要性を判断することが可能と考えられる。 Introduction: The effectiveness of rituximab administration for refractory thrombotic thrombocytopenic purpura (TTP) is well known. To determine the optimal timing of rituximab administration, evaluations of ADAMTS13 activity and its inhibitor are essential. The measurement of these variables, however, is limited in most hospitals. Method: We reviewed eight recent cases of TTP in six years and examined whether a commonly determined clinical parameter, such as platelet count or some other biochemical variable, could be a surrogate marker for the determination of the optimal timing for rituximab administration. Results: Of the eight cases, five were refractory to the standard therapies of plasma exchange and steroid administration. For these five cases, the ADAMTS13 inhibitor was boosted between days 4 to 7. Among blood variables, platelet counts decreased simultaneously on the day of inhibitor boosting. Conclusion: Since measurements of ADAMTS13 activity and its inhibitor, as well as rituximab administration, are limited in Japan, we believe the commonly measured variable of platelet counts could be a surrogate marker for determining the optimal timing of rituximab administration in TTP treatment. 血栓性血小板減少性紫斑病（thrombotic thrombocytopenic purpura: TTP）は多臓器不全に至る重篤な血栓症であり，救命のためには集学的治療を要する。全TTPの95%以上を占める後天性TTPは，血管内皮細胞に発現するvon Willebrand因子（VWF）の特異的切断酵素であるADAMTS13（a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13）に対する自己抗体（インヒビター）が後天的に産生されることで発症する 1。インヒビターにより，ADMATS13の活性が低下すると，VWFが切断されないため，超高分子量多重体（unsually–large VWF multimer: UL–VWF）が血液中を循環する。このUL–VWFが血小板と結合し，大量の血小板血栓を形成することで生じる微小血管障害が本疾患の本態である。 後天性TTPに対する第一選択として行われる治療法は血漿交換である。血漿交換によって，血中のADAMTS13インヒビター・UL–VWFM・サイトカインが除去され，ADAMTS13が補充される 2, 3。同時にADAMTS13インヒビター産生抑制を目的としてステロイドパルス投与（メチルプレドニゾロン1,000mg ×3日間）が経験的に行われてきた 4。血漿交換とステロイドパルス投与が標準的に行われるようになってからは，死亡率は大幅に改善し，現在は10%以下と報告されている 2。しかし，これらの治療を行っても寛解に至らない難治例には，抗がん剤や免疫抑制剤が使用されてきた。抗CD20抗体であるリツキシマブは自己抗体産生細胞を直接破壊することにより，ADAMTS13インヒビター産生を抑制する。リツキシマブがTTPに有効であると報告されて以来，多くの症例集積研究において難治性TTPに対する有効性が示されてきた 4, 5, 6, 7, 8, 9。現在のところ，血漿交換とステロイド投与による標準治療 3に反応しない場合は難治性と判断し，速やかにリツキシマブ投与を考慮するべき，と考えられている。しかしリツキシマブの適切な投与時期に関する一定の見解は本邦にはない。ADMATS13活性およびADAMTS13インヒビター値が連日測定できれば，早期に難治性と判断し，リツキシマブを早期に投与することが可能と考えられるが，これらのリアルタイムな測定は通常の医療機関では困難である。 本研究では，当院で経験したTTP症例の臨床経過から，簡便な血液検査で難治性TTPと判断し得る指標がないか検討し，さらにリツキシマブの適切な投与タイミングについても検討する。 2008年1月から2013年3月の間に奈良県立医科大学附属病院高度救命センターで加療した後天性TTP 8症例を対象とし，後ろ向きに検討した。 血液検査は，血小板数，ヘモグロビン値，血清LDH値，血清クレアチニン値，総ビリルビン値を連日測定した。ADAMTS13活性およびADAMTS13インヒビターは連日採血を行って血液検体を冷凍保存し，EIA法を用いて週に2〜3回の頻度で測定した。ADAMTS13活性は%，ADAMTS13インヒビタータイターはBethesda Unit（BU）を用いて表現する。 当施設でのTTP治療プロトコールは血漿交換（新鮮凍結血漿40～60mL/kg，5日間）とメチルプレドニゾロン；mPSL 1,000mg/day×3日間をまず行い，4日目からはmPSL 1mg/kg/dayの投与を継続し，経過をみながら漸減している。また，血漿交換も必要に応じて追加する。mPSL投与期間と血漿交換の追加は，ADAMTS13活性，ADAMTS13インヒビターおよび血液生化学検査に基づいて判断する。本治療プロトコールにて臨床症状，ADAMTS13活性，ADAMTS13インヒビター，血液生化学的検査が改善した後，再度悪化を認めなかった症例を“標準治療反応群”，プロトコールに沿った治療中に再度臨床症状が悪化した，あるいは治療に反応しなかった群を“難治性TTP群”とし，両群の経過を比較検討した。なお，寛解はステロイドの投与量が10〜20mgの維持量となってから30日間に臨床症状と検査所見に異常がみられない場合と定義する。また当施設での難治性TTPに対するリツキシマブの投与方法は375mg/m2を週1回，計4回投与としている 5。 表記は中央値と四分位区間とし，群間の比較はMann–Whitney U検定を用い，危険率5%未満を有意とした。 標準治療反応群は3例で，難治性TTP群は5例であった。 来院時ADAMTS13活性は両群ともすべての症例で検出感度以下であった。また来院時のADAMTS13インヒビターは難治性TTP群で高い傾向にあるものの，有意差は認めなかった（標準治療反応群vs難知性TTP群；2.1BU [1.7–3.4] vs 4.8BU [4.7–5.0]，p=0.066）。来院時血小板に関しても両群で有意差を認めなかった（標準治療反応群vs難治性TTP群；1.2万/µL [0.9–1.6] vs 1.4万/µL [1.0–1.5]，p=0.279）。 Clinical course of case 1. Case 1 was successfully managed with 4 sessions of plasma exchange and steroid administration. Platelet count (black squares, ×106/μL); ADAMTS13 activity (white circles, %); ADAMTS13 inhibitor (black triangles, BU/mL); mPSL, methylprednisolone. 標準治療反応群の代表症例の臨床経過をFig. 1に示す。症例は76歳女性で，入院当日より血漿交換を連日行った。メチルプレドニゾロン1,000mg/dayを3日間投与し，第4病日以降，50mg（1mg/kg）/dayで投与を続けた。これらの治療により，入院時には4.6BUと比較的高値であったインヒビターは低下し，再上昇を認めなかった。ADAMTS13活性も上昇し，血漿交換終了後の第6病日以降には軽度低下したものの20%程度にとどまった。血小板数は連日上昇し，経過中に低下に転じることはなかった。 難治性TTP群の症例6の経過をFig. 2aに示す。症例は47歳女性で，第3病日までは，標準治療にてADAMTS13インヒビターは低下し，血小板数は上昇したが，ADAMTS13活性は感度以下が続いていた。第3病日も血漿交換を行ったが，第4病日に血小板数は減少し，インヒビターも上昇に転じた。第4病日以降も血漿交換を継続したが，インヒビターは加速度的に上昇した。そこで第7病日にリツキシマブ投与に踏み切ったところ，第8病日の抗体価は減少に転じ，第12病日には血小板も再上昇し，第15病日には血小板数が10万/µLを超え，ADAMTS13活性も15％，ADAMTS13インヒビターも陰転化し，寛解し得た。 Clinical courses of cases 6 and 8. a) Case 6; Inhibitor boosting and platelet counts decreasing occurred at day 4. Rituximab was administered at day 7. b) Case 8; Inhibitor boosting and platelet counts decreasing occurred at day 5. Rituximab was administered at day 8. Platelet count (black squares, ×106/μL); ADAMTS13 activity (white circles, %); ADAMTS13 inhibitor (black triangles, BU/mL); mPSL, methylprednisolone. Fig. 2bに難治性TTPと診断し，リツキシマブ投与を行ったが，投与翌日に死亡した症例8の経過を示す。第4病日までは標準治療にて血小板は上昇，インヒビターも低下していったが，第5病日にインヒビターが上昇に転じ，血小板数も減少し，以後血漿交換を続けるもインヒビターは急激な増加を続け，第8病日には抗体値は20BU/mLを超えた。第8病日にリツキシマブを投与したが，第9病日の頭部CTにて出血を伴う多発脳梗塞を認め，同日に永眠となった。 Fig. 3に標準治療反応群の症例2と3，難治性TTPの症例4，5，7の血小板数とADAMTS13インヒビターの推移を示す。標準治療反応群ではADAMTS13インヒビターの再上昇は認められず，血小板数も治療開始後7日までに減少に転じる症例はなかった。一方，5例の難治性TTP群においては，ADAMTS13インヒビターの再上昇，inhibitor boosting 10, 11が第4〜7病日に起こっており，このinhibitor boostingに一致して血小板数が減少に転じていた。リツキシマブ投与は第5〜10日目に行われていた。 Course of platelet count and ADAMTS13 inhibitor in cases 2, 3, 4, 5 and 7 (a–e, respectively). In cases 2 and 3, platelet count (black squares, ×106/μL) and ADAMTS13 activity (white circles, %) increased and ADAMTS13 inhibitor (black triangles, BU/mL) decreased during plasma exchange and steroid administrations, while in cases 4, 5 and 7, platelet count decreased on the day of the inhibitor boosting (days 7, 5 and 5). 症例8以外ではinhibitor boosting時の血清LDH値と総ビリルビン値にも上昇が認められたが，標準治療反応群においても，血清LDH値や総ビリルビン値は増減している症例もあり，一定の傾向は認められなかった。 血漿交換とステロイド投与に反応した標準治療反応群は，治療開始直後よりADAMTS13インヒビターは減少し，ADAMTS13活性および血小板数が上昇していた。血漿交換終了後はADAMTS13の補充がなくなるため，ADAMTS13活性は若干低下するが，血小板数は上昇を続け，寛解に至る。こうした標準治療に反応する症例はTTPの80%以上を占める 2。一方で難治性TTPでは治療によって一旦は病態が改善する方向へ向かうが，経過中にADAMTS13インヒビターが再度上昇し，治療に抵抗性を示す。この現象は “inhibitor boosting” という概念で知られている 10, 11。“inhibitor boosting” とは，血漿交換当初は一時的にインヒビターのタイターが低下するが，血漿交換開始数日後よりインヒビターが再度増加するという現象である。Isonishiらは52名のTTP cohortのうち32名のTTPにおいて，血漿交換開始4日までに一旦低下したinhibitorが10日までに著明に増加した現象をinhibitor boostingと称している 11。我々はIsonishiらの報告と同様に，血漿交換とステロイド投与によって一旦は減少に転じたinhibitorが治療経過中に再度上昇する現象をinhibitor boostingと定義している。本現象の明確な機序は明らかではないが，ちょうど，ワクチン接種時のブースター効果と同様の現象が血漿交換でADAMTS13を補充することで生じるものと思われる。今回我々が経験した難治性TTP 5例すべてにこのinhibitor boostingが認められた。 この現象は難治性TTPの特徴と考えられ，inhibitor boostingを検知できれば，早期に難治性と判断し，速やかなリツキシマブ投与が可能になると考えられる。しかし，通常の医療機関でADAMTS13インヒビターを連日測定し，inhibitor boostingを検知することは不可能である。当施設でもリアルタイムな測定ではなかったため，結果的に難治性の判断，およびリツキシマブ投与に数日の遅れが生じたと考えられる。TTPに特徴的である破砕赤血球の出現割合，血清LDHやビリルビン値なども病勢の判断の参考とはしているが，TTP発病後の定量性には再現性などに問題もあり，標準治療に反応しているか否かの判断は難しい。今回の当施設での難治性TTP群の検討において，治療開始後のinhibitor boostingに呼応するように，血小板数の減少が認められた。これは血漿交換によって一旦は血小板数が改善するものの，inhibitor boostingによって，補充されたADAMTS13が速やかに失活し，血小板数が再度減少に転じたものと考えられる。ヘモグロビンやCre値にはこうした傾向は認められなかったことから，血漿交換によって上昇もしくは維持されていた末梢血の血小板数の再低下が，inhibitor boostingを示唆するものと考えられた。以上よりTTPに対して標準治療を行っている経過中，7日以内に血小板が再低下した場合には，inhibitor boostingと捉えて，リツキシマブの速やかな投与を考慮することが重要と考えられる。 リツキシマブの投与開始時期に関しては文献的にも一定の見解はない。Scullyらは新たに診断されたTTP患者に，標準治療に加えて3日以内にリツキシマブ投与を行った群では，寛解率，生存率とも有意に改善し，また再発率も低いと報告している 5。さらに54例のRetrospective studyにおいても，急性のTTPに対しては，3日以内の早期投与が早期の寛解に貢献することが示されている 12。本研究でのリツキシマブ投与は5日から10日目となっていた。Fig. 2aで示した症例6のようにinhibitor boostingが起こってから数日後にリツキシマブを投与しても寛解に至っている症例も存在したが，症例8のようにinhibitor boostingの程度が強く，ADAMTS13インヒビターが急激に再上昇し，救命できなかった症例もあった。症例8では，頭部CTで出血を伴う多発脳梗塞を生じたことから，急激に血栓症が悪化したものと考えられる。こうした症例では，もっと早期にリツキシマブを投与することで救命できたのではないかと考えられる。また，リツキシマブの投与が遅れる要因の一つに，本疾患に対するリツキシマブ投与が保険適応でない点が挙げられる。実際に投与する場合，非常に高額な患者個人負担，あるいは病院負担となる。この問題を解決するだけで，数日間の投与の遅れが生じ得る。現在，医師主導型のリツキシマブの治験が進行中であるが，早期の保険適応が待たれるところである。 本研究は単一施設での症例集積研究であり，多施設での症例集積研究ではないため，この点が本研究の限界である。さらに，本邦でのリツキシマブ投与を行ったTTPの症例集積研究は非常に少なく，保険適応が認められることで，今後さらなる症例の集積が可能になると考える。 標準治療に抵抗性を示す難治性TTPの本質は，ADAMTS13インヒビターの再上昇であるinhibitor boostingと考えられる。7日以内の血小板数の推移を指標に，inhibitor boostingを察知することで，早期のリツキシマブ投与が可能になると考えられる。 本研究において，申告すべき利益相反はない。

Efficiency of Intensive Immune -Suppression Treatment with Rituximab in Acquired Thrombotic Thrombocytopenic Purpura: A Chinese Case study

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA) and functional failure of the organs due to ADAMTS13 deficiency. ADAMTS13 inhibitor is the main contributor of pathogenesis in acquired TTP. With the help of retrospective analysis the researchers tried to evaluate the effectiveness of rituximab in preventing the acquired TTP in China. Out of 27 patients with acquired TTP that have registered from 2006 to 2015, eleven cases started rituximab infusion before remission, while sixteen others began rituximab infusion after remission. Twenty-three cases got rituximab with standard doses (375 mg/m2 , weekly for 4 weeks) and four patients were administered reduced doses (100mg, weekly for 4 weeks) after remission. Till date, there was no patient reported with the acquired TTP relapsed during the follow-up sessions, whenever rituximab treatment was initiated (before or after remission), or what dose of rituximab was administrated (standard dose or reduced dose). There were mild controllable side-effects reported in the case of four patients. The study concludes that rituximab can act remarkably in relieving acquired TTP, while preventing the relapse.

