Plasmin cleaves annexin A2 and disrupts the annexin A2 heterotetramer. (A) Plasmin cleaves annexin A2 on the monocyte membrane. For the in vivo studies, 2 × 106 monocytes were incubated for 30 minutes at 37°C in RPMI 1640 without lysine in absence or presence of 0.43 CTA U/mL plasmin or equivalent amounts of plasmin catalytically inactivated by pretreatment with 25 μM VPLCK. The reaction was stopped by the addition of aprotinin (6000 KI/mL), and proteins were separated on 15% SDS-PAGE and visualized by immunoblotting using mAbs against annexin A2 and S100A10. Antisense ODN treatment was performed as described for Figure 4A. (B) Plasmin cleaves annexin A2 at lysine 27. Wild-type (WT) and K27A annexin A2 (Lys27Ala) proteins were translated in vitro in the presence of 35S-methionine and were subsequently incubated for 10 minutes at 37°C in the absence or presence of 0.43 CTA U/mL plasmin. After addition of protease inhibitor, the proteins were separated on 15% SDS-PAGE and the bands were visualized by PhosphoImager. (C) Plasmin induces dissociation of the annexin A2 heterotetramer complex. Monocytes, either unstimulated (lanes 1 and 4) or stimulated for 30 minutes either with 0.43 CTA U/mL plasmin (lanes 2 and 5) or equivalent amounts of catalytically inactivated plasmin (lanes 3 and 6) were lysed and S100A10 was immunoprecipitated (IP). Proteins coimmunoprecipitating with S100A10 were resolved by Tricine-SDS-PAGE and analyzed using mAbs against annexin A2 and S100A10. All data are representative of at least 3 independent experiments.
