RBC-binding and fibrinolytic capacity of anti-CR1/tPA conjugate. (A) Anti-CR1/¹²⁵I-tPA (closed symbols) or IgG/¹²⁵I-tPA (open symbols) binding to washed human RBCs at 37°C, determined after elimination of unbound material (n = 3). Inset: far-Western blotting of RBC plasma membranes. Lysates prepared from RBC ghosts from humans (lanes 3, 6, 9), WT mice (lanes 1, 4, 7), or TgN-hCR1 mice (lanes 2, 5, 8) were subjected to 4% to 12% gradient SDS-PAGE under nonreducing conditions, transferred to nitrocellulose membranes that were cut in 3 pieces, and probed with anti-CR1 (lanes 1-3), mIgG/tPA (lanes 4-6), or anti-CR1/tPA (lanes 7-9). Conjugate binding was detected with polyclonal anti-tPA antibody (2 μg/mL) and anti-CR1 binding was detected with horseradish peroxidase–conjugated anti–mouse IgG. (B) Washed human RBCs loaded with anti-CR1/I-tPA (circles) or anti-CR1 antibody (triangles) for 60 minutes at 37°C were washed with PBS-BSA and incubated in a shaker at 37°C. FACS analysis after staining with labeled anti–mouse IgG was used to monitor detachment of the conjugate or anti-CR1. Inset: typical fluorescent intensity distribution. Two almost overlapping curves on the left depict intact RBC (T = 0 gray solid line, T = 6h dashed black line); 2 almost overlapping curves on the right depict anti-CR1/tPA-RBC complex (T = 0 black dotted line, T = 6h black solid line). (C) ¹²⁵I-fibrin clots were incubated with human RBCs coated with either anti-CR1/tPA (closed circles) or IgG/tPA (open circles) at 37°C and fibrinolysis was measured as in Figure 1 (n = 3).