Laser activated endothelial cells can induce thrombin generation. HUVECs plated on photo-etched coverslips were loaded with Fluo-4 AM and incubated with plasma in presence of calcium and corn trypsin inhibitor for 15 minutes. Cells were either stimulated with laser or left unstimulated in the presence or absence of various antibodies. After incubation with plasma, cells were fixed and immunostained for fibrin (red), phalloidin (green) and DAPI (blue). (A) Representative images of cells not stimulated by laser and incubated with recalcified plasma show minimal fibrin specific Alexa 647 signal (red). (B) Detection of fibrin-specific signal (red) after laser induced injury of a single cell in a field in presence of recalcified plasma. (C) Representative image showing lack of fibrin formation after laser injury when the cells were incubated with recalcified plasma containing 100 μg/mL function blocking tissue factor monoclonal antibody cH36. (D) No signal was detected when laser stimulated cells were immunostained with an isotype matched control IgG instead of fibrin antibody in the presence of plasma. (E) Fibrin meshwork was detected on cells activated with laser and incubated with recalcified plasma in the presence of an isotype matched control human IgG instead of the monoclonal antibody cH36. (F) Mean integrated fluorescence signal intensity of fibrin (n = 29). Data are from 2 independently performed experiments. The mean ± SEM is plotted for each group. A background mask was created for all images from panel D. Mean of the maximum signal intensities from 29 images in panel D was used as a constant background number to create a threshold segment mask for all other conditions and integrated fluorescence intensity was calculated. The means of the integrated fluorescence intensity show a significant decrease in fibrin generation when laser stimulated endothelial cells are incubated with recalcified plasma in presence of function blocking tissue factor antibody.