Search

Thrombotic thrombocytopenia purpura (TTP) and atypical hemolytic uremic syndromes (aHUS) are distinct clinical disorders characterized by hemolytic anemia, thrombocytopenia, microthrombi, and end organ damage. TTP is characterized by a low ADAMTS13 activity level at diagnosis of10 % ADAMTS13 activity, while aHUS is characterized as having10 % ADAMTS13 activity. Despite clinical remission, survivors of thrombotic microangiopathy suffer significant comorbidity and decreased quality of life (QOL) than their healthy counterparts. The reason for this is unclear. Is it a lingering effect from their initial acute episode or ongoing subclinical injury/inflammation despite clinical remission? Common clinical complaints validated in practice include increased depression, deficits in memory, concentration, mood, and mental endurance. We suspect headaches may be an important clinical tool toward understanding patient morbidity and decreased QOL. To date, no studies report headache frequency or severity in this population. To answer this question, adult patients3 months since their last acute episode of TTP or aHUS were approached to take a Headache Impact Test (HIT-6) survey. Between June 1, 2013 and May 30, 2014, 31 patients in remission (21 patients with prior TTP and 10 patients with prior aHUS) completed the HIT-6 survey. The survey scores were then compared to the HIT-6 normative population data established by Qualitymetric incorporated. Overall, TTP patients had a significantly higher average HIT-6 score of 59.9 compared to an average HIT-6 score of 51 seen in sex-matched controls (SD 9.6, p value 0.002). No significant difference was seen in the HIT-6 scores of aHUS patients. Of TTP patients studied, approximately 57 % (12/21) had three or more episodes and were24 months since the last episode. The average time since last acute episode in TTP patients was 37.5 months. There was no significant correlation between TTP survivor HIT-6 scores and the number of prior episodes (1, 2, or3) or timing from the last episode (3-6, 7-12, 13-24, or24 months). About 19/21 (90 %) patients, who are TTP survivors, also had a normal ADAMTS13 activity level (10 %) on the day of the survey. Our study suggests that headaches have significant impact on TTP survivors and should be followed in the clinical setting to prevent undue patient morbidity. Larger studies are needed to understand how headaches impact long-term survival and risk of relapse.

Although steroids are routinely used as an adjunct to plasma exchange (PEX) therapy in the treatment of immune-mediated thrombotic thrombocytopenic purpura (iTTP), limited data regarding their efficacy or effect on ADAMTS13 biomarkers are available. We report the results of a prospective, randomized study that compared the effectiveness of prednisone or cyclosporine (CSA) as adjuncts to PEX in the treatment of iTTP. A total of 26 of the planned 72 subjects were enrolled and treated from November 2007 until February 2014 before the study was halted after a planned interim analysis. Fourteen patients were randomly assigned to the prednisone arm, and 12 to the CSA arm of the study. One patient died in each arm of the study, and 2 patients in the prednisone arm of the study failed to achieve a response and crossed over to the CSA arm, leaving 11 patients in each arm of the study evaluable for the primary end point of exacerbation. One of the 11 prednisone-treated subjects (9%) suffered an exacerbation, whereas 3 of the 11 (27%) patients in the CSA arm suffered an exacerbation. Although there was no significant difference in the exacerbation rate, suppression of the anti-ADAMTS13 antibodies and improvement in ADAMTS13 activity in the first month after stopping PEX were significantly better in the prednisone-treated subjects. Side effects were manageable and comparable in both arms of the study. These data demonstrate the superiority of prednisone over CSA as an adjunct to PEX in the suppression of the anti-ADAMTS13 antibodies and improvement in ADAMTS13 activity. This trial was registered at www.clinicaltrials.gov as #NCT00713193.

Background Thrombotic thrombocytopenic purpura (TTP) requires immediate treatment with plasma exchange (PE) to prevent disease mortality and/or morbidity. Frequently, PE is initiated before blood sample is collected to confirm ADAMTS13 deficiency. However, the effect of PE treatments on the evaluation of ADAMTS13 is uncertain. Moreover, the pertinence of ADAMTS13 activity during PE therapy to prediction of treatment outcomes is unclear. Thus, clarification of the diagnostic and prognostic values of ADAMTS13 activity obtained during PE treatment is an unmet clinical need. Study Design and Methods A total of 212 sequential samples were obtained during the course of daily PE treatment from 19 patients with acquired TTP. ADAMTS13 activity levels were determined in these longitudinal samples for analysis. Results After the initial three daily PE procedures, the sensitivities of ADAMTS13 activity in diagnosis of TTP (

Historically, attempts were made to differentiate acquired thrombotic thrombocytopenic purpura (TTP) from atypical hemolytic uremic syndrome (aHUS) based upon the age at presentation and the presence of neurologic or renal injury. Although these means of differentiating acquired TTP from aHUS have now been demonstrated to be inaccurate, there were no clinical consequences as the treatment for both disorders remained plasma exchange therapy (PEX). With the regulatory approval and remarkable efficacy of eculizumab (Soliris) for the treatment of aHUS, the accurate and timely differentiation of acquired TTP from aHUS now has real clinical consequences. In the following review we will address the emerging methods of clinically differentiating acquired TTP from aHUS using collectively their clinical presentation, laboratory data, and initial response to PEX therapy to differentiate patients more consistent with a diagnosis of aHUS, and therefore more likely to benefit from complement inhibition therapy.

The increased understanding of the pathophysiology of both atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP) in recent years has led to significant therapeutic advances for both conditions. These advances have placed an increased emphasis on a more rapid differentiation of both disorders which remain clinical diagnoses. In particular, recent data demonstrating the effectiveness of complement inhibition in patients with aHUS have increased the need for a more rapid and accurate differentiation of aHUS and TTP. Previously utilized criteria have used the presence or absence of neurologic or renal injury and the pretreatment ADAMTS13 activity to differentiate aHUS from TTP. The use of presenting clinical symptoms and findings alone to differentiate these conditions is problematic given their overlapping clinical presentations. Similarly, the use of the pretreatment ADAMTS13 activity alone to differentiate aHUS from TTP is also problematic, and could lead to the inappropriate witholding of plasma exchange (PEX) therapy. However, when used collectively, the pretreatment clinical findings (symptoms and laboratory data) and ADAMTS13 activity in the context of the patient's response to PEX therapy can allow for a more effective differentiation of these two disorders in a timely fashion that will allow for the prompt initiation of the most appropriate therapy.

An acute increase in the international normalized ratio (INR; a comparison of prothrombin time to monitor the effects of warfarin) over 3 in patients with chronic kidney disease (CKD) is often associated with an unexplained acute increase in serum creatinine (SC) and an accelerated progression of CKD. Kidney biopsy in a subset of these patients showed obstruction of the renal tubule by red blood cell casts, and this appears to be the dominant mechanism of the acute kidney injury. We termed this warfarin-related nephropathy (WRN), and previously reported cases of WRN only in patients with CKD. We now assess whether this occurs in patients without CKD, its risk factors, and consequences. In 15,258 patients who initiated warfarin therapy during a 5-year period, 4006 had an INR over 3 and SC measured at the same time; however, the large data set precluded individual patient clinical assessment. A presumptive diagnosis of WRN was made if the SC increased by over 0.3 mg/dl within 1 week after the INR exceeded 3 with no record of hemorrhage. WRN occurred in 20.5% of the entire cohort, 33.0% of the CKD cohort, and 16.5% of the no-CKD cohort. Other risk factors included age, diabetes mellitus, hypertension, and cardiovascular disease. The 1-year mortality was 31.1% with compared with 18.9% without WRN, an increased risk of 65%. Thus, WRN may be a common complication of warfarin therapy in high-risk patients and CKD doubles this risk. The mechanisms of these risks are unclear.

