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Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare and life-threatening hematologic disorder. The decision to start plasma exchange is challenging in clinical practice, due to aTTP rarity, and the extensive differential diagnosis with other thrombotic microangiopathies (TMA). Although the ADAMTS13 activity measurement is essential to aTTP diagnosis, a rapid quantification is unavailable in most centers, being necessary to develop clinical prediction models to evaluate severe enzymatic deficiency. The first model developed was Bentley score, followed by French score and more recently PLASMIC score. Studies comparing these 3 methods are scarce. The criteria of published scoring models are summarized in Table1 Aim: To compare and evaluate the discriminative power of the Bentley, French and PLASMIC scores in predicting severe ADAMTS13 deficiency (≤10%) in our group of patients (pts). Methods: We performed a retrospective analysis of pts with ADAMTS13 activity tested between 2008 and 2019, in a single center. Pts were divided in two groups: ADAMTS13 activity ≤10% (ADAMTS13≤10%) and >10% (ADAMTS13>10%) and then stratified according to the 3 scores. We evaluate sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) to predict low ADAMTS13 activity. Diagnostic accuracy was tested using the area under the ROC curve. A P-value of Results: ADAMTS13 activity was evaluated in 119 pts with suspected aTTP. There was a female predominance (60.5%; n=72) and the mean age was 54±20 years. Thirty-one pts had ADAMTS13≤10% and 27 (87.1%) of them had been diagnosed with aTTP, two were inherited thrombotic thrombocytopenic purpura and the other two were sepsis associated TMA. Comparing the groups ADAMTS13≤10% and ADAMTS13>10%, pts in the first were younger (46±18 vs 57±21 years; p=0.008), had lower platelet count (23.04±27.1 vs 47.79±37.6x109/L; p PLASMIC score was calculated in all pts. Due to missing data, Bentley score was only applied in 44.5% (n=53) and French score in 54.6% (n= 65) pts. Using a high-risk PLASMIC score as the predictor, the sensitivity was 80.6%, specificity 78.4%, PPV 56.8% and NPV 92.0%. The C-statistics for high PLASMIC score cut-offs (>5 points) was 0.80 (95% confidence interval [CI] 0.70-0.89) (fig.1). Applying high-risk Bentley score vs low-to-intermediate-risk score, the sensitivity was 44.4%, specificity 94.3%, PPV 80.0% and NPV 76.7%. The C-statistics was 0.69 (95% CI 0.53-0.86) using the high Bentley score cut-offs (fig.2). The French score had higher sensitivity (83.3%), but lower specificity (36.2%), with 33.3% of PPV and 85.0% of NPV. Using the high French score cut-offs, the C-statistics was 0.60 (95% CI 0.45-0.75) (fig.3). There were not statistically significant differences between PLASMIC and Bentley scores in the ability to predict low ADAMTS13 activity, and both performed better than French score (p Conclusion: In our cohort, the PLASMIC score showed to be the most balanced and efficient clinical prediction score, with high sensitivity and specificity. Bentley score was also a good predictor of ADAMTS13 activity, but had lower sensitivity and less applicability; French score was comparatively inferior. Pts with low PLASMIC score are highly unlikely to have ADAMTS13 deficiency. PLASMIC score is easy to calculate and readily usable in most emergency settings while the French and Bentley scores require additional laboratory data, which is not always accessible. These results support the previously published data, showing PLASMIC score as a helpful complementary tool in clinical practice recognizing high-risk pts when ADAMTS13 activity measurement is not available. Disclosures No relevant conflicts of interest to declare.

Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare but life-threatening thrombotic microangiopathy, with an untreated mortality rate of >90%. Prompt treatment with therapeutic plasma exchange (TPE) and immunosuppression improves outcomes in patients with aTTP, but 10-20% of patients still die acutely from this disease. The aim of this analysis was to describe in more detail the characteristics and disease courses of the patients who died during the caplacizumab clinical development program. Methods: Patient narratives on all deaths occurring during the phase 2 TITAN and phase 3 HERCULES studies were extracted. Results: In the overall study periods, a total of 6 patients died, 2 patients enrolled in TITAN (Patients 1 and 2) and 4 patients enrolled in HERCULES (Patients 3-6). Five patients received placebo, while 1 (Patient 6) received caplacizumab. Demographics and baseline disease characteristics are summarized in Table 1. The patient narratives are provided below. Patient 1 (placebo) was a 57-year-old male, with a recurrent episode of aTTP. Baseline platelet count was 25 x109/L and ADAMTS13 activity Patient 2 (placebo) was a 49-year-old female, with an initial presumed aTTP episode. Baseline platelet count was 7 x109/L and baseline ADAMTS13 activity was 75%. The patient did not respond to therapy (TPE and corticosteroids), with platelet counts remaining below 35 x109/L over the whole period. On Day 10 of the study, the patient experienced a cerebral hemorrhage, for which study drug treatment was permanently discontinued. The patient was intubated and died the next day of cerebral hemorrhage. Patient 3 (placebo) was a 62-year-old female with an initial aTTP episode. Baseline platelet count was 18 x109/L and ADAMTS13 activity Patient 4 (placebo) was a 72-year-old female with an initial aTTP episode. Baseline platelet count was 21 x109/L and baseline ADAMTS13 activity was Patient 5 (placebo) was a 30-year-old female enrolled with her third aTTP episode. Baseline platelet count was 21 x109/L and ADAMTS13 activity Patient 6 (caplacizumab) was a 77-year-old female with her initial aTTP episode. Baseline platelet count was 38 x109/L and ADAMTS13 activity Conclusion: Although the use of TPE and immunosuppression reduces mortality in patients with aTTP, the disease is still associated with a substantial risk of mortality. The fact that all 5 immediate deaths occurred in the placebo arm suggests that the use of caplacizumab has the potential to reduce acute mortality in patients with aTTP. Table 1. Baseline demographics and disease characteristics. Table 1 Disclosures Cataland: Ablynx/Sanofi: Consultancy, Research Funding; Alexion: Consultancy, Research Funding. Scully:Alexion: Consultancy; Ablynx/Sanofi: Consultancy; Novartis: Consultancy; Shire/Takeda: Consultancy; Shire: Research Funding. Peyvandi:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Grifols: Honoraria; Kedrion: Honoraria; Alnylam: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Octapharma: Research Funding. Knoebl:Roche: Consultancy; Shire/Takeda: Consultancy; Novo-Nordisk: Consultancy, Research Funding; CSL-Behring: Consultancy; Ablynx/Sanofi: Consultancy. Kremer Hovinga:Ablynx/Sanofi: Consultancy, Honoraria, Other: Honoraria go to employer (Insel Gruppe AG, Department of Hematology); Shire: Consultancy, Other: Honoraria go to employer (Insel Gruppe AG, Department of Hematology), Research Funding; CSL-Behring: Honoraria, Other: Honoraria go to employer (Insel Gruppe AG, Department of Hematology); Roche: Honoraria, Other: Honoraria go to employer (Insel Gruppe AG, Department of Hematology); Siemens: Honoraria, Other: Honoraria go to employer (Insel Gruppe AG, Department of Hematology). Coppo:Ablynx/Sanofi: Consultancy; Alexion: Consultancy; Shire: Consultancy. Metjian:Genentech: Consultancy, Research Funding; AblynxNV/Sanofi: Consultancy, Research Funding. De La Rubia:Takeda: Consultancy; AMGEN: Consultancy; Janssen: Consultancy; Celgene Corporation: Consultancy; AbbVie: Consultancy. Pavenski:Ablynx: Honoraria, Research Funding; Bioverativ: Research Funding; Shire: Honoraria; Octapharma: Research Funding; Alexion: Honoraria, Research Funding. De Winter:Ablynx, a Sanofi company: Employment. Callewaert:Sanofi (formerly employed by Ablynx, a Sanofi company): Employment.

Introduction: Increases in plasma von Willebrand Factor (vWF) levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated in diseases of microvascular injury. We hypothesized that following severe trauma, a burst of ultra-large vWF (UL-vWF) is released into the bloodstream by damaged endothelium, resulting in increased thrombogenicity due to circulating vWF multimers. We further hypothesized that traumatic injury would lead to a deficit of ADAMTS13, promoting the accumulation of UL-vWF forms and, ultimately, the increased risk of microvascular disease, such as acute kidney injury (AKI). Methods: A cohort of 37 severely injured trauma patients was analyzed for antigen levels of plasma vWF and ADAMTS13 at 0- and 24-hours after admission. Circulating vWF multimeric composition from both time points was determined by vertical agarose gel electrophoresis. Multivariate analyses were performed with data abstracted from the electronic medical records to identify further dependences. A similar analysis was also performed on plasma from a cohort of 8 patients with trauma induced AKI at 0-, 24-, and 72-hours after admission; these patients were well matched against trauma patients without AKI. Finally, we utilized a murine model of polytrauma and hemorrhage, in conjunction with qRT-PCR of ADAMTS13 in total liver RNA, to specifically address how the expression of ADAMTS13 is altered by the systemic effects of traumatic injury. Results: Circulating vWF levels were increased in severe trauma patients when compared to healthy controls at presentation (189% (110-263) vs. 95% (74-120)) and persisted through 24-hours (213% (146-257) vs. 132% (57-160)). Ultra-large vWF forms were elevated at both 0- and 24-hours when compared to pooled normal plasma ((10.0% (8.9-14.3) and 11.3% (9.1-21.2), respectively, vs 0.6%). The largest vWF forms within trauma patient plasma circulated at 33±4 dimers vs 18±1 dimer in length within pooled normal plasma. Severe trauma patient ADAMTS13 activity was decreased at 0-hours (66% (47-86) vs. 100% (98-100)) and at 24-hours (72.5% (56-87.3) vs 103% (103-103)) when compared to healthy patients. Furthermore, the proportion of circulating low molecular weight multimeric (LMWM) vWF to total circulating vWF forms was directly dependent upon ADAMTS13 activity at 24-hours (Decreased ADAMTS13 Activity: 20.4% (15.0-22.7) LMWM vWF vs Normal Activity: 25.8% (22.7-35.2) LMWM vWF). Strikingly, ADAMTS13 activity independently predicted the development of coagulopathy, correlating with presentation INR (ρ =-0.63), activated clotting time of thromboelastography (TEG) (ρ=-0.36), and TEG maximum amplitude (ρ=0.36). ADAMTS13 activity also closely correlated with injury severity (ISS) (ρ=-0.34) and blood product transfusion (ρ =-.45). The cohort of 8 trauma patients who went on to develop AKI showed a 1.54-fold (1.02-2.05) increase in plasma vWF antigen levels between 0 and 72 hours, while those who did not develop AKI showed no change in vWF levels over this time period. Furthermore, those who developed AKI demonstrated a smaller proportion of LMWM vWF in plasma than those who did not (25.4% (23.4-28.0) vs 31.2% (27.2-35.6)), suggesting the increased thrombogenicity of their circulating vWF forms. Finally, qRT-PCR of total liver RNA in 6 mice demonstrated a 2-fold decrease in ADAMTS13 RNA expression levels between the times immediately before and 24-hours after trauma. Altogether, these data indicate that both circulating ADAMTS13 and its production are deficient in the days following severe injury. Conclusions: Severe traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes thrombosis. Not only is the concentration of circulating ADAMTS13 decreased following traumatic injury, but hepatic expression of the enzyme is lacking as well. In the immediate moments following injury, these mechanisms contribute to life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to burden that may exacerbate microvascular disease. Disclosures Ragni: Bioverativ/Sanofi: Consultancy, Research Funding; Sangamo: Research Funding; Shire/Takeda: Consultancy, Other: Study drug; Alnylam/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; Spark Therapeutics: Consultancy, Research Funding; ICER: Consultancy; OPKO: Research Funding; Biomarin: Consultancy, Research Funding. Rollins-Raval:Bayer, Inc: Membership on an entity's Board of Directors or advisory committees. Raval:Sanofi: Membership on an entity's Board of Directors or advisory committees; Bayer, Inc: Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Background: It is commonly accepted that severely deficient ADAMTS13 activity in remission increases the risk for relapse, but relapse in severely deficient ADAMTS13 activity in patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) is not uniform. Mouse models and published studies have suggested that a "second hit" is required in addition to severely deficient ADAMTS13 activity to lead to clinical relapse. Our initial published experience led us to preliminarily conclude that pregnancy could serve as the second hit in addition to severely deficient ADAMTS13 activity and lead to relapse. The aim of this study was to evaluate the risk of relapse and outcomes of pregnant patients with a diagnosis of iTTP. Methods: Since the initiation of the Ohio State University TTP Research Program in 2003, patients were consented at enrollment and followed longitudinally in our IRB approved iTTP registry, usually every three to six months. During pregnancy, patients were seen and CBC, LDH and ADAMTS13 activity were obtained monthly. Due to prior reports of iTTP relapses during pregnancy, it is our practice to offer prophylaxis with cyclosporine to pregnant patients with a diagnosis of iTTP and severely deficient ADAMTS13 activity. The clinical diagnosis of iTTP (defined as thrombocytopenia, microangiopathic hemolytic anemia, without an alternative explanation was confirmed by severely deficient ADAMTS13 activity ( Results: During the study time, we have followed 11 pregnancies from eight patients with iTTP. Two pregnancies occurred at the time of their initial diagnosis, and nine pregnancies occurred during follow up. Two patients were black, nine were white. Median age was 26 ( range 20-36). Of the nine pregnancies in patients with a previous diagnosis of iTTP, three resulted in relapses, two in the same patient; one at 23 weeks that resulted in fetal demise, and the next one occurred one week after delivery at 36 weeks. The other patient relapsed at 35 weeks and the baby was delivered early at 36 weeks. All relapses were preceded by a documented ADAMTS13 Conclusions: Pregnancy in patients with a diagnosis of iTTP is a risk factor for iTTP relapse. To our knowledge, this is the first report of the relationship between ADAMTS13 and the risk of relapse during pregnancy. Our results demonstrate that severely deficient ADATMS13 activity uniformly leads to relapse in pregnancy. It also highlights the clinical relevance of monitoring ADAMTS13 activity closely in patients with a history of iTTP that become pregnant. Figure 1 Disclosures Masias: Rigel Pharmaceuticals: Consultancy. Cataland:Alexion: Consultancy, Research Funding; Ablynx/Sanofi: Consultancy, Research Funding. OffLabel Disclosure: the use of cyclosporine for prevention of TTP relapses

In this issue of Blood , [de Vries et al][1] report the contribution of genetic variants to plasma ADAMTS13 (disintegrin and metalloproteinase with thrombospondin motifs 13) activity with a hypothesis-free genome-wide association (GWA) approach using the Rotterdam Study, a large population-based

Introduction: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by disseminated thrombus due to the mutations of ADAMTS13, which cleaves its substrate von Willebrand factor(VWF) in shear-induced unfolding condition. Most of the congenital TTP we found is woman with pregnancy. Here, we characterize two children suspected with congenital TTP. Methods:ADAMTS13 activities were analyzed by residual collagen binding assay (R-CBA) plus FRET-VWF substrate. And the inhibitors of ADAMTS13 were analyzed by 9:1 mixture of patient and pooled normal plasma followed by R-CBA. The secretion of recombinant ADAMTS13 mutants was studied. Results: Two children, one aged four months and the other aged three years old, were diagnosed with congenital TTP because their ADAMTS13 activities were less than 5% (both FRET-VWF and R-CBA), and there are short of ADAMTS13 inhibitors. The following mutations were found: Q1385P, Y177C, C522R. In addition to the previously reported mutation of Y177C, the two novel mutations (Q1385P and C522R) failed to secrete from the HEK293 cells. Conclusion: Two children with congenital TTP were determined thanks to screening of ADAMTS13 activity and its corresponding inhibitor assays, and it seems that congenital TTP could occur in different ages although most of congenital TTP we found were women with pregnancy. Disclosures No relevant conflicts of interest to declare.

Background:Thrombotic thrombocytopenic purpura (TTP) is a rare but deadly thrombotic microangiopathy (TMA) that is caused by an acquired deficiency in the ADAMTS13 enzyme. The PLASMIC clinical scoring system was developed and validated in 2014 by the Harvard TMA Research Collaborative in order to determine the pretest probability of severe ADAMTS13 deficiency in cases of suspected TTP. We studied the role of the PLASMIC score in guiding use of the ADAMTS13 activity assay and assessed the cost effectiveness of a score-based diagnostic approach to patients with TMA compared to two strategies currently in use within our consortium. Methods:We utilized an expanded dataset from the Harvard TMA Research Collaborative consisting of all consecutive cases of TMA at three large academic medical centers for which an ADAMTS13 assay was sent between 2004-2015 (n=647). To investigate trends in ADAMTS13 testing over time, we compared the experience at two centers (A and B) with a liberal ADAMTS13 testing policy against a third center in our consortium (C) that carries a more restrictive policy requiring pre-approval by the blood transfusion service. Cost savings analysis was subsequently performed to assess the potential impact of an algorithm incorporating the PLASMIC score for clinical decision support in the workup of these patients. Results:At Sites A and B, we observed after adjusting for changes in inpatient volume that the quantity of ADAMTS13 tests increased greater than eight-fold during the study period (from 11 per 100,000 admissions in 2004 to 94 per 100,000 admissions in 2015, P Conclusions:Taken together, our results demonstrate that a significant number of patients in our consortium who are at minimal risk for TTP nevertheless undergo ADAMTS13 testing and receive expert consultation and/or TPE. An approach incorporating upfront application of the PLASMIC clinical scoring system to risk stratify patients with suspected TTP may help enhance the cost effectiveness of diagnosing and managing these cases. Figure Figure. Table Table. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood , Kremer Hovinga and colleagues demonstrate that a lower level of initial plasma ADAMTS13 activity (< 10%) is associated with higher risk of relapse in patients with TTP. Also, patients with a low plasma ADAMTS13 activity and high-titer inhibitor (≥ 2 BU) appear to have

Rationale: Cirrhosis frequently affects multiple components of hemostasis. Reversal of the coagulopathy of these patients is frequently required in case of bleeding episodes, or as prophylaxis before invasive procedures. A simple and cost-effective way to correct a prolonged bleeding time in patients with cirrhosis is administration of 1-deamino-8-D-arginine vasopressin (DDAVP). Although DD AVP is widely used as a pro-hemostatic agent in patients with cirrhosis, it is unclear whether DDAVP truly enhances the hemostatic potential in these patients. We investigated the hemostatic effects of a single bolus dose of DDAVP in patients with cirrhosis. Methods: Ten patients with cirrhosis (n=6 Child-Pugh score B, n=4 Child-Pugh score C) and ten patients with mild hemophilia A (FVIII 0.05-0.40 IU/ml) were included in this study. All patients received an intravenous single bolus of 0.3 microgram/kg DDAVP. Plasma was collected prior to and at 1, 3, 6, and 24 hours after DDAVP administration. Levels of Von Willebrand factor (VWF:Ag), VWF propeptide, Factor VIII (FVIII), and ADAMTS13 were measured in all plasma samples, whereas VWF multimers and functional VWF-dependent platelet adhesion were determined in the samples pre- and 1 hour after DDAVP administration. VWF-dependent platelet adhesion was studied under flow conditions in a reconstituted blood model. Results: The median age of patients with cirrhosis was 55 years (IQR 47-58) and 46 years (IQR 40-55) in patients with haemophilia. Baseline values of coagulation parameters are shown in table 1. Following DDAVP administration, VWF:Ag, FVIII levels and VWF propeptide increased in patients with hemophilia, while patients with cirrhosis only showed a modest increase in VWF propeptide and FVIII levels (figure 1). Only in those patients with low Child-Pugh scores, VWF:Ag slightly increased following DDAVP. The proportion of high molecular weight VWF multimers and VWF-dependent platelet adhesion increased in patients with hemophilia one hour after DDAVP administration, but did not change in the patients with cirrhosis. Levels of ADAMTS13 were unaffected in both patient groups after DDAVP. Conclusion: DDAVP administration in patients with cirrhosis did not have effect on VWF:Ag and ADAMTS13, but VWF propeptide and FVIII did increase. The lack of relevant hemostatic effects of DDAVP in patients with cirrhosis may be the explanation for the limited value in previous clinical studies in these patients. Table 1. Baseline levels of VWF:Ag and related proteins Patient group Hemophilia A Cirrhosis p-value VWF:Ag (IU/dl) 114 (94.5-164.3) 521 (238-643.8) Data are presented as medians with IQR; VWF:Ag= Von Willebrand factor antigen; ADAMTS13= a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; FVIII= Factor VIII Figure 1. Coagulation parameters in patients with hemophilia (panel A) or cirrhosis (panel B) Figure 1. Coagulation parameters in patients with hemophilia (panel A) or cirrhosis (panel B) Disclosures No relevant conflicts of interest to declare.

Comment on Crawley et al, page [1085][1] Left unchecked, ADAMTS13 activity may inhibit platelet adhesion at sites of vascular injury under high shear stress. Crawley and colleagues present evidence that the proteolytic cleavage of ADAMTS13 by coagulation proteinases inhibits its ability to cleave

Comment on Studt et al, page [542][1] Free hemoglobin inhibits ADAMTS13 activity in vitro, demonstrating a potential source of artifact affecting ADAMTS13 activity assays and inhibitor screens. Deficiency of the ADAMTS13 metalloprotease causes thrombotic thrombocytopenic purpura (TTP). The rare

Introduction While the activity level of ADAMTS13 can be helpful in diagnosing patients with thrombotic thrombocytopenic purpura (TTP), the current long turnaround time of this test for most institutions limits its role in early clinical decision-making about the initiation of plasma exchange. Levels of ADAMTS13 Methods Patients with a diagnosis of TTP were identified from hospital records of the major hospitals in Rhode Island which perform therapeutic apheresis in calendar years 2011 and 2012. From a chart review and blood bank records, baseline clinical parameters were collected, the number of therapeutic plasma exchanges (TPE) performed and the volume of plasma utilized. Pre-treatment ADAMTS13 activity was recorded if available in addition to the number of days from the initiation of TPE to test result availability. An analysis was performed to examine if patients who had a pre-treatment ADAMTS13 activity level ≤10% differed in baseline characteristics or response to TPE from those with activity levels >10%. Based on the normality of the distribution of the data, independent t-tests or Wilcoxon rank-sum tests were performed using SAS version 9.3. Results During this two year period, 24 patients received plasma exchange in Rhode Island for a presumptive diagnosis of TTP. The mean age was 47 years (range 20-89 years) and 38% were male. ADAMTS13 activity was available for 20 patients and 7 (30% of those exchanged) had documented pre-treatment activity levels ≤10% consistent with TTP. The median turnaround time for the ADAMTS13 assay was 10 days (range 2-52). Mean baseline parameters at the time of presentation are shown in the table. As expected, creatinine levels were lower in those patients with true TTP (p=0.0410). ADAMTS13 activity level was predictive of the number of days to a platelet count ≥150 x 109/L (Pearson correlation 0.56; p-value 0.0458). Overall, 4238 units of plasma were utilized for exchange. Of these 4238 units, 1886 were transfused to patients who were subsequently shown to have an ADAMTS13 activity >10%, and 813 of the 1886 units (20% of all plasma exchanged) were transfused after the results of enzyme activity were available in this population. Conclusions Based on an ADAMTS13 >10%, a significant volume of plasma was unnecessarily transfused. Reducing the turnaround time for the ADAMTS13 assay in tertiary care centers could help clinicians better determine which patients will benefit from plasma exchange, avoiding the morbidity and expense associated with large volume plasma exchange. Disclosures: No relevant conflicts of interest to declare.

HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY ![Figure][1] Current beliefs are that familial thrombotic thrombocytopenic purpura (TTP) is caused by mutations of the ADAMTS13 gene and that acquired TTP is caused by autoantibodies that inhibit ADAMTS13 activity. These concepts are consistent

Abstract 4666 The clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is unclear. Therefore, we investigated the characteristics of severe ADAMTS13 deficiency and evaluated its clinical significance in Thrombotic thrombocytopenic purpura (TTP). The Korean TTP Registry includes 66 patients from 13 teaching hospitals in Korea who received the diagnosis of TTP from January 2005 to December 2008. Blood samples obtained upon admission were sent for ADAMTS13 analysis (multimer analysis by sodium dodecyl sulfate electrophoresis and/or ELISA) to a central laboratory along with patient clinical information. After 6 months, patient data regarding treatment, response, and prognosis were collected on standardized report forms. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001) and WBC counts (P=0.050) than patients with non-severe ADAMTS13 deficiency. Although severe ADAMTS13 deficiency was associated with better response rate (75% vs 53%, P=0.145), remission rate (81% vs 61%, P=0.209), and mortality rate (19% vs 31%, P=0.508) than non-severe ADAMTS13 deficiency, treatment outcomes did not differ significantly between groups. After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. Patients with severe ADAMTS13 deficient had lower serum creatinine levels and WBC counts at presentation but Severe ADAMTS13 activity deficiency at TTP diagnosis does not appear to have prognostic significance. Disclosures: No relevant conflicts of interest to declare.

Abstract 1414 Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. We showed previously by in vitro and ex vivo studies that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine and rat ADAMTS13, whereas ADAMTS13 from rabbits and cynomolgus monkeys is able to cleave human rVWF. Here we tested the pharmacological behavior of human rVWF in rabbits and cynomolgus monkeys. The animals were infused once with different doses of human rVWF. VWF antigen rose sharply in a dose-dependent manner (∼25 IU/ml VWF:Ag for the highest dose, 15 min after injection) and then declined gradually (∼7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). By contrast, the ADAMTS13 activity did not show relevant changes throughout the entire test period in the rabbit or in the monkey samples, indicating that an excess of intravenously administered rVWF as the substrate does not exhaust its enzyme ADAMTS13. Most importantly, neither rabbits nor cynomolgus monkeys showed any exaggerated pharmacological or toxic effects upon rVWF administration. Even the administration of 14 daily doses of rVWF to cynomolgus monkeys did not lead to any toxicological effect. Our data indicate that rabbits and cynomolgus monkeys, two species with a sufficient rVWF cleavage capacity by endogenous ADAMTS13, are appropriate animal models for a meaningful preclinical evaluation of the rVWF product, which allows the therapeutic safety margins for human patients to be determined. Disclosures: Muchitsch: Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment.

Plasma exchange (PE) with plasma infusion is the treatment of choice for Thrombotic Thrombocytopenic Purpura (TTP) but doubts remain as to whether all kinds of plasma are equally effective. Since October 2004, a multicentric prospective cohort study is being conducted in Spain to compare Methylene Blue Photoinactivated Plasma (MBPIP) with Fresh Frozen Plasma (FFP) in the treatment of TTP. Sixty-three first episodes of idiopathic TTP were included. MBPIP was used in 38 and FFP in 25. The treatment schedule consisted of daily PE (> 40 mL/kg of body weight) and costicosteroids (1.5 mg/kg/d). Response was defined as the achievement of a platelet count >= 150 x 109/L for at least three consecutive days, normal LDH level and absence of TTP-related symptoms and signs. Recurrence was defined as a fall in platelet count below 50 x 109/L or below 50% of the highest count achieved after response. Response lasting for more than 15 days was considered as remission. The prognostic significance of the kind of plasma used was investigated by logistic regression analysis after adjustment for other variables that had previously been found to influence on response to PE (gender; the Rock score; days from first medical attendance to PE; volume of plasma infused in the first 7 days of treatment). Both groups were comparable with regard to clinical and biological parameters at diagnosis. A severe deficit in ADAMTS13 activity was found in 9 out 12 (75%) patients treated with MBPIP and in 12 out of 16 (75%) patients treated with FFP. When compared to FFP, patients treated with MBPIP required a higher number of PEs (16±13 vs 9±7, p=0.004) and a larger volume of plasma (763±678 ml/kg vs 413±326 ml/kg, p=0.02) to achieve a remission and presented more recurrences while on PE treatment (21 out of 38 vs 6 out of 25, p=0.02). Splenectomy or rituximab was required in eight patients (21%) treated with MBPIP to achieve a remission vs in none out of the 25 patients treated with FFP (p= 0.02). After adjustment for other prognostic variables, patients in the MBPIP group had a lower likelihood of remission by the 8th treatment day (OR: 5.1; 95% CI: 1.6 – 15.9) and a higher risk of recurrence while on PE treatment (OR: 4.2; 95% CI: 1.3 – 13.5). In conclusion, MBPIP is less effective than FFP in the treatment of TTP.

