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Annexin II tetramer (AIIt) is a major Ca2+-binding protein of endothelial cells which has been shown to exist on both the intracellular and extracellular surfaces of the plasma membrane. In this report, we demonstrate that AIIt stimulates the activation of plasminogen by facilitating the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. Fluid-phase AIIt stimulated the rate of activation of [Glu]plasminogen about 341-fold compared with an approximate 6-fold stimulation by annexin II. AIIt bound to [Glu]plasminogen(S741C-fluorescein) with a Kd of 1. 26 +/- 0.04 microM (mean +/- S.D., n = 3) and this interaction resulted in a large conformational change in [Glu]plasminogen. Kinetic analysis established that AIIt produces a large increase of about 190-fold in the kcat, app and a small increase in the Km,app which resulted in a 90-fold increase in the catalytic efficiency (kcat/Km) of t-PA for [Glu]plasminogen. AIIt also stimulated the t-PA-dependent activation of [Lys]plasminogen about 28-fold. Furthermore, other annexins such as annexin I, V, or VI did not produce comparable activation of t-PA-dependent conversion of [Glu]plasminogen to plasmin. The stimulation of the activation of [Glu]plasminogen by AIIt was Ca2+-independent and inhibited by epsilon-aminocaproic acid. AIIt bound to human 293 cells potentiated t-PA-dependent plasminogen activation. AIIt that was bound to phospholipid vesicles or heparin also stimulated the activation of [Glu]plasminogen 5- or 11-fold, respectively. Furthermore, immunofluorescence labeling of nonpermeabilized HUVEC revealed a punctated distribution of AIIt subunits on the cell surface. These results therefore identify AIIt as a potent in vitro activator of plasminogen.