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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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A rat lung homogenate was centrifuged on a sucrose  density gradient. On the Y axis, the percentage per fraction of  tPA antigen (•) and of vWf antigen (▪) are shown. Note that the  distribution of tPA antigen is practically identical to the distribution on a Nycodenz density gradient (Figs. 1 a and 2). The distribution of vWf on the sucrose gradient is, however, very different,  due to an extensive loss of vWf from the density range 1.10–1.12  g/ml (compare Figs. 1 b and 2). The density of the fractions from  the sucrose gradient is indicated as well (♦) and is very similar to  the density profile of Nycodenz gradients.
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Figure 3: A rat lung homogenate was centrifuged on a sucrose density gradient. On the Y axis, the percentage per fraction of tPA antigen (•) and of vWf antigen (▪) are shown. Note that the distribution of tPA antigen is practically identical to the distribution on a Nycodenz density gradient (Figs. 1 a and 2). The distribution of vWf on the sucrose gradient is, however, very different, due to an extensive loss of vWf from the density range 1.10–1.12 g/ml (compare Figs. 1 b and 2). The density of the fractions from the sucrose gradient is indicated as well (♦) and is very similar to the density profile of Nycodenz gradients.

Mentions: Using a sucrose gradient of d = 1.03–1.16 g/ml, tPA (both antigen and activity) was found at the same density (1.11– 1.12 g/ml) as in Nycodenz gradients (Fig. 3). vWf, however, showed a different distribution than in Nycodenz gradients, as no vWf peak was present in fractions 6 and 7 of sucrose density gradients. This resulted (Fig. 3) in a clear separation of tPA (peak in fractions 6 and 7) and vWf (peak in fractions 10 and 11). Marker enzymes for subcellular fractions, including the mitochondrial marker glutamate dehydrogenase, showed, in sucrose gradients, a similar distribution as in Nycodenz gradients (not shown).


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)

A rat lung homogenate was centrifuged on a sucrose  density gradient. On the Y axis, the percentage per fraction of  tPA antigen (•) and of vWf antigen (▪) are shown. Note that the  distribution of tPA antigen is practically identical to the distribution on a Nycodenz density gradient (Figs. 1 a and 2). The distribution of vWf on the sucrose gradient is, however, very different,  due to an extensive loss of vWf from the density range 1.10–1.12  g/ml (compare Figs. 1 b and 2). The density of the fractions from  the sucrose gradient is indicated as well (♦) and is very similar to  the density profile of Nycodenz gradients.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: A rat lung homogenate was centrifuged on a sucrose density gradient. On the Y axis, the percentage per fraction of tPA antigen (•) and of vWf antigen (▪) are shown. Note that the distribution of tPA antigen is practically identical to the distribution on a Nycodenz density gradient (Figs. 1 a and 2). The distribution of vWf on the sucrose gradient is, however, very different, due to an extensive loss of vWf from the density range 1.10–1.12 g/ml (compare Figs. 1 b and 2). The density of the fractions from the sucrose gradient is indicated as well (♦) and is very similar to the density profile of Nycodenz gradients.
Mentions: Using a sucrose gradient of d = 1.03–1.16 g/ml, tPA (both antigen and activity) was found at the same density (1.11– 1.12 g/ml) as in Nycodenz gradients (Fig. 3). vWf, however, showed a different distribution than in Nycodenz gradients, as no vWf peak was present in fractions 6 and 7 of sucrose density gradients. This resulted (Fig. 3) in a clear separation of tPA (peak in fractions 6 and 7) and vWf (peak in fractions 10 and 11). Marker enzymes for subcellular fractions, including the mitochondrial marker glutamate dehydrogenase, showed, in sucrose gradients, a similar distribution as in Nycodenz gradients (not shown).

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