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.

Thrombotic thrombocytopenic purpura(TTP) is a serious, low morbidity and high mortality disease, which can simultaneously affect multiple systems in the patients’ body. In the event that the patients cannot be treated by plasma exchange(PE), mortality will be 95–100%(1). Between September, 2000 and May, 2003, thirteen patients with TTP were treated mainly by heparin, vincristine, dexamethasone, six of whom have acceptted one or two PE. The results were excellent. Twelve of the thirteen patients survived. One patient was dead. The ADAMTS13 activity was measured in 10 patients using a Residual-Collagen Binding Assay(R-CBA).

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

INTRODUCTION: Many cases of idiopathic thrombotic thrombocytopenic purpura (TTP) are characterized by very low levels of the metalloprotease ADAMTS13, presumably resulting in dysregulation of platelet/vWF interactions and microvascular thrombosis. Treatment for TTP begins with therapeutic plasma exchange (TPE), allowing replacement of ADAMTS13 and removal of autoantibody, when present. Fresh frozen plasma (FFP) and plasma cryoprecipitate reduced (also known as cryoprecipitate poor plasma [CPP]) have been used as replacement fluids. While current AABB standards allow thawed FFP and CPP components to be stored at 1–6°C for up to 5 days before use, the amount of ADAMTS13 activity in these products has not previously been measured nor has its stability been evaluated over the 5 days. Given the labor-intensive nature of our standard collagen-binding assay for ADAMTS13 activity, we chose to analyze ADAMTS13 activity in FFP and CPP components using a modified FRETS-VWF73 assay recently developed in our laboratory. Preliminary data indicate that the two assays correlate well. METHODS: Ten whole blood (WB) units (5 group O and 5 group A) were centrifuged within 8 hours of collection. Prior to freezing of the FFP, an aliquot of plasma was obtained from each component. The aliquots and FFP components were frozen at

Patients: The 3 patients detailed in this report presented to Ohio State University Hospitals and were enrolled on our prospective study of plasma exchange as initial therapy of TTP. Given the close outpatient follow-up during the first month after the tapering of plasma exchange, 2 patients were found to have an early recurrence of their TTP. The third patient had a recurrence 8 weeks after completing 6 months of CSA after being treated with concurrent CSA and PE for a previous acute episode of TTP. She completed the planned 6 months of cyclosporine therapy but relapsed 8 weeks after discontinuing the drug. Her recurrence was characterized by numbness and tingling in her right hand in addition to laboratory findings consistent with TTP. Complete demographic and clinical details of these 3 patients are shown in Table 1. Methods: All 3 patients received oral cyclosporine (2–3 mg/kg) daily for six months in a divided dose. None of the patients underwent plasma exchange, infusion, or transfusion of blood products of any kind. Patients were seen daily to monitor for any clinical or laboratory findings that would warrant the re-initiation of plasma exchange therapy. Remissions were defined as a normal platelet count, normalization of LDH for at least 2 consecutive days, and improvement or stabilization of any renal and neurologic abnormalities. Serial measurements of ADAMTS13 activity and inhibitors were performed at regular intervals throughout therapy and follow-up. Results: All three patients showed clear laboratory and clinical signs of improvement within the first 7 days of therapy and met criteria for clinical remission 14, 24, and 34 days respectively from the time they began treatment with cyclosporine. The third patient that presented with numbness and tingling in her right hand also had complete resolution of her neurologic symptoms within one week of restarting cyclosporine. Pretreatment ADAMTS13 activity was determined to be 33%, 25%, and 10% in all 3 patients respectively with all 3 patients having documented inhibitors of ADAMTS13. Conclusions: Treatment with cyclosporine alone resulted in the suppression of ADAMTS13 inhibitors and normalization of ADAMTS13 activity. Figure Figure All three patients have completed the planned 6 months of cyclosporine and have maintained their remission 4, 3, and 1 month respectively after discontinuing cyclosporine. Patient Demographic and Clinical Data Laboratory Studies at the TIme CSA Initiated Age Sex Race Therapy Preceeding Recurrence Time to Relapse After Most Recent Therapy Platelet Count (150-450 K/uL) LDH (100-190 U/L) PE=Plasma Exchange, CSA=Cyclosporine, S=Corticosteroids Patient 1 48 F C PE + S 17d 113 212 Patient 2 39 F C PE 6d 135 400 Patient 3 56 F AA CSA 56d 51 418