Successful Eculizumab Therapy in Thrombotic Thrombocytopenic Purpura (TTP) Refractory to Plasma Exchange, Steroids and Rituximab

IntroductionWe present a case of TTP refractory to usual therapies treated successfully with eculizumab.Case presentationA 64 year-old African American female with history of diabetes and hypertension presented with left sided weakness and seizure-like activity preceded by 2 days of headaches and 2 weeks of easy bruising. On admission her vital signs and physical exam were unremarkable. Her basic metabolic panel, ANA panel, hepatitis panel, HIV, and anti-phospholipid panel was negative. Her hemoglobin was 9.0 g/dL, platelets were 13K/uL, LDH was 954IU/L, haptoglobin <30 mg/dL, eGFR was 50, and blood smear showed 4-5 schistocytes/high power field. ADAMTS13 activity was <5% and there was an inhibitor level of 1.1BU. C3 level was 75.3 (normal range 90-180mg/dL), C4 was <10 mg/dL (normal range 15-45 mg/dL). On day 1, she was started on oral prednisone (1mg/kg/day) and daily plasma exchange (TPE). changes. After 6 days of TPE without signs of improvement, the first dose of rituximab 375 mg/msq/dose was given on day 7. During the second week of hospitalization, patient also developed delirium and mental status changes. Vincristine 2 mg was given on day 13 for refractory TTP. Due to clinical worsening of microangiopathy and mental status, a decision was made to administer eculizumab 900 mg on day 17. Within a few hours of eculizumab administration, her platelets improved from 8k/uL to 21k/uL and to 47k/uL 12 hours later. After the second dose of eculizumab on day 24, platelets improved to 117k/uL and continued to rise. Eculizumab 900 mg was given weekly for 4 weeks, at which time ADAMTS13 activity was 75% and ADAMTS13 inhibitor was undetectable. Eculizumab 1200 mg was then given every two weeks, for a total of 3 more doses, during which the CBC, LDH, and haptoglobin remained in the normal range. Eculizumab was stopped, and after four months of close follow up, platelets, LDH, renal function and mental status were still normal range. C3 level was 203 (normal range 90-180 mg/dl), C4 was 35 mg/dl (normal range 15-45 mg/dl), ADAMTS 13 activity assay is >100% and there is no inhibitor.DiscussionTTP and aHUS are both thrombotic microangiopathies (TMA). TTP is caused by acquired or inherited deficiency of the ADAMTS 13 protein or due the formation of an inhibitor to the ADAMTS13 protein. aHUS results, in most cases, from a genetic mutation in complement factor H in the alternative pathway of the complement cascade. Recent studies have described the importance of the complement system in all forms of TMA (Update on the role of the complement system in the pathogenesis of thrombotic microangiopathies. Prilozi. 2014;35(1):115-22). While plasma exchange is the cornerstone of TTP treatment, complement inhibition with drugs such as eculizumab is the treatment of choice for aHUS. The use of complement inhibiting drugs has not yet been studied in TTP.We report an unusual case of TTP, with confirmed ADAMTS 13 inhibitor and low activity, which did not respond to conventional treatment for TTP. Theorizing that this patient either had concurrent aHUS or had TTP with a dysregulated alternate complement pathway, we used eculizumab, to which the patient had a remarkable and persistent response.There are other reports in literature of patients with aHUS having reduced levels of ADAMTS13 protein; however, these patients did not have an inhibitor as was seen in our patient (Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome. Blood. 2013 Aug 22;122(8):1487-93). There is one other case in the literature where TTP was treated successfully with eculizumab. In that case, discontinuation of eculizumab was associated with a relapse of TTP and so had to be continued (Eculizumab in the treatment of refractory idiopathic thrombocytopenic purpura. Br J Haematology. 2012 June;157 (6):772-4). To our knowledge, we report the first case of a confirmed diagnosis of TTP treated successfully with eculizumab with remission continuing 3 months after discontinuation of the drug.The use of eculizumab and the role of the complement pathway in TTP deserves further study. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Importance of Eculizumab in Patients with Atypical Hemolytic Uremic Syndrome: Single Center Experience

