Novel Situations of Endothelial Injury in Stroke — Mechanisms of Stroke and Strategy of Drug Development: Novel Mechanism of the Expression and Amplification of Cell Surface–Associated Fibrinolytic Activity Demonstrated by Real-Time Imaging Analysis

Abstract

Abstract.: Vascular endothelial cells (VECs) secrete tissue plasminogen activator (tPA) in an active form and thus its facilitated secretion directly enhances fibrinolytic activity. We have recently demonstrated its unique secretory dynamics in GFP-tagged tPA expressing VECs using total internal reflection fluorescence microscopy. tPA-GFP appeared to remain on the cell surface after secretion. Studies using a domain-deleted mutant of tPA-GFP suggested that its binding to the cell surface was heavy-chain dependent. PA inhibitor-1 (PAI-1) facilitated dissociation of tPA-GFP by forming a high molecular weight complex. Lack of dissociation from the cell surface of catalytically inactive mutant tPA-GFP, which does not complex with PAI-1, supported PAI-1 dependence of the disappearance of tPA from the VEC surface. To confirm the possibility that retained active tPA modified cell surface fibrinolytic activity, we analyzed binding of Alexa Fluor 568–labeled plasminogen (plg-568) in tPA-GFP expressing cells. Plg-568 appeared to accumulate at tPA-GFP–retained spots as well as in pericellular/matrix adhesive areas. Either modification of the active site or deletion of the tPA-GFP heavy chain resulted in decreased accumulation of plg-568. Prolonged retention appeared essential for tPA to effectively express and amplify fibrinolytic activity on VECs, which may also be responsible for development of deleterious effects in the case of stroke. Keywords:: tissue plasminogen activator, vascular endothelial cell, real-time imaging, fibrinolysis, plasmin, endothelial injury