Background The assay for ADAMTS13 activity helps clinicians to confirm the clinical diagnosis of idiopathic thrombotic thrombocytopenic purpura. The clinical value of testing for the antigen level of ADAMTS13 protein is, however, less clear.Design and Methods In this study, both ADAMTS13 antigen and activity levels were measured in 835 sequential samples from 40 consecutive patients who were followed for an average of 29 months throughout the course of acute episode plasma exchange treatment and clinical remission.Results During acute episodes, ADAMTS13 activity was severely deficient while ADAMTS13 antigen levels were more variable, ranging from severely deficient to as high as within the reference range. A severe depletion of ADAMTS13 antigen level during acute disease was, however, statistically associated with disease mortality (P=0.0322). For patients who achieved initial clinical responses, ADAMTS13 antigen levels appeared to be restored faster than ADAMTS13 activity to the normal range. Further analysis demonstrated that the ADAMTS13 antigen level at the time of initial clinical recovery was significantly higher in the patients who subsequently achieved a sustained clinical remission than in the group who soon after had an exacerbation (P=0.0187).Conclusions Our data suggest that evaluation of ADAMTS13 antigen levels during the course of therapy and follow-up may offer additional useful information for the management of patients with thrombotic thrombocytopenic purpura.

Approximately 50% of patients with venous thromboembolism also present with hereditary predisposition. The most common genetic factors are single nucleotide polymorphisms (SNPs) of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T. Genotyping these SNPs helps clinicians to correctly diagnose the disease and properly manage patients. In this study, we report a novel method using surface-enhanced laser desorption and ionization time of flight mass spectrometry to rapidly genotype, in a multiplex fashion, 3 SNPs that predispose patients to thrombosis. First, patient DNA samples were subjected to polymerase chain reaction to amplify and extend the DNA products with masses corresponding to specific genotypes. Polymerase chain reaction products were then applied to Q10 anionic protein chips, undergoing on-chip sample enrichment and clean-up. Finally, the genotypes of the SNPs were determined by surface-enhanced laser desorption and ionization time of flight mass spectrometry. This method offers a rapid turnaround time of less than 5 hours from sample collection to result reporting. The analytical accuracy of each SNP genotyping result has been confirmed by DNA sequencing. In addition, the genotype results produced by this method were validated by comparing them with results obtained by the approved method in the clinical reference laboratory. This novel method is fast, accurate, and reproducible, and thus provides an excellent platform to promote personalized medicine in the management of clotting disorders.

In terms of managing thrombotic disorders, genotype-based individualized patient care emerged as early as 1994 when the association of factor V Leiden (G1691A), and later, prothrombin (G20210A), with thrombotic phenotypes were discovered. Since then, genetic tests for specific thrombophilic SNPs have been routinely incorporated into daily practices in both thrombotic risk assessment and clinical decision-making with respect to prophylactic anti-thrombotic therapy. Recently, the area of pharmacogenomics in major anti-thrombotic drugs, such as warfarin and clopidogrel, has been the principal driver for personalized therapy based on one’s own individual characteristics.

Several reports have been published regarding the use of cyclosporine (CSA) in the treatment of idiopathic thrombotic thrombocytopenic purpura (TTP). We hypothesized that prophylactic CSA therapy may prevent recurrences in patients with a history of multiple relapses of TTP. Nineteen patients with idiopathic TTP were enrolled on prospective studies at Ohio State University between September 2003 and May 2007. Patients achieving remission remained on CSA therapy for 6 months, allowing us to evaluate the efficacy of CSA as prophylactic therapy. CSA was administered orally at a dose of 2–3 mg/kg in a twice a day divided dose in all patients and continued for a total of 6 months. Long-term clinical follow-up with serial analysis of ADAMTS13 biomarkers during and after CSA therapy were performed to evaluate the efficacy of CSA as a prophylactic therapy. 17/19(89%) patients completed 6 months of CSA therapy in a continuous remission. Two patients relapsed during therapy with CSA and 7 patients relapsed after discontinuing CSA therapy. Ten patients have maintained a continuous remission a median of 21 months (range, 5 to 46) after discontinuing CSA. The ADAMTS13 data suggest that CSA resulted in a significant increase in the ADAMTS13 activity during therapy with CSA. 8/9(89%) relapsing patients had severely deficient ADAMTS13 activity (< 5%) suggesting this is a significant risk factor for relapse of TTP. These data support the hypothesis that prophylactic CSA improves the ADAMTS13 activity and may be effective at preventing relapses in patients at risk for recurrences of TTP.

Idiopathic thrombotic thrombocytopenic purpura (TTP) is characterized by frequent recurrences. Effective screening for relapses will enable intervention prior to overt episodes of TTP. The present study used a modified assay to detect ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, 13) activity as low as 0.5%. This analytical improvement permits adequate measurement of ADAMTS13 activity levels in 97% of remission samples used for statistical modelling. ADAMTS13 activity and ADAMTS13 antibody (IgG) were measured in 157 serial samples prospectively collected from 24 TTP patients during periods of clinical remission. These patients were followed-up quarterly for an average of 23 months, during which time nine episodes of TTP relapse occurred among six patients. Finally, logistic regression modelling was used to define the relationship between ADAMTS13 activity levels (0.5-100%) and the probability of TTP relapses. Our data demonstrated that lower ADAMTS13 activity and younger age were significantly associated with higher risk of relapse in the 3 months after specimens were taken. In contrast, ADAMTS13 antibody IgG levels were not predictive of TTP relapses. Identification of low ADAMTS13 activity during clinical remission as a key risk factor for TTP relapses provides a new screening strategy to identify patients who may benefit from prophylactic therapy prior to disease relapses.

We hypothesized that cyclosporin (CSA) as adjunct to plasma exchange (PE) improves the efficacy of PE in idiopathic thrombotic thrombocytopenic purpura (TTP) via suppression of the antibody inhibitor of ADAMTS13. Our preliminary findings with CSA and PE as the upfront treatment of TTP suggested that the addition of CSA to PE significantly decreased the exacerbation (disease recurrence within 30 d of the last PE) rates compared to a cohort that received corticosteroids and PE as their upfront therapy of TTP. We present an updated analysis with long-term follow-up of 18 patients with idiopathic TTP treated with concurrent CSA and PE with analysis of serial measurements of ADAMTS13 activity, antigen and inhibitor concentration in the context of clinical outcome data. Overall, 16/18 (89%) patients achieved remission, similar to historical remission rates in idiopathic TTP with PE with only one patient suffering an exacerbation. Clinical responses correlated with improvements in ADAMTS13 activity and suppression of the antibody inhibitor of ADAMTS13. These data suggest that the efficacy of CSA is at least in part related to its suppression of the antibody inhibitor of ADAMTS13 and a subsequent improvement in ADAMTS13 activity and antigen.

There has clearly been an evolution in the treatment of thrombotic thrombocytopenic purpura (TTP) over the past several years based upon a better understanding of the pathophysiology of this disorder. An autoantibody inhibitor of the ADAMTS13 protease and the resulting severe deficiency of ADAMTS13 function seem to be significant risk factors for the development of TTP, a disease increasingly recognized as an autoimmune disorder. Immune-based therapy aimed at suppressing the inhibitor of ADAMTS13 has been applied to the treatment of patients with TTP who are refractory to the standard treatment of plasma exchange and to patients with multiple recurrences of TTP. More recently, immunosuppressive therapy has been used alone as prophylactic therapy to prevent recurrences of acute TTP. This review focuses on the recent developments in the use of rituximab and ciclosporin in the treatment of TTP.

Summary We present the results of two consecutively performed cohort studies that evaluated the clinical effects of corticosteroids or ciclosporin as an adjunct to plasma exchange (PE) for the treatment of an acute episode of thrombotic thrombocytopenic purpura (TTP). In comparing 12 corticosteroid-treated patients with eight ciclosporin-treated patients, none of the ciclosporin-treated patients suffered an exacerbation or recurrence of TTP in the first 30 d after discontinuing PE compared with 6/10 (60%) of the corticosteroid-treated patients (P = 0·042). These data suggest that ciclosporin may have advantages over corticosteroids as an adjunct to PE therapy in the initial treatment of idiopathic TTP.

