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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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Immunocytochemical staining for tPA in first  passage control HUVEC  (left) and in HUVEC after  induction of regulated secretion by 1 NIH U/ml of human α-thrombin for 3 min  (right). Cells were fixed and  stained as described in Materials and Methods. The figures show a semiquantitative  measurement of fluorescence  intensity, using pseudocolor  banding. Note that after  thrombin treatment, some  cells have lost all granular  tPA staining, while the other  cells show a reduced staining  intensity. Bar, 25 μm.
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Figure 6: Immunocytochemical staining for tPA in first passage control HUVEC (left) and in HUVEC after induction of regulated secretion by 1 NIH U/ml of human α-thrombin for 3 min (right). Cells were fixed and stained as described in Materials and Methods. The figures show a semiquantitative measurement of fluorescence intensity, using pseudocolor banding. Note that after thrombin treatment, some cells have lost all granular tPA staining, while the other cells show a reduced staining intensity. Bar, 25 μm.

Mentions: Although the differences in tPA concentration in the fractions induced by thrombin were relatively small, immunocytochemistry of tPA antigen in these cells showed that, after 3 min of thrombin stimulation, the granular tPA staining had disappeared from most cells (Fig. 6), while in other cells a weak granular staining persisted. This loss of staining is in agreement with biochemical data, which showed that, after 1 NIH U/ml thrombin stimulation, no further acute release of tPA could be obtained by ionomycin (van den Eijnden-Schrauwen et al., 1995). By Western blotting, P-selectin was detected (using the 300-cm2 HUVEC cultures) in the density range 1.10–1.15. After thrombin stimulation, the distribution of P-selectin shifted (one to two fractions) to higher densities (Fig. 7) and became more intense.


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)

Immunocytochemical staining for tPA in first  passage control HUVEC  (left) and in HUVEC after  induction of regulated secretion by 1 NIH U/ml of human α-thrombin for 3 min  (right). Cells were fixed and  stained as described in Materials and Methods. The figures show a semiquantitative  measurement of fluorescence  intensity, using pseudocolor  banding. Note that after  thrombin treatment, some  cells have lost all granular  tPA staining, while the other  cells show a reduced staining  intensity. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Immunocytochemical staining for tPA in first passage control HUVEC (left) and in HUVEC after induction of regulated secretion by 1 NIH U/ml of human α-thrombin for 3 min (right). Cells were fixed and stained as described in Materials and Methods. The figures show a semiquantitative measurement of fluorescence intensity, using pseudocolor banding. Note that after thrombin treatment, some cells have lost all granular tPA staining, while the other cells show a reduced staining intensity. Bar, 25 μm.
Mentions: Although the differences in tPA concentration in the fractions induced by thrombin were relatively small, immunocytochemistry of tPA antigen in these cells showed that, after 3 min of thrombin stimulation, the granular tPA staining had disappeared from most cells (Fig. 6), while in other cells a weak granular staining persisted. This loss of staining is in agreement with biochemical data, which showed that, after 1 NIH U/ml thrombin stimulation, no further acute release of tPA could be obtained by ionomycin (van den Eijnden-Schrauwen et al., 1995). By Western blotting, P-selectin was detected (using the 300-cm2 HUVEC cultures) in the density range 1.10–1.15. After thrombin stimulation, the distribution of P-selectin shifted (one to two fractions) to higher densities (Fig. 7) and became more intense.

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