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An endothelial storage granule for tissue-type plasminogen activator.

Emeis JJ, van den Eijnden-Schrauwen Y, van den Hoogen CM, de Priester W, Westmuckett A, Lupu F - J. Cell Biol. (1997)

Bottom Line: A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC.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.

View Article: PubMed Central - PubMed

Affiliation: Gaubius Laboratory TNO-PG, Leiden, The Netherlands. JJ.Emeis@pg.tno.nl

ABSTRACT
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.

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Percentage per fraction of tPA antigen (•) and of vWf  (▪) in Nycodenz gradients after centrifugation of homogenate  prepared from 60 cm2 HUVEC cells. Shown are mean ± SD of  five homogenates from separate cell cultures.
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Figure 4: Percentage per fraction of tPA antigen (•) and of vWf (▪) in Nycodenz gradients after centrifugation of homogenate prepared from 60 cm2 HUVEC cells. Shown are mean ± SD of five homogenates from separate cell cultures.

Mentions: First passage HUVEC (60 cm2/experiment) were fractionated on Nycodenz gradients. In Fig. 4, the averaged distribution of tPA antigen and of vWf is shown for five such fractionations. Slight differences in density distribution, especially for tPA, were found between cultures, but the overall distribution pattern (Fig. 4) resembled that found in rat lung. For tPA, the major peak was found at d = 1.105 g/ml (fraction 6), with a (variably pronounced) shoulder at d = 1.13 g/ml (fraction 4). A second, smaller peak was seen at d = 1.06 g/ml (fraction 10). vWf had a single major peak (d = 1.105–1.115; fractions 5 and 6), and, in three out of five cultures, a small shoulder at d = 1.06 g/ml (fraction 11). No sucrose density gradient centrifugation was performed on HUVEC.


An endothelial storage granule for tissue-type plasminogen activator.

Emeis JJ, van den Eijnden-Schrauwen Y, van den Hoogen CM, de Priester W, Westmuckett A, Lupu F - J. Cell Biol. (1997)

Percentage per fraction of tPA antigen (•) and of vWf  (▪) in Nycodenz gradients after centrifugation of homogenate  prepared from 60 cm2 HUVEC cells. Shown are mean ± SD of  five homogenates from separate cell cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139817&req=5

Figure 4: Percentage per fraction of tPA antigen (•) and of vWf (▪) in Nycodenz gradients after centrifugation of homogenate prepared from 60 cm2 HUVEC cells. Shown are mean ± SD of five homogenates from separate cell cultures.
Mentions: First passage HUVEC (60 cm2/experiment) were fractionated on Nycodenz gradients. In Fig. 4, the averaged distribution of tPA antigen and of vWf is shown for five such fractionations. Slight differences in density distribution, especially for tPA, were found between cultures, but the overall distribution pattern (Fig. 4) resembled that found in rat lung. For tPA, the major peak was found at d = 1.105 g/ml (fraction 6), with a (variably pronounced) shoulder at d = 1.13 g/ml (fraction 4). A second, smaller peak was seen at d = 1.06 g/ml (fraction 10). vWf had a single major peak (d = 1.105–1.115; fractions 5 and 6), and, in three out of five cultures, a small shoulder at d = 1.06 g/ml (fraction 11). No sucrose density gradient centrifugation was performed on HUVEC.

Bottom Line: A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC.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.

View Article: PubMed Central - PubMed

Affiliation: Gaubius Laboratory TNO-PG, Leiden, The Netherlands. JJ.Emeis@pg.tno.nl

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus