The human in vitro BBB is highly sensitive to t-PA–mediated in situ plasmin generation. (A) t-PA (25nM) and plasminogen (Plgn; 50nM) were added to the luminal chamber of the BBB either alone or in combination and permeability assessed after 24 hours (n = 4; ***P < .001 compared with all other groups). (B) Inhibition of t-PA–mediated in situ plasmin generation fully protects the BBB. t-PA (25nM) and Plgn (50nM) were added to the luminal chamber of the BBB with or without aprotinin (2μM), and permeability was assessed 24 hours later (n = 5; ***P < .001 compared with all other groups). (C) Time course analysis of BBB disruption at 4 and 24 hours after addition of t-PA (25nM) and Plgn (50nM; n = 5, open squares) or t-PA (250nM) alone (n = 4, full circles). Compared with 250nM t-PA alone, t-PA + Plgn mediate a faster progression of BBB opening that is maximal already at 4 hours. In all panels, bars/data points represent mean ± SEM.