(a) Thrombin generation assay using fluorescent thrombin substrate. Thrombin was generated following addition of platelet poor plasma to static monolayers of tissue factor expressing, TNF-α activated HUVEC. Treatment of cells with hTM/R6.5, followed by washing to remove non-specifically bound fusion protein, was more effective than addition of shTM to the plasma. Inset shows AUC (area under curve) analysis: * = p < 0.05 vs. TNF only, # = p < 0.05 for hTM/R6.5 vs. shTM at each concentration. (b) Fibrin generation (measured using AF647-conjugated fibrin antibody) in whole blood microfluidic assay. 3D-confluent endothelial monolayers were grown within flow chambers and activated with TNF-α prior to the infusion of whole blood at 5 dynes/cm2. hTM/R6.5 shows more prolonged inhibition of coagulation than shTM. Inset shows fluorescence microscopy images at 30 min after infusion of whole blood and AUC analysis: * = p < 0.05 vs. TNF only. (c) Fibrin deposition in control vs. PC deficient blood. PC supplementation with plasma-derived concentrate restores the anti-thrombotic activity of hTM/R6.5. Inset shows fluorescence microscopy images for each condition at 7.5 min after infusion of whole blood. AUC analysis is also shown: ** = p < 0.05 vs. all other conditions.
