Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defi ned, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore , whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) e xhibited immunofluorescent staining for t-PA, which was observed only in We ibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected i n Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation In contrast, plasminogen activat or inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsul in recombinant adenovirus resulted in the storage of insulin in Weibel-Pala de bodies, indicating that these organelles can also store nonendothelial p roteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the local ization of t-PA in adrenocorticotropic hormone-containing granules, indicat ing that t-PA can be diverted to secretory granules independently of vWf. C oinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenov irus resulted in the colocalization of t-PA and insulin in the same granule s. Taken together, these results suggest that HUVEC have protein sorting me chanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.