FIGURE 3.

C4BP forms complexes with plasminogen. A and B, interaction between C4BP and plasminogen. Polyclonal sheep anti-plasminogen (10 μg/ml, A) or polyclonal anti-C4BP Ab (10 μg/ml, B) were coated on 96-well microtiter plates. A mixture of plasminogen with plasma-purified C4BP-PS or rC4BP (A), or normal plasma or serum (B) were added. Bound C4BP-plasminogen complexes were detected with a polyclonal rabbit anti-C4BP Ab (A) and sheep anti-plasminogen Ab (B) and peroxidase-conjugated respective secondary Abs and substrate. A one-way analysis of variance test was used to calculate statistical significance. ns, not significant; ***, p < 0.001. C, rC4BP (0.2 mg/ml) and plasminogen (0.2 mg/ml) were incubated separately or together overnight at 37 °C and analyzed on a Superose 12 gel filtration column. The A_{280 nm} for rC4BP (black solid line), plasminogen (gray dotted line), and rC4BP with plasminogen (black dotted line) are shown. Complexes of rC4BP and plasminogen were measured using a sandwich ELISA and plotted as percentage of complex present in normal human serum (gray squares). Furthermore, ELISA was used to detect C4BP-plasminogen complexes in pooled human plasma (black dots). ELISA data represent mean ± S.D. of three independent experiments performed in duplicates. D, protein G-coated-Sepharose beads where incubated with a monoclonal anti-plasminogen Ab, and then incubated with IgG-depleted normal serum. C4BP was detected by Western blotting under reduced conditions using a polyclonal anti-C4BP Ab. Isotype control Ab was used as negative control.