Objectives: Recent studies have shown that complement hyperactivation contributes to development of thrombotic microangiopathy. The evaluation of complement biomarkers is known to be influenced by inappropriate specimen handling. However, there has been no study fully addressing this topic. Methods: Blood from each donor was subjected to 62 different handling conditions prior to complement assays. Results: Complement biomarkers (C4d/C3a/factor Bb/C5a/C5b-9) are stable at room temperature (RT) for up to 4 hours in whole blood containing citrate or EDTA. However, under similar conditions, levels of C4d and C3a were significantly higher in serum than those in plasma. Thawing of the samples on ice or at RT had no significant effect on complement levels. In contrast, thawing at 37°C resulted in striking increases in levels of the complement system in serum and citrated plasma but not in EDTA plasma. Up to four freeze/thaw cycles on ice or RT did not substantially increase the levels of C3a, factor Bb, C5a, and C5b-9 but had a significant effect on C4d. Long-term storage of citrated plasma at −80°C for up to 6 years had no significant effect on levels of complement factors. Conclusions: The results from this study thus provide crucial guidelines for future investigations using complement biomarkers to define the role of complement system in disease.

Surface Enhanced Laser Desorption/Ionization Time Of Flight (SELDI-TOF) mass spectrometry is characterized by integration of mass spectrometry with surface chemistry, which gives rise to rapid purification and subsequent determination of protein/peptide analytes. There are several surface matrices, named proteinChips, available for analyzing a particular analyte or a subset of biomolecules in biological samples. Each proteinChip has a unique surface property suitable for fractionation of a specific group of molecules. This article demonstrates the application of SELDI-TOF for the analysis of a cleaved peptide (Mr7739 daltons) from von Willebrand Factor by a metalloproteinase, ADAMTS13. Deficiency of ADAMTS13 is a known primary risk factor for the devastating hematological disorder, Thrombotic thrombocytopenic purpura (TTP). Rapid determination of ADAMTS13 activity helps clinicians tremendously in making the correct diagnosis and initiating timely therapy. Most patients with TTP are acquired cases who exhibit a production of autoimmune antibodies against ADAMTS13 protease. TTP's clinical course is critically controlled by the autoantibody's ability to inhibit ADAMTS13 function. Thus, a second SELDI-TOF based test has been devised to measure ADAMTS13 autoantibody activity for the evaluation of TTP disease activity. In conclusion, the unique features of SELDI-TOF which allow for the examination of the role of key proteases in disease processes have opened up new doors for the clinical application of mass spectrometer based techniques.

This review focuses on recent advances in the use of immune-based therapy to treat patients with refractory and relapsing acquired thrombotic thrombocytopenic purpura.Advances in understanding of the pathophysiology of idiopathic thrombotic thrombocytopenic purpura have provided the rationale for immune-based treatment approaches to refractory and relapsing cases of thrombotic thrombocytopenic purpura. The demonstration that a severe deficiency of von Willebrand factor-cleaving protease activity in acquired thrombotic thrombocytopenic purpura is due to an autoantibody inhibitor of the von Willebrand factor-cleaving protease argues for targeting the antibody inhibitor of the von Willebrand factor-cleaving protease with immunosuppressive therapy as a means of increasing protease activity and inducing a sustained remission of the disease. Recent publications, largely case reports and small series of patients, have suggested a role for rituximab and cyclosporine in cases refractory to plasma exchange or resistant to the tapering of plasma exchange therapy.In the future, a better understanding of the relation between the von Willebrand factor-cleaving protease activity, the nature and titer of von Willebrand factor-cleaving protease inhibitors, and the risk of relapse of thrombotic thrombocytopenic purpura will be important to further validate the strategy of targeting the von Willebrand factor-cleaving protease inhibitory antibody as an effective means of treating acquired thrombotic thrombocytopenic purpura. Prospective data from carefully designed clinical trials are needed to define the effectiveness of specific immunosuppressive therapies at suppressing antibody inhibitors of von Willebrand factor-cleaving protease, improving von Willebrand factor-cleaving protease activity, and inducing a sustained remission of the disease.

Alveolar macrophages (AM) belong to a phenotype of macrophages with distinct biological functions and important pathophysiological roles in lung health and disease. The molecular details determining AM differentiation from blood monocytes and AM roles in lung homeostasis are largely unknown. With the use of different technological platforms, advances in the field of proteomics have made it possible to search for differences in protein expression between AM and their precursor monocytes. Proteome features of each cell type provide new clues into understanding mononuclear phagocyte biology. In-depth analyses using subproteomics and subcellular proteomics offer additional information by providing greater protein resolution and detection sensitivity. With the use of proteomic techniques, large-scale mapping of phosphorylation differences between the cell types have become possible. Furthermore, two-dimensional gel proteomics can detect germline protein variants and evaluate the impact of protein polymorphisms on an individual's susceptibility to disease. Finally, surface-enhanced laser desorption and ionization (SELDI) time-of-flight mass spectrometry offers an alternative method to recognizing differences in protein patterns between AM and monocytes or between AM under different pathological conditions. This review details the current status of this field and outlines future directions in functional proteomic analyses of AM and monocytes. Furthermore, this review presents viewpoints of integrating proteomics with translational topics in lung diseases to define the mechanisms of disease and to uncover new diagnostic and therapeutic targets.

The interaction of lactoferrin with endogenous heparin-like molecules modulates glycosaminoglycan-mediated biological processes. We performed site-specific mutagenesis and expressed recombinant lactoferrin and lactoferrin mutants by the baculovirus insect cell expression system. Five basic residues at the lactoferrin N terminus; Arg 5, Arg 25, Arg 28, Lys 29, and Arg 31, were individually replaced by alanines. Heparin chromatography on fast-performance liquid chromatography system showed that the NaCl concentrations corresponding to the peak of each eluted recombinant protein from the column were 665, 620, 540, 550, 630, or 650 mM for wild-type recombinant lactoferrin, Arg 5, Arg 25, Arg 28, Lys 29, or Arg 31 recombinant lactoferrin mutant, respectively. We compared the ability of each mutated lactoferrin derivative to neutralize glycosaminoglycans in the thrombin serpin inhibition assays. In comparison to wild-type recombinant lactoferrin, all the mutants showed decreased ability to neutralize glycosaminoglycan in a dose-dependent manner. The mutations of lactoferrin at Arg 25 and Arg 28 demonstrated the most striking decrease in lactoferrin’s ability to neutralize various glycosaminoglycans in both enzymatic and plasma clotting-based experiments. Therefore, our results suggest that Arg 25 and Arg 28 are the critical basic residues at the lactoferrin N terminus responsible for heparin binding. The other basic residues on the N terminus, Arg 5, Lys 29, and Arg 31, also contribute to heparin binding by presenting an additional cationic motif.

Atypical hemolytic uremic syndrome (aHUS) is characterized by dysregulated complement activity, the development of a thrombotic microangiopathy (TMA), and widespread end organ injury. aHUS remains a clinical diagnosis without an objective laboratory test to confirm the diagnosis. We performed a retrospective analysis of 103 patients enrolled in the Ohio State University TTP/aHUS Registry presenting with an acute TMA. Nineteen patients were clinically categorized as aHUS based on the following criteria: (1) platelet count100 × 10(9)/L, (2) serum creatinine2.25 mg/dL, and (3) a disintegrin and metalloprotease with thrombospondin type 1 motif, 13 (ADAMTS13) activity10%. Sixteen of 19 patients were treated with plasma exchange (PEX) therapy, with 6/16 (38%) responding to PEX. Nine patients were treated with eculizumab with 7/9 (78%) responding to therapy. In contrast to thrombotic thrombocytopenic purpura (TTP) patients, no aHUS patients demonstrated ultralarge von Willebrand factor multimers at presentation. Median markers of generalized complement activation (C3a), alternative pathway (Bb), classical/lectin pathway (C4d), and terminal complement activation (C5a and C5b-9) were increased in the plasma of these 19 patients. Compared with a cohort of ADAMTS13-deficient TTP patients (n = 38), C5a and C5-9 were significantly higher in the 19 patients clinically characterized as aHUS, suggesting that pretreatment measurements of complement biomarkers C5a and C5b-9 may confirm the diagnosis of aHUS and differentiate it from TTP.

