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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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Localization of  tPA and vWf in endothelial  cells: immunogold labeling in  HUVEC of tPA (a and b)  and of vWf (c), and immunogold labeling of tPA in  murine capillary endothelial  cells in lung (d) and diaphragm (e). HUVEC were  fixed in 3% p-formaldehyde  in PBS, and mouse endothelial cells were perfusion  fixed with the same fixative.  The samples were dehydrated at progressively  lower temperature, embedded in Lowicryl K4M resin at  −35°C, and immunolabeled  on grid with 10-nm colloidal  gold, as described in Materials and Methods. tPA is  found, both in situ and in  vitro, in vesicular structures  with an electron-dense content (a, b, d, and e, arrows).  For comparison, a coated  vesicle is indicated on the  surface of a HUVEC (cv).  vWf was detected in structures with the typical characteristics of Weibel-Palade  bodies (c, W-Pb): elongated  structures containing densely  packed fibrilar material. f–h  show double immunofluorescent labeling of HUVEC with  antibodies specific for tPA (f)  and vWf (g). The images collected from both fluorescence channels were superimposed in h. Note that the red  and green granules are clearly  different, and that in the superimposed figure of h almost  no yellow (red over green)  grains are observed. Bars:  (a–e) 0.2 μm; (f–h) 25 μm.
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Figure 8: Localization of tPA and vWf in endothelial cells: immunogold labeling in HUVEC of tPA (a and b) and of vWf (c), and immunogold labeling of tPA in murine capillary endothelial cells in lung (d) and diaphragm (e). HUVEC were fixed in 3% p-formaldehyde in PBS, and mouse endothelial cells were perfusion fixed with the same fixative. The samples were dehydrated at progressively lower temperature, embedded in Lowicryl K4M resin at −35°C, and immunolabeled on grid with 10-nm colloidal gold, as described in Materials and Methods. tPA is found, both in situ and in vitro, in vesicular structures with an electron-dense content (a, b, d, and e, arrows). For comparison, a coated vesicle is indicated on the surface of a HUVEC (cv). vWf was detected in structures with the typical characteristics of Weibel-Palade bodies (c, W-Pb): elongated structures containing densely packed fibrilar material. f–h show double immunofluorescent labeling of HUVEC with antibodies specific for tPA (f) and vWf (g). The images collected from both fluorescence channels were superimposed in h. Note that the red and green granules are clearly different, and that in the superimposed figure of h almost no yellow (red over green) grains are observed. Bars: (a–e) 0.2 μm; (f–h) 25 μm.

Mentions: The cellular localization of tPA in HUVEC was visualized by indirect immunofluorescence labeling and confocal microscopy, as well as at the ultrastructural level by postembedding immunogold labeling and electron microscopy. In formaldehyde-fixed, saponin-permeabilized HUVEC, tPA showed, by immunofluorescence, well defined granular staining in the cytoplasm of the cells (Fig. 8 f; compare with Fig. 6). To enable precise localization of the fluorescence, some specimens were analyzed by serial optical sectioning followed by computer-assisted reconstruction of the image. Serial optical sectioning along the Z-axis revealed that granular tPA staining was localized in the perinuclear, organelle-rich area as well as in the attenuated areas at the cell edges (Fig. 9).


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)

Localization of  tPA and vWf in endothelial  cells: immunogold labeling in  HUVEC of tPA (a and b)  and of vWf (c), and immunogold labeling of tPA in  murine capillary endothelial  cells in lung (d) and diaphragm (e). HUVEC were  fixed in 3% p-formaldehyde  in PBS, and mouse endothelial cells were perfusion  fixed with the same fixative.  The samples were dehydrated at progressively  lower temperature, embedded in Lowicryl K4M resin at  −35°C, and immunolabeled  on grid with 10-nm colloidal  gold, as described in Materials and Methods. tPA is  found, both in situ and in  vitro, in vesicular structures  with an electron-dense content (a, b, d, and e, arrows).  For comparison, a coated  vesicle is indicated on the  surface of a HUVEC (cv).  vWf was detected in structures with the typical characteristics of Weibel-Palade  bodies (c, W-Pb): elongated  structures containing densely  packed fibrilar material. f–h  show double immunofluorescent labeling of HUVEC with  antibodies specific for tPA (f)  and vWf (g). The images collected from both fluorescence channels were superimposed in h. Note that the red  and green granules are clearly  different, and that in the superimposed figure of h almost  no yellow (red over green)  grains are observed. Bars:  (a–e) 0.2 μm; (f–h) 25 μm.
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Figure 8: Localization of tPA and vWf in endothelial cells: immunogold labeling in HUVEC of tPA (a and b) and of vWf (c), and immunogold labeling of tPA in murine capillary endothelial cells in lung (d) and diaphragm (e). HUVEC were fixed in 3% p-formaldehyde in PBS, and mouse endothelial cells were perfusion fixed with the same fixative. The samples were dehydrated at progressively lower temperature, embedded in Lowicryl K4M resin at −35°C, and immunolabeled on grid with 10-nm colloidal gold, as described in Materials and Methods. tPA is found, both in situ and in vitro, in vesicular structures with an electron-dense content (a, b, d, and e, arrows). For comparison, a coated vesicle is indicated on the surface of a HUVEC (cv). vWf was detected in structures with the typical characteristics of Weibel-Palade bodies (c, W-Pb): elongated structures containing densely packed fibrilar material. f–h show double immunofluorescent labeling of HUVEC with antibodies specific for tPA (f) and vWf (g). The images collected from both fluorescence channels were superimposed in h. Note that the red and green granules are clearly different, and that in the superimposed figure of h almost no yellow (red over green) grains are observed. Bars: (a–e) 0.2 μm; (f–h) 25 μm.
Mentions: The cellular localization of tPA in HUVEC was visualized by indirect immunofluorescence labeling and confocal microscopy, as well as at the ultrastructural level by postembedding immunogold labeling and electron microscopy. In formaldehyde-fixed, saponin-permeabilized HUVEC, tPA showed, by immunofluorescence, well defined granular staining in the cytoplasm of the cells (Fig. 8 f; compare with Fig. 6). To enable precise localization of the fluorescence, some specimens were analyzed by serial optical sectioning followed by computer-assisted reconstruction of the image. Serial optical sectioning along the Z-axis revealed that granular tPA staining was localized in the perinuclear, organelle-rich area as well as in the attenuated areas at the cell edges (Fig. 9).

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