Previous studies have estimated the U.S. incidence of TTP to be 3.7/106/year, based on U.S. death certificates (Am J Hem 1995;50:84), and 3.8/106/year, based on claims submitted to a national health insurer (Epidemiol 2004;15:208). These methods are indirect and require extrapolation to determine incidence rates. We estimated the incidence directly from the Oklahoma TTP-HUS Registry. The Registry accrues all patients treated with plasma exchange (PE) for a clinical diagnosis of TTP or HUS (therefore excluding only children with typical HUS) in central, western, and southeastern Oklahoma since the Oklahoma Blood Institute (OBI) is the sole provider for PE in this region of 2.3 million people. Our estimates are based on patients who had their initial episode from 1/1/1996, after routine ADAMTS13 measurements began, to 6/30/2004. During these 8.5 years ADAMTS13 activity was measured in 189 (90%) of 209 consecutive patients. Prior to 1996, the number of patients accrued in the Oklahoma TTP-HUS Registry increased each year, similar to other data (JAMA 2003; 290:1351); since 1996, there has been no consistent change in the annual number of patients accrued. | Categories | Incidence (new cases/10 6 population/year) 1 | |:---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | -------------------------------------------- | ------------ | | | All patients | Black race 1 | Other race 1 | | | Cases | Incidence 1 | Cases | Incidence 1 | Cases | Incidence 1 | | 1Population data are from the 2000 US Census for the Oklahoma counties served by the OBI. Black race includes Black alone and in combination with one or more other races. Other race refers to all other racial designations. 2Urban or rural designations were determined by county of residence. Urban is the Oklahoma City Metropolitan Statistical Area; all other counties are rural. 3Excludes patients after BMT, pregnant/postpartum, drug-associated, bloody diarrhea prodrome, and additional or alternative diagnoses. | | All patients | 209 | 10.64 | 40 | 24.08 | 169 | 9.40 | | Urban 2 | 117 | 12.71 | 29 | 27.63 | 88 | 10.79 | | Rural 2 | 92 | 8.82 | 11 | 17.99 | 81 | 8.25 | | Idiopathic 3 | 77 | 3.92 | 21 | 12.64 | 56 | 3.12 | | ADAMTS13 deficient (

Patients with a clinical diagnosis of TTP presenting to The Ohio State University were enrolled and treated on a prospective trial of concurrent plasma exchange (PE) and adjuvant immunosuppression. Patients: Between April 2003 and August 2004, 15 patients were entered and followed on this study. The median age was 46 years (range, 19–59), 11/15 were female, 7/15 were African American, and 9/15 were obese (body mass index >30 kg/m2). Nine patients had been previously treated for TTP. Method : All patients with TTP were treated initially with concurrent PE and corticosteroids. Daily exchanges of one plasma volume using cryosupernatant plasma were performed with concurrent prednisone at a dose of 1 mg/kg/day. PE was continued until a clinical remission was achieved as defined by: 1) normal platelet count 2) normalization of LDH for 2 consecutive determinations, and 3) improvement or stabilization of renal or neurologic abnormalities. After criteria for remission were achieved patients had 2 additional procedures performed every other day and the prednisone tapered over the next 4 weeks. Patients with exacerbations (defined as the need to reinstate PE within 30 days discontinuation) of TTP had prednisone discontinued and began oral cyclosporine (CSA) at a dose of 2–3 mg/kg/day orally concurrent with the resumption of PE. After achieving a second remission, PE was tapered as described previously and CSA was continued for a total of 6 months of therapy. Serial measurements of ADAMTS13 activity and inhibitor assays were obtained prior to the initiation of PE and at regular intervals during follow-up. Results : ADAMTS13 data is available for the first 6 patients treated with PE + prednisone. A summary of clinical outcomes and ADAMTS13 activity may be found in Table 1. Of the patients suffering exacerbations after initial therapy with PE + prednisone, ADAMTS13 data is available from the first 2 of 5 patients treated with PE + CSA and is shown in Figure 1. Clinical Responses and ADAMTS13 Activity Inhibitor Remission Exac. Relapse Med. ADAMTS13-Pre Med. ADAMTS13-Remission Med. Remission (mo) * samples tested to date **median ADAMS13 PE+Pred. 5/6* 14/15 7/15 2/15 5% (range, 2-7%)** 5% (range, 2-7%)** 1(range, 1-9 ) PE+CSA 2/2* 5/5 0/5 0/5 Figure 1 Figure 1 6 (range, 3-10) Conclusions : In this prospective study, no patients had significant improvement in ADAMTS13 activity despite achieving remission with PE and corticosteroids. These data showing persistently low ADAMTS13 activity at the time of remission may have contributed to the high rate of exacerbations in this group of patients. In the analysis of the first 2 patients treated with concurrent PE and CSA, there was clear and sustained improvement in ADAMTS13 activity that began after the discontinuation of PE and correlated with the clinical remission of TTP. Figure Figure