Abstract
Background: VECs express and secrete various anti-thrombotic molecules to maintain vascular patency. tPA, a key enzyme in fibrinolysis, is one of them and secreted as an active form. Employing total internal reflection fluorescence microscopy (TIRF-M), we clarified a unique dynamics of slow disappearance of tPA-GFP (half time of peak fluorescence: TF1/2=9.86s) from plasma membrane (PM) after opening of its containing granules. Here we discuss possible role of PA inhibitor-1 (PAI-1) on both exocytotic dynamics of tPA and fibrinolytic activity on VECs. Method: An established cell-line of VECs was cultured and transfected with tPA-GFP, tPA(S478A)-GFP (Ser478 at active center is replaced by Ala, so as not to form complex with PAI-1) and tPA(CD)-GFP (deleted in FEK-domains). The exocytotic dynamics of these tPAs-GFP near PM and the associated changes in their activities were analyzed by TIRF-M and chromogenic assay, respectively. Results: 1) Supplemented PAI-1 shortened TF1/2 of tPA-GFP and suppressed membrane-bound tPA activity. tPA-PAI-1 complex but not free tPA increased in cultured medium after PAI-1 addition. 2) TF1/2 of tPA(S478A)-GFP was slower than that of tPA-GFP and was not modified by the addition of PAI-1. 3) TF1/2 of tPA(CD)-GFP was faster than that of tPA-GFP. Conclusion: tPA stays on PM after exocytosis by binding through FEK domains. PAI-1 facilitates tPA dissociation from PM by forming an inactive complex, which suppresses cellular fibrinolytic activity on VECs. [J Physiol Sci. 2007;57 Suppl:S217]
