Figure 8. Effect of DNA on fXI-dependent thrombin generation in plasma.

(A) Thrombin generation measured in normal plasma after addition of 25 μg/ml DNA without (left hand panel) or with (right hand panel) 0.5 pM TF. Assays were run in the presence of vehicle (solid line), the anti-fXI IgG O1A6 (dotted line) or the fXIIa inhibitor CTI (dashed line). (B) Images of neutrophils stimulated with vehicle (control) or 100 ng/ml phorbol myristate acetate (PMA) with or without 100 U/mL DNAse. Cell permeable Hoechst stain (top row) indicates DNA within intact cell nuclei. Sytox Green staining (bottom row) demonstrates extracellular DNA or DNA within dying cells that have lost the ability to exclude the dye. PMA treated cells form neutrophil extracellular traps (NETs, middle column), which are not observed in the presence of DNase (right column). Images were made on an Inverted Fluorescence Microscope (89404-464) using a V10MP camera (VWR), and Motic Images Plus 2.0 software. (C) Thrombin generation in normal plasma supplemented with neutrophils treated with vehicle (gray lines, –PMA) or PMA (black lines, + PMA), without (solid lines) or with (dashed lines) DNAse in the absence of an inhibitor (No inhibitor), or in the presence of O1A6 or CTI. For reactions containing O1A6, no thrombin generation was detected with or without DNAse treatment. For reactions with CTI, some activity was detected in samples not treated with DNAse.