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* Gaubius Laboratory TNO-PG, 2301 CE Leiden, The Netherlands; In previous studies we have shown that, after
stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human
umbilical vein endothelial cells (HUVEC). However, the
mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules.
By density gradient centrifugation of rat lung homogenates, a particle 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 Nycodenz density
gradients and in sucrose density gradients. A similar
density distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretion, the amount of
tPA present in high-density fractions decreased, concomitant 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 Weibel-Palade bodies,
showed an identical distribution to tPA in Nycodenz
gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf
were not present in the same particle.
Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy. The distribution of tPA also differed from the distribution of
tissue factor pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicroscopy, immunoreactive
tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies. It
is concluded that tPA in endothelial cells is stored in a
not-previously-described, small and dense (d = 1.11-
1.12 g/ml) vesicle, which is different from a Weibel-Palade
body.
Electron Microscopy Unit, Clusius Laboratory, University
of Leiden, 2333 AL Leiden, The Netherlands; and § Thrombosis Research Institute, SW3 6LR London, United Kingdom
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