抄録
Background: Vascular endothelial cells (VECs) contribute to keep the patency of vasculature through the regulated expression and secretion of various molecules having either anti-coagulatory or high fibrinolytic activity. tPA, the primary PA in the vasculature, is secreted from VECs as an active form and express fibrinolytic activity in blood. In blood there also exist its specific inhibitor of PAI-1. Though the impaired tPA secretion as well as the elevated plasma concentration of PAI-1 are considered to be risk factors for thrombosis, precise mechanism underlying in tPA secretion is not clarified. Here, we analyzed the dynamics of tPA secretion from its containing granules and its modulation by PAI-1 using total internal reflection fluorescence microscopy(TIRFM). Method: An established cell-line of VECs was cultured and transfected with tPA-GFP. The dynamics of tPA-GFP secretory granules near the plasma membrane was analyzed by TIRFM. Results: 1) The dynamics of tPA-GFP granules including its opening and tPA-GFP secretion were successfully monitored by TIRFM. 2) Once tPA-GFP granules open, they kept open and tPA-GFP was released slowly. The secreted tPA-GFP was detected as tPA-GFP-PAI-1 complex in cultured medium. 3) The velocity of tPA release was facilitated by supplementary added PAI-1, which resulted in the increase in tPA-PAI-1 complex in supernatant. Conclusion: tPA-GFP is beneficial tool to investigate its exocytotic dynamics. PAI-1 seems to facilitate tPA release as an inactive complex-form, which suppresses fibrinolytic activity on VECs. [J Physiol Sci. 2006;56 Suppl:S76]
