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2229 Background: Acquired thrombotic thrombocytopenic purpura (TTP) is still associated with a 10–20% death rate, which did not significantly improve for more than 20 years despite a better awareness about this diagnosis. Usually, death occurs within the first days of management and so far, early prognostic factors of death could not be clearly identified. In this context, the accurate understanding of factors associated with a fatal outcome at the acute phase of the disease would help to better tailor initial treatment and further improve these results. Objective: To identify prognostic factors associated with 1-month death in TTP patients with acquired severe (< 10% of normal activity) ADAMTS13 deficiency. Design: Prospective national cohort of adult (≥ 18 year-old) patients included between October, 2000, and December, 2008. A validation cohort of patients was set up from January, 2009 to March, 2010. Participants: 248 (analysis cohort) and 39 (validation cohort) consecutive adult TTP patients with acquired severe ADAMTS13 deficiency from 39 French centers. Measurements: 30-day mortality after treatment initiation according to characteristics at inclusion. Results: When compared to survivors, non-survivors (11%) were older (54.0 ± 19.4 versus 39.0 ± 15.5 year-old, respectively, P <.001) and had more frequently a past history of arterial hypertension (37% versus 10%, respectively, P <.001) and ischemic heart disease (19% versus 4%, respectively, P =.002). Prognosis was increasingly poor with age (p <.004), particularly in patients ≥ 60 year-old. On presentation, cerebral manifestations were more frequent in non-survivors (81% versus 55%, respectively, P =.009) and serum creatinine level was higher (172 ± 123 versus 117 ± 91 μmol/L, respectively, P =.037). Death occurred after a few days (mean 7 days, interquartile range = [3, 12]), in a context of one or multiple organ failure in relation with an uncontrolled TTP. Platelet count between diagnosis and death did not significantly increase (20 ± 25×109/L versus 28 ± 46×109/L, respectively, P =.44). The most significant independent variables for determining death were age (P <.001), cerebral involvement (stupor and seizure) and LDH level > 10 times normal value (P <.04 for both). After computing the risk scores using these 3 variables (Table), patients were stratified into 3 distinctive risk groups regarding death: low, 0 and 1; intermediate, 2; and high, 3 and 4. The proportion of positive predictive value for 30-day death was 11–13% in the low risk group, 20% in the intermediate group, and 39–50% in the high risk group. This score was confirmed in the validation cohort using these variables, with higher values corresponding to increased risk of early death (P <.01). Conclusions: A risk score for early death was defined in patients with TTP and validated on an independent cohort. This score should help to stratify early treatment and intensify patients with a worse prognosis. Importantly, we provide clear evidence that age is an important prognostic factor of TTP. Consequently, old patients with a diagnosis of TTP should benefit from more intensive supportive care and should be monitored more closely in intensive care units with more aggressive attention to cardiac and renal function. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment.

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To cite this article: Grillberger R, Gruber B, Skalicky S, Schrenk G, Kn€ obl P, Plaimauer B, Turecek PL, Scheiflinger F, Rottensteiner H. A novel flow-based assay reveals discrepancies in ADAMTS-13 inhibitor assessment as compared with a conventional clinical static assay. J Thromb Haemost 2014; 12: 1523-32.Summary. Background: Several static Bethesda-type assays are routinely used to determine ADAMTS-13-neutralizing autoantibodies in acquired thrombotic thrombocytopenic purpura (TTP), but the inhibitory activity of these antibodies has not been thoroughly evaluated under the more physiologic condition of flow. Objectives: We investigated whether ADAMTS-13 inhibitor assessment with the FRETS-VWF73 assay is predictive for evaluation under flow. Methods: Anti-ADAMTS-13 autoantibodies were purified from patients with acquired TTP by chromatography involving an ADAMTS-13 affinity matrix and/or protein G. ADAMTS-13 activity was measured with the FRETS-VWF73 assay and a novel flow assay determining the ADAMTS-13-mediated decrease in platelet aggregate surface coverage, caused by perfusion of a suspension containing platelets, erythrocytes and von Willebrand factor (VWF) over a surface coated with extracellular matrix components. The neutralizing activities of ADAMTS-13 inhibitors were compared under static conditions and under flow by use of the two assays. Results: The suitability of the flow-based ADAMTS-13 activity assay for quantification of ADAMTS-13 inhibitors could be demonstrated by reversibility of the ADAMTS-13-dependent decrease in surface coverage upon addition of goat AD-AMTS-13 antiserum. Testing the neutralizing activity of purified autoantibodies from six patients in the flow assay according to their FRETS-VWF73-based inhibitor titers gave rise to vastly different inhibitory effects, indicating a discrepancy in inhibitor assessment between static and flow conditions. Conclusions: Anti-ADAMTS-13 autoantibodies may show inhibitory properties in vivo that are not consistent with the ADAMTS-13 inhibitor levels determined in routine static assays, possibly because certain epitopes are selectively exposed under shear. Consequently, the course of disease and treatment efficacy may vary among TTP patients, despite common inhibitor titers.

Thrombotic thrombocytopenic purpura (TTP) is characterized by a functional deficiency in the plasma metalloprotease ADAMTS13, caused by mutations in the ADAMTS13 gene or by autoantibody inhibition. ADAMTS13 is the key regulator of the hemostatic activity of von Willebrand factor (VWF), accomplished by cleavage of a single site within the A2 domain of VWF. The catalytic domain of ADAMTS13 possesses various binding sites for metallic cations including one Zn2+ binding site composed of three histidine residues within the sequence HEXXHXXGXXHD and up to three putative calcium ion-binding sites. The dependence of ADAMTS13 activity on zinc and calcium ions is reflected in its inactivation by chelating agents such as EDTA and doxycycline. Although replenishment with an appropriate metallic cation is thought to fully restore the proteolytic activity of the enzyme, the stability of ADAMTS13 in a Ca2+-depleting environment has not yet been explored. This aspect, however, is clinically relevant, as citrated human plasma serves as the standard source for testing ADAMTS13-specific parameters, where the chelator citrate not only prevents activation of the coagulation cascade, but also renders ADAMTS13 inactive. Here, we addressed the stability of plasma ADAMTS13 in the presence or absence of citrate (0.38%) at various temperatures. While ADAMTS13 proved stable at 4-8°C and at room temperature for up to 24 h irrespective of the presence of citrate, a time-dependent decrease in activity was observed at 37°C in the presence but not in the absence of citrate. No decrease in activity was seen when heparinized plasma was used as source of ADAMTS13, but the addition of 0.38% citrate again caused ADAMTS13 instability at 37°C. Similar results were observed when using the purified recombinant protein as source for ADAMTS13. Higher order structural analyses using Fourier-transformed infrared spectroscopy and dynamic light scattering demonstrated citrate-dependent structural changes in ADAMTS13 at 37°C that are typical for less-ordered protein structures. Addition of 5 mM Ca2+, Zn2+, or both metal ions prior to incubation completely restored ADAMTS13 stability, but these compounds failed to restore activity when added post incubation even after prolonged periods, suggesting that the loss in activity was irreversible. We conclude that Zn2+ and/or Ca2+ ions are required to stabilize the structure of ADAMTS13 at physiological temperature. This finding needs to be considered when using citrated plasma as source of ADAMTS13 in clinical settings such as for plasma exchange to treat acute episodes of TTP. Disclosures Grillberger: Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Kink:Baxalta Innovations GmbH: Employment. Matthiessen:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment.