Plasma derived FVIII concentrates induce neutrophil activation and neutrophil extracellular trap formation.

B. E. Kehrel, A. Bertling, M. F. Brodde (Muenster, Germany)

Bleeding disorders - Basic science
Date: 17.02.2017,
Time: 08:00 - 09:15

Objective: In this project, we studied whether pdFVIII may boost inflammation by the activation of neutrophils.

Methods: Three pdFVIII products (Octanate®, HaemateP® and Haemoctin®) and two rFVIII products (Kogenate®FS and Advate®) were studied. Neutrophils were gently isolated from fresh human blood by counter-flow elutriation. Low activation status after isolation was controlled by fibrinogen binding to CD11b. Whether FVIII concentrates induced generation of the reactive oxygen species (ROS) superoxide anion (O2.-) from nonactivated neutrophils was determined as the linear rate of superoxide dismutase-inhibitable reduction of cytochrome C. Activation of CD11b was analyzed by flow cytometry using an activation specific monoclonal antibody against CD11b labeled with PE. Associate formation in whole blood between platelets and neutrophils was measured by flow cytometry. Effect of factor VIII products on DNA release from isolated human neutrophils was measured by two methods, a fluorogenic assay using the fluorescent dye Syto13 binding to nucleic acids, and a sandwich ELISA against histone-DNA complexes detecting neutrophil extracellular traps (NETs) from FVIII treated human neutrophils. Net formation was in addition analysed by light microscopy.

Results: Significant activation of CD11b (MAC-1) was only observed on neutrophils treated with the used (0.1-1 U/ml) pdFVIII, but not on neutrophils treated with rFVIII. pdFVIII induced neutrophil ROS production as well as the release of extracellular DNA (neutrophil extracellular traps, NETs), which was not observed with rFVIII. Fluorescence microscopy showed that pdFVIII induced neutrophil nets present alpha defensins (HNP1-3) and myeloperoxidase. In addition to experiments with isolated neutrophils, we studied the effect of FVIII on neutrophils in melagatran anticoagulated whole blood. Two of three tested pdFVIII clearly and significantly induced the formation of associates between neutrophils and platelets. In contrast, rFVIII had no effect. The activation effects of pFVIII on neutrophils were more pronounced in the presence of platelets.

Conclusion: Based on these in vitro results, pdFVIII products, in contrast to rFVIII products, may be proinflammatory by activating neutrophils. Further research aims to investigate the effect of factor VIII products ex vivo. This work was supported by a grant of Bayer Vital GmbH, 51368 Leverkusen, Germany