Thrombotic thrombocytopenic purpura (TTP) requires immediate treatment with plasma exchange (PE) to prevent disease mortality and/or morbidity. Frequently, PE is initiated before blood sample is collected to confirm ADAMTS13 deficiency. However, the effect of PE treatments on the evaluation of ADAMTS13 is uncertain. Moreover, the pertinence of ADAMTS13 activity during PE therapy to prediction of treatment outcomes is unclear. Thus, clarification of the diagnostic and prognostic values of ADAMTS13 activity obtained during PE treatment is an unmet clinical need.A total of 212 sequential samples were obtained during the course of daily PE treatment from 19 patients with acquired TTP. ADAMTS13 activity levels were determined in these longitudinal samples for analysis.After the initial three daily PE procedures, the sensitivities of ADAMTS13 activity in diagnosis of TTP (10%) were 89, 83, and 78%, respectively. To determine prognostic value, patients with (n = 7) and without (n = 12) a recovery of ADAMTS13 activity to more than 10% within seven sessions of daily PE treatment were compared. Recovery of ADAMTS13 activity to more than 10% within 7 days is significantly associated with a timely achievement of clinical response (p 0.01). In contrast, the patients without more than 10% ADAMTS13 within 1 week appear at risk for worse treatment outcomes manifested as TTP exacerbation, treatment refractoriness, or death.The data suggest that ADAMTS13 activities measured during the initial period of PE therapy offer both diagnostic and prognostic values in acquired TTP.

Published data demonstrating the efficacy of complement inhibition therapy in patients with atypical hemolytic uremic syndrome (aHUS) are remarkable in contrast to the historically poor long-term prognosis for aHUS patients treated with plasma-based therapy. Although both aHUS and acquired thrombotic thrombocytopenic purpura (TTP) remain clinical diagnoses, an increased understanding of both conditions has improved our ability to differentiate aHUS from acquired TTP. These same data have also demonstrated the importance of a more rapid identification and diagnosis of aHUS as the recovery of end-organ injury present appears to be related to the time to initiate therapy with eculizumab. The diagnosis of acquired TTP can be confirmed by the finding of severely deficient ADAMTS13 activity (

Despite improvements in our understanding of the pathophysiology of thrombotic thrombocytopenic purpura (TTP), little data exist regarding the long-term sequelae following a diagnosis of TTP. We present the results of a comprehensive evaluation of neurologic injury that included a magnetic resonance imaging (MRI), a neurocognitive testing, and an evaluation of health-related quality of life. Twenty-seven patients with a history of idiopathic TTP functioning normally in their activities of daily living were recruited from existing patient cohorts at both the Ohio State University (n 5 12) (Columbus) and the University College London Hospitals (n 5 15) (London, UK). Nine of 23 (39%) of the MRI studies were abnormal; 17/27 (63%) patients demonstrated neurocognitive impairment, particularly in tests of visual learning and memory. Health-related quality of life scores were also significantly lower than age- and gender-matched US norms for both the composite mental component score and physical component score. These data suggest that the prevalence of neurologic findings in TTP patients in remission is quite high and is largely undetected by routine clinical evaluations. Further longitudinal study will be required to define the risk for neurologic injury and the long-term prognosis in patients previously diagnosed with TTP.

To the editor: Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia, hemolytic anemia, and microvascular thrombosis with ischemic injury to organs. Multiple myeloma is a B-cell malignancy with a terminally differentiated plasma cell phenotype characterized by lytic bone lesions, renal insufficiency, anemia, hypercalcemia, and humoral as well as cell-mediated immunodeficiency. In this report, we provide a longitudinal study of two cases of acquired TTP associated with multiple myeloma. Both patients had severe ADAMTS13 deficiency and an elevated ADAMTS13 inhibitor titer at diagnosis, but the diagnosis of TTP and response to plasma exchange (PE) appeared to be independent of the patient’s myeloma disease state. The first patient is a 50 year old African American male with no significant past medical history that complained of low back pain for three months prior to inpatient admission for multiple thoracic and lumbar compression fractures, serum calcium of 10.4 mg/dL, and creatinine 3.6 mg/dL. Bone marrow biopsy showed 75% plasma cells and a normal karyotype. Thalidomide and weekly dexamethasone led to a very good partial response (VGPR)(1), but a monoclonal protein plateau and the development of peripheral neuropathy led to a switch to lenalidomide and weekly dexamethasone for six months. At a time when his monoclonal protein level was stable, he was admitted to the hospital for three episodes of syncope, chest pain, mental status changes, and admission studies showed a non ST-segment elevation myocardial infarction with a troponin of 6 mg/dL, LDH 2046 U/L, platelets of 8,000/mm3, hemoglobin of 9.9 g/dL, and 3+ schistocytes per high power field. Dipyridamole sestamibi cardiac perfusion study and transthoracic echocardiogram were both normal and it was felt the NSTEMI was due to microthrombi from TTP. He initially improved with daily PE for six days with normalizing platelet and LDH, but when PE was held for one day, he suffered mental status changes, a 50% rise in LDH, and a 50% drop in his platelet count. He was started on cyclosporine 2 mg/kg twice daily as we have previously reported (2–3) and the volume of his exchanges were increased. He subsequently achieved clinical response after 7 additional daily exchange treatments. At the time of discharge his LDH and platelet count were both normal and he continued cyclosporine twice daily. Five months later his monoclonal protein continued to rise without symptoms, his TTP remained in remission, and cyclosporine was discontinued. As shown in the figure panels A–C, we examined ADAMTS13 activity, ADAMTS13 antigen levels, ADAMTS13 antibody (IgG) concentration, as well as the antibody inhibitory titer, over the entire course of the disease and follow up visits. Figure Longitudinal evaluation of laboratory values in patients with multiple myeloma and TTP. Panels A–C reference a patient who has TTP diagnosed during a period of disease stability of his myeloma and responds to plasma exchange (PE) and cyclosporine ... The second patient is a 50 year old female with a history of major depression who developed TTP in 1993 with a platelet count of 37,000/mm3 and an LDH of 376 U/L. She was successfully treated with an infusion of three units of fresh frozen plasma per day for five consecutive days. Because of a monoclonal IgA-kappa paraprotein (IgA 903 mg/dL) without other signs or symptoms of myeloma, she was thought to have an IgA monoclonal gammopathy of undetermined significance (MGUS). Thirteen years later she was seen in our medical center with a platelet count of 25,000/mm3, LDH of 390 U/L, and she complained of abdominal pain. Her ADAMTS13 activity was found to be 0.7% with an inhibitory titer of 1.0 BU. Daily infusions of fresh frozen plasma were attempted with an improvement in her abdominal pain, but without a complete clinical recovery of her TTP. Her platelet count remained at approximately 80,000/mm3 with an LDH at 300 U/L. Cyclosporine was added at 2 mg/kg twice daily (2–3) and over the next two months her platelet count stabilized at 60,000–80,000/mm3 but with an ADAMTS13 activity less than 5.5%. Her cyclosporine was then increased to 2.5 mg/kg twice daily, but there was a gradual decrease over the next six months in her platelet count with an increasing creatinine and calcium. She then presented with abdominal pain, petechiae, and bruising with a platelet count 28,000/mm3, hemoglobin 8.8 g/dL, creatinine 2.1 mg/dL, LDH of 398 U/L, and new onset hypercalcemia (12.1 mg/dL); schistocytes were not present on her peripheral smear. Urgent PE was initiated. Further testing revealed new thoracic and lumbar compression fractures and an IgA of 2623 mg/dL due to an IgA kappa myeloma with 40% kappa light chain restricted plasma cells on bone marrow biopsy. Daily PE for three sessions did not yield an improvement by any parameter and, after bone marrow biopsy results were available, she was started on bortezomib 1.3mg/m2 with 20 mg intravenous dexamethasone prior to each infusion. After one cycle her platelets had doubled and creatinine normalized. After three cycles her platelets were normal and remained normal afterward as she received a total of eight cycles of bortezomib and dexamethasone. In the meantime, her ADAMTS13 antibody (IgG) concentration and inhibitory titer were remarkably reduced but her ADAMTS13 activity and antigen remained low. Her serum immunofixation remained positive and approximately 10 months later she went on to autologous hematopoietic stem cell transplantation with melphalan 200 mg/m2. She had a very good partial response (VGPR)(1) to autologous transplant, but, for the first time, her ADAMTS13 activity level normalized along with a parallel increase of her ADAMTS13 antigen level. Her serial ADAMTS13 activity, antigen, autoantibody, and inhibitor titers are shown in the figure panels D–F. Five months after transplant, her monoclonal protein level was barely detectable, signifying stable myeloma disease, but her ADAMTS13 activity disappeared with a rise in her ADAMTS inhibitor titer. In summary, this study reports the first two cases of a comprehensive longitudinal evaluation of acquired TTP arising in association with multiple myeloma. We have carefully documented the clinical evolution of TTP, collected serial specimens from these patients, and performed a comprehensive study of the ADAMTS13 biomarkers with respect to the clinical course of TTP. The development of idiopathic TTP is related to the impairment of the activity of ADAMTS13, a key metalloprotease that cleaves von Willebrand factor (vWF) multimers and prevents vWF-induced platelet thrombosis (4). The majority of idiopathic adult TTP patients have detectable levels of autoantibodies to ADAMTS13 (5) and respond well to PE. Differing from idiopathic TTP, secondary TTP associated with stem cell transplantation, also known as transplant-associated thrombotic microangiopathy (TA-TMA), is not associated with severe ADAMTS13 deficiency (6) and typically does not respond to PE (7). In the two reported cases of secondary TTP after autologous stem cell transplant in patients with myeloma, no ADAMTS13 deficiency was documented and PE failed (8). In our first patient, during a period of disease stabilization, the patient’s immune system was able to mount a sufficient ADAMTS13 autoantibody response that required treatment with both PE and cyclosporine. In the second patient, at the time of progression from MGUS to multiple myeloma, her clinical presentation had features of both TTP and myeloma. Her symptoms and hematologic parameters did not resolve until she received effective treatment for her myeloma, but her ADAMTS13 activity remained low. Interestingly, bortezomib, a drug known to cause significant thrombocytopenia in patients with relapsed myeloma, normalized her platelet count within two cycles. After high dose melphalan followed by autologous stem cell rescue, her ADAMTS13 activity level normalized. We hypothesize that high dose chemotherapy prior to autologous stem cell transplantation depleted the B-cell clone responsible for ADAMTS13 autoantibody production, theoretically in agreement with the immune reset hypothesis for autologous transplant in refractory autoimmune disease (9–10). Unfortunately high dose intravenous melphalan followed by stem cell rescue did not prevent a biochemical recurrence of her TTP five months after transplant. In conclusion, the response to treatment of each of the patient’s TTP was independent of the myeloma disease state. The second patient’s course reveals that high dose intravenous melphalan followed by autologous stem cell rescue has activity in both myeloma and TTP.

