Urokinase activity of free and cell-bound anti-PECAM scFv-uPA. (A) Amidolytic activities of fusion protein generated at different molar ratios of plasmin to scFv-uPA. (B) Fibrinolytic activity using a fibrin plate. From left to right 1:3 serial dilutions of lmw-tcuPA (50 ng), lmw-scuPA (100 ng), and scFv-lmw scuPA (200 ng) were incubated on a fibrin-coated plate at 37°C. Lytic zones were measured after staining fibrin with trypan blue. (C) Amidolytic activity associated with the cell surface of control REN (○) versus PECAM-transfected (•, REN/PECAM) REN cells was determined by conversion of chromogenic substrate after incubation with various amounts of fusion protein. (D) Preincubation of REN/PECAM cells with parental anti-PECAM IgG, mAb 390, reduces binding of enzymatically active scFv-uPA. Kinetics of disappearance of cell-bound scFv-uPA (□) and its amidolytic activity (▦) was determined by using H5V mouse endothelioma cells (E) and human REN/PECAM-1 cells (F). Basal levels of uPA antigen (▪) and amidolytic activity (▨) were determined by using intact cells. Amounts of fusion protein and amidolytic activity anchored to cell surface were significantly different from basal levels at 3 hours in mouse cells (P < .002) and 24 hours in human cells (P < .01). Error bars indicate SEM.