Figure 4.

MPO-dependent tyrosine nitration of ECM fibronectin. (a) NO₂Tyr formation in cultured endothelial cell ECM proteins. Confluent BAEC monolayers were exposed to MPO (13 nM), and washed prior to NO₂^– (100 μM) and H₂O₂ (50 μM) addition. In some cases, cells were exposed to enoxaparin (Enox, 150 μg·ml^–1) and washed, followed by MPO exposure and no washing, before NO₂^– and H₂O₂ were added. Matrix-enriched protein fractions were isolated as described in Methods, separated by 4–20% SDS-PAGE gradient gels, and probed with mouse monoclonal anti-fibronectin and rabbit anti-NO₂Tyr. Protein staining with Coomassie blue confirmed that equal amounts of protein were electrophoretically resolved in each exposure condition. (b–d) NO₂Tyr formation in purified human fibronectin and its major fragments. Fibronectin (100 μg·ml^–1) (b) and the 30-, 45-, and 70-kDa fragments of fibronectin (d) were incubated with MPO (26 nM), μM NO₂^– (20–80 μM), and H₂O₂ (50 μM) in HBSS for 90 minutes. In some cases, fibronectin (c) and fibronectin fragments (d) were preincubated with enoxaparin (15 and 150 μg·ml^–1) for 45 minutes before MPO, NO₂^–, and H₂O₂ addition. Proteins were separated by SDS-PAGE electrophoresis (7.5% gels for fibronectin and 10% gels for fibronectin fragments) and then immunoblotted with rabbit polyclonal anti-NO₂Tyr.