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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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Double staining of first passage HUVEC for tPA (left column, a, d, and g) and either TFPI (b), caveolin (e), or endothelin-1  (h). The panels in the right column represent the superposition of staining for tPA (green) with staining (red) for either TFPI (c), caveolin (f), or endothelin-1 (i). In case of colocalization of two proteins, the resultant superimposed image in the right hand column is yellow.  Note that only in the case of tPA and endothelin-1 (i) in some cells is superimposition observed. For technical details, see Materials and  Methods. Bar, 25 μm.
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Figure 10: Double staining of first passage HUVEC for tPA (left column, a, d, and g) and either TFPI (b), caveolin (e), or endothelin-1 (h). The panels in the right column represent the superposition of staining for tPA (green) with staining (red) for either TFPI (c), caveolin (f), or endothelin-1 (i). In case of colocalization of two proteins, the resultant superimposed image in the right hand column is yellow. Note that only in the case of tPA and endothelin-1 (i) in some cells is superimposition observed. For technical details, see Materials and Methods. Bar, 25 μm.

Mentions: Using identical techniques of indirect immunofluorescence and confocal microscopy as described above for vWf, we looked for colocalization of tPA with three other endothelial proteins: endothelin-1 and tissue factor pathway inhibitor, which are both secreted by HUVEC, and caveolin, a component of caveolae (Lisanti et al., 1994). As shown in Fig. 10, no evidence for colocalization of these proteins with tPA in HUVEC was obtained. We show elsewhere (Lupu et al., 1997) that caveolin and TFPI, but not tPA, colocalize in HUVEC in caveolae.


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)

Double staining of first passage HUVEC for tPA (left column, a, d, and g) and either TFPI (b), caveolin (e), or endothelin-1  (h). The panels in the right column represent the superposition of staining for tPA (green) with staining (red) for either TFPI (c), caveolin (f), or endothelin-1 (i). In case of colocalization of two proteins, the resultant superimposed image in the right hand column is yellow.  Note that only in the case of tPA and endothelin-1 (i) in some cells is superimposition observed. For technical details, see Materials and  Methods. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Double staining of first passage HUVEC for tPA (left column, a, d, and g) and either TFPI (b), caveolin (e), or endothelin-1 (h). The panels in the right column represent the superposition of staining for tPA (green) with staining (red) for either TFPI (c), caveolin (f), or endothelin-1 (i). In case of colocalization of two proteins, the resultant superimposed image in the right hand column is yellow. Note that only in the case of tPA and endothelin-1 (i) in some cells is superimposition observed. For technical details, see Materials and Methods. Bar, 25 μm.
Mentions: Using identical techniques of indirect immunofluorescence and confocal microscopy as described above for vWf, we looked for colocalization of tPA with three other endothelial proteins: endothelin-1 and tissue factor pathway inhibitor, which are both secreted by HUVEC, and caveolin, a component of caveolae (Lisanti et al., 1994). As shown in Fig. 10, no evidence for colocalization of these proteins with tPA in HUVEC was obtained. We show elsewhere (Lupu et al., 1997) that caveolin and TFPI, but not tPA, colocalize in HUVEC in caveolae.

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