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease characterized by acute episodes of widespread microvascular thrombosis. The discovery that the plasmatic activity of the von Willebrand factor cleaving protease, ADAMTS13, is severely deficient in patients with TTP partially clarified the pathophysiology of the disease. However, the finding of severe deficiency of ADAMTS13 alone is unable to fully explain the clinical heterogeneity of the disease. The recent development of methods that measure ADAMTS13 activity with great analytical precision offers the opportunity to define the relationships between levels of ADAMTS13 activity below 10% (herein defined as "residual ADAMTS13 activity") and the clinical manifestations of the disease. Recent studies suggest that the amount of residual activity of ADAMTS13 may be a major determinant of the clinical heterogeneity of TTP. Herein, we review the recent findings on residual ADAMTS13 activity and their implications for research and clinical practice in the field.

INTRODUCTION: A prospective, randomized study (NCT00713193) was undertaken to compare the efficacy and safety of cyclosporine (CSA) and prednisone (P) as adjuncts to plasma exchange therapy (PEX) in the treatment of acquired TTP. The primary objective was to determine if CSA or P given as an adjunct to PEX has a lower exacerbation rate. Secondary endpoints included the effect of CSA and P on the ADAMTS13 activity and anti-ADAMTS13 antibody concentration and their relationship to clinical outcomes. METHODS: Patients were randomized 1:1 to CSA (2-3mg/kg/d orally) or P (1mg/kg/d orally) with daily PEX until response was achieved (normal platelets , LDH). After stopping PEX CSA was continued for 6 months; P was continued at full dose for the first 4 weeks, then tapered over 4 weeks. Exacerbation was defined as recurrent thrombocytopenia requiring PEX 30 days after the last PEX. ADAMTS13 biomarkers were obtained pretreatment, weekly for 4 weeks after the last PEX, monthly for 5 months, and then every 3 months until 42 months of follow-up. RESULTS: Since November 2007, 26 patients were randomized to CSA (n=12) or P (n=14) and followed for a median 42 m. (range, 1 -42). The study was halted after a planned interim analysis. Response rates were comparable in both arms (CSA: 11/12(92%); P:11/14,(79%);p=0.2), with one patient dying on each arm before achieving a response, and 2 P treated patients crossing over to the CSA arm after failing to achieve a response. The exacerbation rate on the CSA arm was 3/11(27%) v. 1/11(9%) on the P arm (p=0.3). Relapses in patients that achieved remission (without exacerbation) occurred in 5/8 CSA treated patients and 4/10 P treated patients at a median of 7 m. (range, 3-39) and 16m. (range, 4-26), respectively. Serial measurements of the ADAMTS13 activity and IgG inhibitor concentrations over the first 30 days after the last PEX and over the 6 m. CSA treatment course are show in Figures 1 and 2. CONCLUSIONS: There was no significant difference in the exacerbation rate between the CSA and P arms. However, improvement in the ADAMTS13 activity and suppression of the anti-ADAMTS13 antibodies were significantly better in the P arm compared to the CSA-treated patients. These ADAMTS13 activity and inhibitor concentration data for the first time provide support for the efficacy of P as an adjunct to PEX, and the superiority of P over CSA as an adjunct to PEX therapy in the initial treatment of acquired TTP. Figure 1 Pretreatment and serial measurements of the median ADAMTS13 activity and ADAMTS13 autoantibodies over the first weeks after stopping PEX therapy Figure 1. Pretreatment and serial measurements of the median ADAMTS13 activity and ADAMTS13 autoantibodies over the first weeks after stopping PEX therapy Figure 2 Serial measurement of the median ADAMTS13 activity over the 6 months CSA dosing period of patients with a continuous remission. Patients were censored at the time of clinical events (crossover, exacerbation, relapse). Figure 2. Serial measurement of the median ADAMTS13 activity over the 6 months CSA dosing period of patients with a continuous remission. Patients were censored at the time of clinical events (crossover, exacerbation, relapse). Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

The quantification of residual plasmatic ADAMTS13 activity in congenital thrombotic thrombocytopenic purpura (TTP) patients is constrained by limitations in sensitivity and reproducibility of commonly used assays at low levels of ADAMTS13 activity, blunting efforts to establish genotype-phenotype correlations. In the present study, the residual plasmatic activity of ADAMTS13 was measured centrally by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (limit of detection = 0.5%) in 29 congenital TTP patients. The results were used to study correlations among ADAMTS13 genotype, residual plasmatic activity, and clinical phenotype severity. An ADAMTS13 activity above 0.5% was measured in 26 (90%) patients and lower levels of activity were associated with earlier age at first TTP episode requiring plasma infusion, more frequent recurrences, and prescription of fresh-frozen plasma prophylaxis. Receiver operating characteristic curve analysis showed that activity levels of less than 2.74% and 1.61% were discriminative of age at first TTP episode requiring plasma infusion < 18 years, annual rate of TTP episodes > 1, and use of prophylaxis. Mutations affecting the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity and a more severe phenotype in an allelic-dose dependent manner. The results of the present study show that residual ADAMTS13 activity is associated with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.

