t-PA causes marked morphologic and functional changes in both brain endothelial cells (BECs) and astrocytes via plasmin. (A) Representative phase contrast images of human BECs 12 hours after stimulation with t-PA (250nM) alone or t-PA (25nM) + Plgn (50nM) in the absence or presence of aprotinin (2μM) or TXA (10mM). Aprotinin and TXA fully block t-PA/Plgn–mediated changes in endothelial cell morphology, suggesting that cell surface–associated active plasmin is responsible for this effect. Scale bars represent 40 μm. (B-E) Permeability analysis of BECs alone on porous membranes 4 hours after stimulation by luminal addition of t-PA (25nM) and Plgn (50nM) with or without aprotinin (2μM; n = 3-7; B), TXA (10mM added to the luminal and abluminal chambers; n = 5; C), or RAP (0.5μM; n = 6; D). The effect of t-PA (250nM) alone or in combination with RAP (0.5μM) after 24 hours is shown in panel E (n = 4). Hence, endothelial cell permeability is also strongly influenced by cell surface–bound, active plasmin. (F) Phase-contrast images of SVG human astrocytes 16 hours after treatment with vehicle, t-PA (25nM) + Plgn (50nM) with or without aprotinin (2μM) or TXA (10mM) or with t-PA (250nM) alone, or in combination with 250nM α2AP. Scale bars represent 40 μm. (G) Phase-contrast images of primary mouse astrocytes 16 hours after treatment with vehicle or t-PA (250nM) alone. Both human SVG and mouse astrocytes undergo marked morphologic changes after treatment with t-PA/Plgn. Scale bars represent 40 μm.