INTRODUCTION:The atypical hemolytic uremic syndrom (aHUS) is a rare disease especially seen in childhood. It can be differentiated from HUS with the absence of diarrhea history in the prodromal phase. Although the disease is rarely seen in children, it is even rarer in adults and is usually misdiagnosed as TTP (thrombotic thrombocytic purpura). The most prominent differential findings of the disease compared to TTP, are the development of chronic renal failure requiring hemodialysis and persistant hypertension attacks. Plasmapheresis and hemodialysis has been the choice of therapy for a long time until Eculizumab. In this abstract we will report 20 cases of aHUS that are treated with Eculizumab in our clinic.MATERIALS AND METHOD:Plasma samples of the patients were analysed for the ADAMTS13 activity and inhibitor levels before plasmapheresis. One patient out of 20 responded to plasmapheresis treatment in one week and started to be followed on out – patient clinic without any further medication, of the remaining 19 were planned to be given Eculizumab therapy. All of of the patients were vaccinated for Neisseria meningitides, Pneumococcus and Hemophilus influenza type B prior to 2 weeks of initiation of Eculizumab. We used antibacterial prophylaxis with amoxicillin – clavulanic acid in patients requiring urgent Eculizumab. Eculizumab was administered 900mg/day for 4 weeks and dosage was elevated to 1.200 mg/day in week 5, then 1.200 mg/day every other week as maintenance therapy. The response to therapy was evaluated by the fragmentation level in the peripheral blood samples, blood cell counts, serum LDH, reticulocytes ratio, creatinine levels and proteinuria.FINDINGS AND RESULTS:We enrolled 20 subjects to our study who were diagnosed as aHUS and followed in our clinic since 2012. Seventeen of the 20 patients were female and the median age was 37,5 years (ranging from 23 – 80). One of the patients responded to plasmapheresis treatment and no further medication was needed. Of the remaining 19 patients eculizumab treatment was planned. However two of them died due to acute respiratory distress prior to therapy in intensive care unit. Three of the 17 patients were diagnosed with aHUS recently (in one month) and they are currently on the induction phase of eculizumab.Fourteen of our patients are using Eculizumab for 2 to 26 months (median 8 months). One of them has been diagnosed during pregnancy and eculizumab treatment was started immediately. She had a successful delivery with preserved renal functions. Four patients did not require hemodialysis at the time of diagnosis. While four of the patients who were on dialysis, dialysis requirements were gradually decreased and was stopped in median 10 months (in 4 to 13 months). The remaining eight patients who were on eculizumab in median 2.5 months still require dialysis. 8 patient who had been on eculizumab therapy in median 2,5 months (in 1 to 7 months) still on dialysis. We terminated eculizumab therapy in patient after 13 months due to full clinical and laboratory improvement. We lost one of our patient due to severe respiratory failure in her third month of therapy.DISCUSSION AND CONCLUSION:Although aHUS is quite a well – established clinical presentation in childhood it is extremely rare in adults without settled diagnostic criteria and serologic features. The clinical outcome is unfavorable in this group with death rates as high as 25% during the acute phase and up to 50% of cases progressing to end-stage renal failure. It is confirmed that progress in intensive care and dialysis opportunity has contributed to the decrease of mortality. Death rate was 15% in our cohort but it would be higher without awareness of the syndrome. Even in induction period of the therapy, both clinical and laboratory findings were improved significantly with eculizumab. Renal functions were restored in nine of the 17 patients. Eculizumab has changed the future perspectives of patients with aHUS and both FDA and the EMA have approved it as life-long treatment. But, there are still some unresolved issues about the follow-up such as the optimal duration of eculizumab treatment and whether it can be stopped or how to stop the therapy. All these questions can be resolved with the data from large international prospective cohort studies. DisclosuresNo relevant conflicts of interest to declare.