While our understanding of the pathophysiology of both congenital and acquired forms of thrombotic thrombocytopenic purpura (TTP) has increased, it is only over the past couple of years that this understanding has been translated into clinically significant advances in the diagnosis, management, and treatment of TTP. More specifically, our understanding of the central role of the ADAMTS13 protease in the pathophysiology of TTP has allowed ADAMTS13 testing to have a more prominent role in confirming or re-evaluating the clinical diagnosis of idiopathic TTP with the finding of severely deficient or measurable ADAMTS13, respectively. Additionally, measurement of ADAMTS13 activity at presentation and during remission may be useful to predict both the risk of exacerbation and relapse in patients with idiopathic TTP. There are also several novel approaches to the therapy of TTP including a recombinant ADAMTS13 protease and agents that target the A1 domain of von Willebrand factor (VWF), blocking its interaction with platelet glycoprotein (GP)Ib to prevent the formation of microthrombotic disease in patients with TTP. The addition of these exciting new therapies to conventional plasma-based and/or immune modulating therapies provide hope for improved treatment outcomes for TTP patients.

Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy.

Objective: Approximately 40% of idiopathic thrombotic thrombocytopenic purpura (TTP) patients will suffer an exacerbation (recurrence of TTP within 30 d after their last plasma exchange (PE) procedure), but there are no data to predict who is at greater risk. We studied the clinical utility of demographic and ADAMTS13 biomarker data to predict the risk for exacerbation. Patients: Forty-four acute episodes of idiopathic TTP from 26 patients were studied. Methods: PE was performed plus either prednisone (1 mg/kg/d) or cyclosporin (2–3 mg/kg/d) as adjuncts. PE was continued daily until response (platelet count >150 000/μL and normalized lactate dehydrogenase) and tapered uniformly in all patients. ADAMTS13 biomarkers were studied prior to PE and after achieving a response, but within 7 d of the last PE. Results: African American race (AA) was associated with an increased risk for exacerbation (P = 0.046). ADAMTS13 at presentation was also significantly lower in patients experiencing an exacerbation (P = 0.0364). After adjusting for the race effect, ADAMTS13 remained marginally significant (P = 0.0569). Conclusions: AA is significantly associated with an increased risk for exacerbations of TTP. These data also suggest that decreasing pretreatment ADAMTS13 activity was associated with an increased risk for exacerbation, even after accounting for the effect of race.

Plasma microparticles (MPs) are spherical cell membrane fragments derived from either apoptotic or activated cells. Characterized by a rich phospholipid moiety and many protein constituents, MPs normally circulate in the blood and contribute to numerous physiological processes. In disease states, MPs derived from the injured organ likely contain valuable markers for determining the site, type, and extent of disease pathology. However, the basic protein characteristics of plasma MPs have yet to be described. In this study, MPs from a pooled plasma sample derived from 16 healthy donors, all of group A blood type, were prepared by ultracentrifugation. Flow cytometry confirmed that a majority of these MPs are smaller than 1 microm. Factor Xa generation assay revealed the presence of tissue factor activity in these MPs, confirming MPs' role in initiating blood coagulation. The MP proteome was analyzed by two-dimensional (2-D) gel electrophoresis performed in triplicate, and compared with a 2-D gel of pooled whole plasma and blood platelets. Overall, plasma MPs displayed distinct protein features and a greater number of protein spots (1021-1055) than that detected in whole plasma (331-370). Protein spots expressed in high abundance in the MP proteome were then excised and submitted for protein identity determination. This process provided protein identification for 169 protein spots and reported their relative protein quantities within the MP proteome. These 169 protein spots represented 83 different proteins and their respective isoforms. Thirty of these proteins have never before been reported in previous proteome analyses of human plasma. These results provide unprecedented information on the MP proteome and create a basis for future studies to understand MP biology and pathophysiology.

Alveolar macrophages (AMs) are a subset of tissue macrophages situated in the alveolar milieu. Compared with their precursor blood monocytes, AMs exhibit distinct physiologic functions unique to their anatomic location. However, the molecular details that control monocyte differentiation into AMs remain unknown. This study employed a proteomic approach to define protein characteristics that distinguish AMs from monocytes. AMs and monocytes were obtained from six nonsmoking, healthy donors. Whole cell lysates from each donor's AMs and monocytes were analyzed by two-dimensional (2D) gel electrophoreses. The protein density for each protein spot in a 2D gel was compared between these two cell types. Proteins that demonstrated consistent level changes of greater than 2.5-fold in all six donors were subjected to tandem mass spectrometry for protein identity. Using this process, we revealed proteome changes in AMs that relate to their physiologic roles in proteolysis, actin reorganization, and cellular adaptation in the unique alveolar milieu. By comparison, blood monocytes displayed higher levels of the proteins involved in transcription, metabolism, inflammation, and in the control of proteolysis. These results provide new insights into the biology of mononuclear phagocytes and set a basis for future causality studies.

Platelet transfusion is known for carrying a high incidence of clinically significant transfusion reactions such as febrile nonhemolytic transfusion reaction. The mechanism responsible for these transfusion-associated adverse events, however, is poorly understood. In this study, we hypothesize that prolonged in vitro storage activates the complement system in the platelet product that in turn causes a high frequency of transfusion reactions. Fresh platelet units obtained from three blood donors were stored on a temperature controlled platelet rotator between 22-24 C°. An aliquot of platelet product was obtained using sterile techniques from each unit on day 2 through day 7. The platelet product from each collection was then immediately centrifuged to obtain platelet poor plasma for the study of complement activation levels. For all study samples, C4d levels were assayed to evaluate the activation of the classical pathway, factor Bb levels were measured to determine the status of the complement alternative pathway, C3a levels were used to examine common pathway activation, and C5a and C5b-9 were assayed for determination of the terminal pathway activation of the complement system. The reference range for each complement factor was determined using citrated plasma from 40 healthy donors. As shown in table 1, both C4d and C3a demonstrated time-dependent increases relevant to storage time. On day 7, C4d and C3a levels were five-fold higher than their baseline levels measured on day 2. In contrast, factor Bb levels remained stable and within the normal range throughout the study. Over a storage span of seven days, the terminal complement factors C5a and C5b-9 were also significantly increased, although not as dramatically as C4d and C3a. Figure 1 illustrates a progressive increase of C3 activation in all three study donors over the time of storage (2-7 days). This report, for the first time, provides strong evidence that substantial complement activation occurs in the platelet products under standard storage conditions. A longer storage time of platelet product in vitro is accompanied by a remarkable elevation of complement activation biomarkers. By examining the pattern of complement profiles in the stored platelets, we further demonstrated that the activation of the classic pathway, rather than alternative pathway, appears to be the driving event that leads up to a level of over-reactivity of the complement system. Given the fact that complement hyperactivation is known to disrupt host homeostasis and cause disease, the adverse reactions seen in platelet recipients is likely related to the infusion of C3a and C5a which are known to be potent inflammatory cytokines. The observations from this study therefore provide a new perspective in understanding the pathophysiology responsible for adverse reactions from platelet transfusions. Further studies will be required to fully evaluate the clinical impact of complement activation in transfused platelet products. Figure 1 Figure 1. Disclosures Cataland: Alexion Corporation: Honoraria, Research Funding, Speakers Bureau. Wu:Alexion Corporation: Honoraria, Research Funding, Speakers Bureau.

Neurological injury is associated with an acute episode of microangiopathy, but only recently have persistent deficits in memory, concentration, mood and mental endurance been reported in patients who have long recovered from an acute episode of Thrombotic Thrombocytopenia Purpura (TTP). The mechanism for these findings remains unclear. To date, no studies have addressed the frequency or severity of headaches in patients with a previous history of TTP or atypical hemolytic uremic syndrome (aHUS). Based upon the longitudinal follow-up of our TTP and aHUS patients, we hypothesize that headaches are more incapacitating in patients with a history of TTP or aHUS than has been appreciated previously. Adult patients >3 months since their last acute episode of TTP/aHUS were approached to take a Headache Impact Test (HIT-6) survey in an outpatient office setting. HIT-6 was developed to screen and monitor patients with headaches in both clinical practice and clinical research. Yang et al. has shown that HIT-6 is a reliable and valid tool for discriminating headache impact across episodic and chronic migraine headaches. The questionnaire covers 6 aspects of functioning most impacted by headache (pain, role functioning (the ability to carry out usual activities), social functioning, energy/fatigue, cognition and emotional distress). Scores range from 36 to 78, with higher scores reflecting greater impact and severity (Table 1). Our objective was to determine if headaches were more severe in patients with a previous diagnosis of TTP or aHUS compared to the general population. We used clinical and demographic features of patients including age, race, sex, number of microangiopathy hemolytic anemia episodes and time since last thrombotic microangioagropathy episode for subset analysis. Between June 1, 2013 and May 30, 2014, 31 patients completed the HIT-6 survey. Of the patients surveyed, 28 were female and 3 were male. Median age was 51 years (range 19-69) and 8 of 31 (26%) subjects were African American. The average HIT-6 score for all 31 subjects was 57.3, significantly higher than sex-matched controls with a HIT-6 score of 51.7 (SD 10.2, p value 0.005, Table 2). Overall, TTP patients had a significantly higher average HIT-6 score of 59.9 vs 51.7 for sex matched controls (SD 9.6, p value 0.002, Table 3). There was no significant difference in HIT-6 scores of aHUS patients and sex matched controls (HIT-6 of 51.9 vs 51.7). Of TTP patients studied, the majority had 3 or more acute episodes and were >24 months since last acute TTP episode. The study was not powered to detect the significance of episodes among only TTP patients; however these data suggest that the number of episodes and time since the last episode did not predict higher scores (Table 4). Survivors of TTP in remission have increased headache severity compared to the general female population. There was no difference in headache severity among aHUS patients. An awareness of increased headache severity in patients with a previous history of TTP may lead to earlier interventions to minimize the morbidity associated with chronic headaches. Additional research is needed to help elucidate the cause of headaches in times of remission.Table 1:HIT-6 interpretationHIT-6 ScoreInterpretation60 or moreHeadaches are severely impacting the patient’s ability to function in everyday life.56 to 59Headaches are substantially impacting the patient’s ability to function in everyday life.50 to 55Headaches are having some impact on the patient’s life but are probably not causing the patient to miss time from family, work, school, or social activities because of headache.49 or lessHeadaches are having little or no impact on the patient’s life. Table 2: Norms for the general US population (N=1,016) HIT -6 Score Mean 50.00 Females (N=795) HIT Scale Mean 51.74 Males (N=221) HIT Scale Mean 47.55 Table 3: Summary of HIT-6 score by disease (aHUS vs. TTP) TTP-aHUS N mean sd p -value1 aHUS 10 51.9 9.9 TTP 21 59.9 9.6 0.002 Total 31 57.3 10.2 0.005 1 p-value based on one-sample t-test comparing the HIT-6 score to the female US norm value of 51.74 for HIT-6. P-values have been adjusted in ordered to conserve the overall type I error rate at 0.05. Table 4: Summary of TTP HIT-6 score by episodes and by months since last episode Months since last episode N mean sd 3-6 3 59.3 9.0 7-12 3 57.0 6.6 13-24 3 69.3 2.3 >24 12 58.4 10.6 Total 21 59.9 9.6 Episodes N mean sd 1 7 61.3 10.3 2 2 52.5 7.8 3 or >3 12 60.3 9.5 Total 21 59.9 9.6 Disclosures No relevant conflicts of interest to declare.

Introduction Recent studies demonstrating the effectiveness of eculizumab in the treatment of atypical hemolytic uremic syndrome (aHUS) have increased the need for more rapid and objective diagnostic tests to help confirm the diagnosis of aHUS over other thrombotic microangiopathies (TMA). Methods We performed a retrospective study of all patients enrolled in the Ohio State University TTP/aHUS Registry since 2003. From this cohort of patients diagnosed with an acute TMA (platelets2.25 mg/dL and measureable ADAMTS13 activity (>10%) were analyzed. The clinical and demographic data for these 19 patients are shown in Table 1. Banked plasma samples from these study subjects were used to measure complement activation factors Bb (alternative pathway (AP)), C4d (classical pathway), C3a, (generalized complement activation), C5a, and C5b-9 complex (terminal complement pathway (TCP)). These complement biomarker data were analyzed in the context of a documented mutation in complement regulatory proteins and response to therapy with PEX and/or eculizumab. Response was defined as a normal platelet count and improved serum creatinine (at 6 months) independent of PEX therapy or with continued eculizumab therapy. Results The median complement biomarker data at presentation are shown in Table 2. Complement activation products are generally sensitive biomarkers for aHUS patients. Notably, elevation of the AP (Bb) and the TCP (C5b-9) biomarkers were more prominent and were seen in 16/19 and 19/19 patients respectively. This is in contrast to previously published data from acquired TTP patients from our group that demonstrated less pronounced activation of the AP and TCP at presentation (accepted for publication, JTH). Similar levels of alternative, classical and terminal complement activation were seen among patients regardless of mutation status or response to PEX. In a preliminary analysis using a one sample T-test model, (Bb) activation was shown to be significantly lower in the non-responder to eculizumab compared to the 8 responding patients. TCP activation was also lower in the non-responder but did not show statistical significance. Conclusions Complement biomarkers of the alternative and terminal complement pathway may more precisely define the diagnosis of aHUS. These hypothesis generating data suggest that pre-treatment complement biomarkers provide valuable information in the diagnosis of aHUS and could be useful in the prediction of response to eculizumab Disclosures: No relevant conflicts of interest to declare.

Thrombotic thrombocytopenic purpura (TTP) is a devastating hematological disease that requires immediate treatment with daily plasma exchange (PE) to prevent mortality and morbidity. In many cases, PE procedures were initiated prior to a collection of patient specimen for laboratory testing. ADAMTS13 deficiency (activity Table 1 shows the percentage of patients who exhibited ADAMTS13 activity 10% within one week of PE treatment while others did not show >10% ADAMTS13 activity until day 10, later or expired. We evaluated the correlation between the improved ADAMTS13 activity with patient characteristics at presentation and PE treatment outcomes. As shown in Table 2, a prompt rise of ADAMTS13 activity during PE therapy is not influenced by age, gender, race, type of TTP episode, platelet count, LDH, or ADAMTS13 activity levels at the time of acute presentation. However, the patients with a higher inhibitor titer or abnormal renal function prior to PE treatment are significantly related with the failure to obtain ADAMST13 >10% after 7 daily PE treatments. After further analysis of the treatment outcomes in this cohort, it was determined that the group with an adequate rise of ADAMTS13 activity is associated with an achievement of clinical response within one week of PE therapy. The group that failed to achieve >10% ADAMTS13 activity within one week is related to the PE treatment failure manifested either as TTP exacerbation, disease refractoriness, or death, but the correlation did not reach a statistical significance (p=0.147). There is no correlation between ADAMTS13 recovery status during PE and future risk of TTP relapses. We concluded from this study that the pre-PE samples obtained prior to PE#4 provide diagnostic value in most cases of acquired TTP. Sequential examination of ADAMTS13 activity during the course of PE treatment may offer some prognostic information with regard to treatment outcomes. Disclosures: No relevant conflicts of interest to declare.

