ROTEM assay conditions sensitive to concizumab

Augustsson C, Ridell A, Fernandez-Bello I, Butta N, Kjalke M, Astermark J, Eichler H, Jimenez Yuste V, Chowdary P

Augustsson C, Ridell A, Fernandez-Bello I, Butta N, Kjalke M, Astermark J, Eichler H, Jimenez Yuste V, Chowdary P. ROTEM assay conditions sensitive to concizumab . Haemophilia. 2021;27(S2): https://doi.org/10.1111/hae.14236

Date of Publication

Introduction: Concizumab is a humanized monoclonal antibody currently under phase 3 clinical investigation for the subcutaneous prophylaxis treatment of patients with haemophilia A (HA) or B with or without inhibitors. Concizumab has the potential to prevent bleeding episodes by binding tissue factor (TF) pathway inhibitor (TFPI) thereby preventing the inhibition of factor Xa (FXa) and the TF?factor VIIa?FXa complex. Thus, TF?dependent global coagulation assays are necessary to measure the coagulation potential of patients treated with concizumab. However, standard TF?initiated rotational thromboelastometry (ROTEM) is not sensitive to HA?like conditions. Here, we present a modified ROTEM method to measure the coagulation potential of whole blood exposed to concizumab.


Methods: To generate HA?like blood, neutralising factor VIII antibody (Hematological Technologies Inc) was added to citrate?stabilised normal whole blood. ROTEM (Tem innovations GmbH) analysis was performed according to manufacturer’s protocol except for a 2940?fold predilution of the TF reagent (r ex?tem® [Tem innovations GmhH]) resulting in 1:50,000 fold dilution of the reagent relative to the total volume of the sample cup including blood (300 uL), calcium reagent (20 uL) and trigger (20 uL). Dade Innovin (Siemens) and Recombiplastin 2G (Instrumentation Laboratories) were evaluated as alternative TF triggers. TF contents of individual reagents were quantified by adding excess rFVIIa and measuring the cleavage of a chromogenic substrate (Chromogenix S2288) using lipidated TF1?244 (25:75 PS:PC) as a calibrator.


Results: TF concentrations were ~4 nM in Innovin and Recombiplastin 2G and 2 nM in r ex?tem (0.13 pM and 0.07 pM final concentrations in plasma, assuming a hematocrit of 45%). The largest difference between normal blood and HA?like blood was observed when using r ex?tem® diluted 1:50,000 as a trigger. When concizumab was spiked into the HA?like blood, a concentration?dependent shortening of clot time (CT) and increase in clot development (??angle) were observed at plasma concentrations of 200–4,000 ng/mL. Further increase of concizumab concentration did not additionally affect ROTEM parameters.


Discussion/Conclusion: ROTEM analysis of HA?like blood using diluted r ex?tem® reagent resulted in concentration?dependent responses between 200–4000 ng/mL concizumab, a range corresponding to the target exposure in the ongoing phase 3 clinical trials.

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