A Phase II Study Evaluating the Efficacy of Rituximab in the Management of Patients with Relapsed or Refractory TTP

Background: As many as 30-50% of patients diagnosed with Thrombotic Thrombocytopenic Purpura (TTP) either relapse or become refractory to therapeutic plasma exchange (TPE). There remains a pressing need to evaluate novel treatments for these patients. Rituximab is a chimeric anti-CD20 monoclonal antibody that has demonstrated efficacy potentially by eliminating active B-lymphocytes and production of ADAMTS 13 inhibitor considered central to the pathogenesis of TTP.Intervention: We conducted a prospective multi-center phase II open-label trial in Canadian hospital-based apheresis units to evaluate the efficacy of rituximab in the management of adult patients with relapsed or refractory TTP. Functional and antigenic ADAMTS13 enzyme, inhibitor levels and anti-CD 20 were measured at initiation, 8, 12, 24 and 52-weeks as well as routine hematologic parameters, complete remission, rate of relapse, overall mortality and adverse events. Rituximab was administered intravenously once weekly for a total of 4 doses at 375 mg/m2 in eligible consenting patients.Results: Twenty refractory and 20 relapsing TTP patients were enrolled (N=40). A complete response (CR) was defined by a platelet count >150 x 109/L, an LDH <1.5 times the upper limit of normal, absence of progressive neurologic symptoms and no further need for TPE. At week 8, 77% of refractory patients and 90% of relapsed patients were in CR. At week 52, 15/20 refractory patients (3 deaths, 1 lost to follow-up) and 18/20 of relapsed patients (1 withdrew, 1 lost to follow-up) were alive and in CR. Of the 40 patients enrolled a total of 3 patients died; all in the refractory arm: two with functional ADAMTS13 <10% and detectable inhibitor. Two deaths were related to fungal infection (week 2) and active TTP (week 8). A third death was attributed to thrombotic complications at week 29. All refractory patients with normal ADAMTS13 enzyme at baseline had a complete response at week 8 and remained in remission for the remainder of follow-up (52 weeks). Despite being in complete hematological and clinical remission, relapsing TTP patients still had a positive inhibitor at 24 (50% of patients) and 52 weeks (10% of patients). All patients had < 3% CD 20+ B-lymphocytes at 8 and 12 weeks post rituximab. Median platelet counts in the refractory group recovered more slowly than in the relapsing TTP patients. By week 8 refractory TTP patients received a median of 26 plasma exchanges and relapsed patients received a median of 11(P<0.001).Conclusion: At the conclusion of this study the overall mortality rate was 8%, 15% (95% CI: 5% to 36%) for the refractory group and 0% (95% CI: 0% to 16%) for the relapsed group. Nine refractory patients were dialysis-dependent at baseline and only 2 had a serum creatinine >250 µmol/L and remained on dialysis at week 52. These patients had normal ADAMTS13 enzyme measurements throughout the time period of the study. Results of this study indicate that TPE and rituximab is an effective combination in patients with relapsed or refractory TTP. DisclosuresFoley:Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Hoffman-LaRoche: Consultancy, Honoraria, Research Funding; Jansen: Consultancy, Honoraria; Alexion: Consultancy, Honoraria. Off Label Use: Rituxan is a CD20-directed cytolytic antibody indicated for the treatment of patients with: -Non-Hodgkin’s Lymphoma (NHL) (1.1) -Chronic Lymphocytic Leukemia (CLL) (1.2) - Rheumatoid Arthritis (RA) in combination with methotrexate in adult patients with moderately-to severely-active RA who have inadequate response to one or more TNF antagonist therapies (1.3) . Arnold:GSK: Honoraria, Research Funding; Hoffman-LaRoche: Research Funding; Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Clark:Alexion: Honoraria; CSN: Honoraria; ASN: Honoraria; Octapharm: Honoraria; Danone Research: Honoraria.