AN ENDOTHELIAL STORAGE GRANULE FOR TISSUE-TYPE PLASMINOGEN-ACTIVATOR

In previous studies we have shown that, after stimulation by a recepto r ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbil ical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, sugges ting that the two proteins may be stored in different secretory granul es. By density gradient centrifugation of rat lung homogenates, a part icle was identified that contained nearly all tPA activity and antigen . This particle had an average density of 1.11-1.12 g/ml, both in Nyco denz density gradients and in sucrose density gradients. A similar den sity distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretio n, the amount of tPA present in high-density fractions decreased, conc omitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin. vWf, known to be stored in We ibel-Palade bodies, showed an identical distribution to tPA in Nycoden z gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, sug gesting that tPA and vWf were not present in the same particle. Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing p articles showed a different distribution by confocal microscopy. The d istribution of tPA also differed from the distribution of tissue facto r pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicr oscopy, immunoreactive tPA could be demonstrated in small vesicles mor phologically different from the larger Weibel-Palade bodies. It is con cluded that tPA in endothelial cells is stored in a not-previously-des cribed, small and dense (d = 1.11-1.12 g/ml) vesicle, which is differe nt from a Weibel-Palade body.