Abstract 2219 ABSTRACT Congenital thrombotic thrombocytopenic purpura (TTP) (OMIM #274150) is a rare, recessively inherited thrombotic microangiopathy characterized by the congenital deficiency of ADAMTS13 due to mutations in the corresponding gene. The clinical phenotype of congenital TTP is variable, encompassing neonatal-onset disease and adult-onset disease, forms with a single disease episode and chronic-relapsing forms. A review of the ∼100 published cases of congenital TTP showed similar age of first disease episode in patients carrying the same ADAMTS13 gene suggesting that different ADAMTS13 mutations might influence the severity of clinical phenotype, conceivably by determining different patterns of residual plasmatic activity of ADAMTS13. However, the quantification of residual ADAMTS13 activity in severely deficient patients was blunted by the relatively high limit of detection (LOD) of commonly used ADAMTS13 activity assays (varying between 3% and 6%). We report 29 cases of congenital TTP from 4 European cohorts, with 32 mutations of ADAMTS13 including 8 not previously published. To assess if residual ADAMTS13 activity was detectable in these patients and correlated with their clinical history, ADAMTS13 activity was measured by SELDI-TOF mass spectrometry (LOD=0.5%). ADAMTS13 activity above 0.5% was measurable in 26 (90%) patients, and lower levels of activity correlated with an earlier age of disease onset (r=0.51; p=0.006) and with the need for regular fresh frozen plasma prophylaxis (2.24% vs 3.88%; p=0.03). Mutations at the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity (1.48% vs 4.89%, p=0.02) and earlier disease onset (3.2 years vs 24.2 years, p=0.003). Our results show that residual ADAMTS13 activity correlates with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations. Disclosures: No relevant conflicts of interest to declare.

Background: Although nearly all patients with idiopathic TTP will achieve remission, a majority of patients will suffer a recurrence at some point in the future. Our group has had an ongoing interest in using laboratory biomarkers to predict the risk of recurrence of idiopathic TTP after patients achieve remission and complete their initial therapy. Method: Twenty patients (Table 1) with idiopathic TTP were analyzed beginning from the time of their initial presentation to our institution. 8 patients had a history of previous episodes of TTP. Patients were followed prospectively and monitored for relapse of TTP. Nine patients have maintained a continuous remission (Non-relapse) and 11 patients (Relapse) had a relapse of TTP. 139 serial samples from both groups, a median of 6.5/patient, were analyzed and compared in terms of ADAMTS13 activity, ADAMTS13 antibody concentration, and ADAMTS13 antigen. Median follow-up for the "Non-relapse" group was 39 months (range, 12 to 50). The median time to relapse was 11 months (range, 1.5 to 34) in the "Relapse" cohort. Data for the relapse group was censored at the time of relapse accounting for the shorter median follow-up of that cohort. The serial biomarkers from the "Relapse" group presented below reflect only samples obtained during continuous remission. Results: Serial measurements of laboratory biomarkers are presented for both the "Relapse" and "Non-relapse" patients in Figure 1 below. During the first year of follow-up, the "Non-Relapse" patients had significantly higher ADAMTS13 Ag levels (p = 0.0065) compared to the patients who relapsed. There was no significant difference in the ADAMTS13 activity and antibody concentration between the two groups during the first year of follow up. In the 11 patients who relapsed, the mean ADAMTS13 activity was 1.2% (range, 0.5 – 3.2%), the mean antibody concentration was 958 ug/ml (range, 147 – 4,988), and the mean ADAMTS13 antigen was 221 ug/ml (range, 73 – 468) at the time of disease recurrence. Conclusion: These data suggest that ADAMTS13 antigen determinations in the first year of follow-up may predict which patients will have a sustained remission of their disease. ADAMTS13 antigen measurement, compared to ADAMTS13 activity or antibody concentrations, may provide additional information to more accurately predict a patient’s future clinical course after achieving remission. Demographic and Laboratory Data at Presentation Mean Laboratory Data at Presentation (range) N Age Sex Previous TTP ADAMTS13 % ADAMTS13 Ab(ug/ml) ADAMTS13 Ag. (ng/ml) Relapse 11 37 (19–56) 4M/7F 4/11 2.5% (0.6 – 7.9) 1,221 (190 – 3,397) 360 (99 – 1,195) Non-Relapse 9 47 (21–59) 2M/7F 4/9 4.2% (

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a devastating disorder characterized by frequent recurrences. A predictive biomarker that allows for the early identification and intervention prior to the overt relapse of disease is needed. In the present study, the assay for ADAMTS13 activity was modified to adequately detect ADAMTS13 activity as low as 0.5%. Serial samples (n=157) collected from 24 TTP patients during periods of sustained clinical remission were then studied. These patients were followed up quarterly for an average of 23 months, during which time 9 episodes of TTP relapses from 6 patients occurred. We measured ADAMTS13 activity in these samples and then employed logistic regression modeling to evaluate the relationship between ADAMTS13 activity levels and the risk of TTP relapses. Our data demonstrated that lower ADAMTS13 activity and younger age are significantly associated with a higher probability of relapse in the 3 months following the date the specimens were taken. In contrast, ADAMTS13 antibody IgG levels are not predictive of TTP relapses. Identification of low ADAMTS13 activity during remission as a key risk factor for TTP relapses provides a new screening strategy to identify patients who may benefit from prophylactic therapy prior to disease relapses.

We previously reported that concurrent cyclosporine and plasma exchange(CSA/PE) is superior to corticosteroids and PE for acute TTP in terms of a decreased exacerbation rate (0% v. 47%) after stopping PE.(ASH 2005,Abstract# 1235) We now present similar data and serial analysis of ADAMTS13 activity and inhibitor concentration from two patient cohorts treated with CSA/PE. Patients: Ten patients failing initial therapy with corticosteroids and PE (exacerbations or refractory) were treated with CSA/PE. This study led to a second cohort study of upfront therapy with CSA/PE in 11 patients with acute TTP. Methods: CSA (2–3 mg/kg) orally was administered concurrent with daily PE. After remission (normal platelet count and LDH), 2 additional exchanges were performed on an every other day schedule. CSA was administered for 6 months and discontinued. Results: In the exacerbation/refractory patients, 9/10 achieved remission after a median of 11 exchanges. One patient suffered an exacerbation and required one additional exchange to achieve a sustained (>30 d.) remission. Four patients relapsed 2,3,7, and 10 months after completing 6 months of CSA. In the CSA/PE upfront group, 10/11 achieved remission. One patient died of infectious and disease-related complications. Remission occurred after a median of 5 exchanges. No patients(0/10) suffered an exacerbation. Three patients recurred, 2 while taking CSA and one 2 weeks after stopping CSA. Median ADAMTS13 activity and the change in inhibitor concentration over time for all CSA/PE patients is shown in Figure 1. Conclusions: CSA/PE improves clinical outcome and prevents exacerbations after tapering PE. CSA/PE also increased in vitro ADAMTS13 activity and suppressed the inhibitor, with both occurring well after the last PE. These findings coupled with the decline in median ADAMT13 activity after discontinuing CSA(Fig. 2) suggest the improvement in ADAMTS13 activity was a result of CSA therapy. Table 1. Presenting ADAMTS13 and Inhibitor Data N Med. ADAMTS13% Prior to Initial Therapy Med. ADAMTS13% Prior to CSA and PE Med. Inhibitor Conc. Prior to Initial Therapy(ug/mL) Med. Inhibitor Conc. Prior to CSA and PE(ug/mL) Remission Rate Exacerbation (30 d. Recurrence) Rate CSA and PE Exacerbation and Refractory 10 2 (0–8) 4 (0–20) 587 (275–1,111) 3,173 (372–9,845) 9/10 1/10 CSA and PE Upfront Therapy 11 N/A 2 (0–60) N/A 571 (147–4,988) 10/11 0/10 Table 2. Effect CSA and PE on ADAMTS13 Activity and Inhibitor Concentration. Time (weeks) 0 4 10 24 CSA and PE Exac. and Refractory N 9 9 7 8 Median ADAMTS13 % 4 (0–20) 24 (4–39) 42 (5–72) 49 (7–116) Median Inhibitor Conc. (ug/mL) 3,173 (372–9,845) 294 (165–6,548) 191 (41–4,228) 204 (55–756) CSA and PE Upfront Therapy N 10 8 5 4 Median ADAMTS13% 2 (0–60) 20 (5–104) 32 (4–37) 49 (7–116) Median Inhibitor Conc. (ug/mL) 571 (147–4,988) 270 (82–2,637) 302 (110–378) 204 (55–756) Figure Figure