Fibrinolytic cross-talk: activation of fibrin- and fibronectin-bound plasminogen by cellular MPs. (A) Native or recombinant active site–inactivated Glu-plasminogen (Glu-Pg, r-Pg-Ala741) at 1μM was bound to fibrin-coated beads for 1 hour at 37°C. Fibrin-coated beads with bound plasminogen were then incubated with 106 EMPs in a final volume of 200 μL. After overnight incubation at 22°C, the fibrin-coated beads were sedimented by centrifugation and resuspended in 10mM Tris-HCl, pH 6.8, containing 10% SDS to elute fibrin-bound plasminogen derivatives. The supernatant was electrophoresed under nonreducing conditions, proteins were transferred to PVDF membranes and revealed with a horseradish peroxidase–conjugated mAb (150 ng/mL) directed against plasminogen K1. The Western blot shows Glu-plasmin formation by EMPs. Purified plasmin is shown as reference. (B-C) Glu-plasminogen (1μM) was bound to fibrin (B) or fibronectin (C) surfaces. After 3 washes with PBS, EMPs were added at various concentrations. (D) Glu-plasminogen (1μM) was bound to fibrin surfaces (main graph) or to fibrin-coated beads (inset). THP-1 cells were then added to fibrin surfaces at various concentrations (main graph) and 2.5 × 105 fibrin-coated beads were incubated with 105 adherent monocytes or neurons (inset). The formation of plasmin was detected by measuring the release of p-nitroaniline from the chromogenic substrate CBS0065 added at 0.75mM. Bars represent the amount of plasmin formed (mOD/minute; mean ± SEM; n = 3) by THP-1 cells on fibrin (B,D) and fibronectin (C), and by adherent monocytes or neurons on fibrin-coated beads (D inset). Amil indicates amiloride; IgG, antibody against uPA and its nonimmune IgG control. *Significant changes compared with activation without THP-1 (A) or EMPs (B-C) or activation on neurons (P < .05); §changes with inhibitors compared with activation at 5 × 105 EMPs (P < .05).
