Fig. 4

2B4 and 1D12 inhibit spontaneous metastasis but have no effect on platelet activation and hemostatic function. Effect of 2B4 (50 μg/mouse), 1D12 (50 μg/mouse), and their Fabs (50 μg/mouse) on (a) mice weight, (b) tumor weight, (c) tumor volume, and (d) pulmonary metastasis on spontaneous metastasis of 4T1 (n = 6 mice in each group). Metastasis was analyzed 3 weeks after injection of tumor cells. Representative examples of the lungs (one of each group) with metastatic foci were depicted. Average number of lung metastasis in each of the groups was shown in right graphs (± SD, P value is indicated; *P < 0.05; **P < 0.01; ***P < 0.001). e 2B4 and 1D12 did not affect platelet activation. Increased expression of APC-conjugated P-selectin indicated the degree of platelet activation. Washed platelets were treated with 10 μg/ml purified 2B4 (blue), 1D12 (orange), negative control (rat IgG, gray), or 0.05 U/ml thrombin (green) as positive control, then probed with APC-conjugated anti-P-selectin Ab. The florescence intensity was detected by flow cytometry. f 2B4 and 1D12 did not induce platelet aggregation. The aggregation of PRP pretreated with 10 μg/ml 2B4 (blue) and 10 μg/ml 1D12 (orange) was detected. Negative control was in the form of rat IgG. g Bleeding time did not prolonged 2 h after the injection of intact 2B4 or 1D12 (50 μg/mouse) (n = 4 mice in each group). h Platelet survival curves for mice injected with 2B4 (50 μg/mouse), 1D12 (50 μg/mouse), or negative control (normal rat IgG, 50 μg/mouse) (n = 6 mice in each group). i Platelet survival curves for mice injected with 2B4 Fab (50 μg/mouse), 1D12 Fab (50 μg/mouse), or negative control (normal rat Fab, 50 μg/mouse) (n = 6 mice in each group). Blood was drawn from mice at the time of antibody injection (0 h), 2 h after injection, and every 24 h following until 96 h after antibody injection. Platelet count determined by flow cytometry. The histograms are representative of three independent experiments