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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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Distribution of tPA  antigen (pg/ml) from a control HUVEC culture (•) and  from a culture treated with 1  NIH U/ml of human α-thrombin for 3 min (○). Data from  two separate experiments are  shown (a and b). HUVEC  homogenate was prepared for  each condition from a 300-cm2  HUVEC culture (first passage), pretreated for 24 h  with 1 mM sodiumbutyrate  (see Materials and Methods). Both control and thrombin-treated cells had been obtained from the same primary HUVEC culture. On  the X axis the fraction numbers are indicated. The difference in density between corresponding fractions from the thrombin-treated and  control Nycodenz gradient fractionations was always <0.004 g/ml. Note that in Fig. 6 a, tPA is lost mainly from fractions 6–10, while in Fig.  6 b, tPA is lost from fractions 4–8. See text for details.
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Figure 5: Distribution of tPA antigen (pg/ml) from a control HUVEC culture (•) and from a culture treated with 1 NIH U/ml of human α-thrombin for 3 min (○). Data from two separate experiments are shown (a and b). HUVEC homogenate was prepared for each condition from a 300-cm2 HUVEC culture (first passage), pretreated for 24 h with 1 mM sodiumbutyrate (see Materials and Methods). Both control and thrombin-treated cells had been obtained from the same primary HUVEC culture. On the X axis the fraction numbers are indicated. The difference in density between corresponding fractions from the thrombin-treated and control Nycodenz gradient fractionations was always <0.004 g/ml. Note that in Fig. 6 a, tPA is lost mainly from fractions 6–10, while in Fig. 6 b, tPA is lost from fractions 4–8. See text for details.

Mentions: For experiments involving thrombin stimulation of regulated secretion (acute release) of tPA, 300-cm2 HUVEC cultures were used that had been preincubated for 24 h with 1 mM sodiumbutyrate to increase tPA synthesis and storage (Kooistra et al., 1987). HUVEC were stimulated with 1 NIH U/ml of human α-thrombin for 3 min (van den Eijnden-Schrauwen et al., 1995), scraped into cold buffer, and homogenized. Control cultures, not treated with thrombin, were run in parallel. After thrombin, a loss of tPA antigen from the high density range of the gradient (fractions 5–8) was observed, compared to the parallel control cultures. Two typical cultures (out of five) are shown in Fig. 5, a and b. In the experiment shown in Fig. 5 a, tPA disappeared mainly from density range 1.11–1.12 g/ml (fractions 6–8), while in the experiment shown in Fig. 5 b, tPA was reduced in fractions 4–6 (density 1.14 g/ml). The differences between cultures possibly reflect greater experimental variation in cultured HUVEC than in experiments involving rat lungs (note the small SDs in Fig. 1, a and b).


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)

Distribution of tPA  antigen (pg/ml) from a control HUVEC culture (•) and  from a culture treated with 1  NIH U/ml of human α-thrombin for 3 min (○). Data from  two separate experiments are  shown (a and b). HUVEC  homogenate was prepared for  each condition from a 300-cm2  HUVEC culture (first passage), pretreated for 24 h  with 1 mM sodiumbutyrate  (see Materials and Methods). Both control and thrombin-treated cells had been obtained from the same primary HUVEC culture. On  the X axis the fraction numbers are indicated. The difference in density between corresponding fractions from the thrombin-treated and  control Nycodenz gradient fractionations was always <0.004 g/ml. Note that in Fig. 6 a, tPA is lost mainly from fractions 6–10, while in Fig.  6 b, tPA is lost from fractions 4–8. See text for details.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139817&req=5

Figure 5: Distribution of tPA antigen (pg/ml) from a control HUVEC culture (•) and from a culture treated with 1 NIH U/ml of human α-thrombin for 3 min (○). Data from two separate experiments are shown (a and b). HUVEC homogenate was prepared for each condition from a 300-cm2 HUVEC culture (first passage), pretreated for 24 h with 1 mM sodiumbutyrate (see Materials and Methods). Both control and thrombin-treated cells had been obtained from the same primary HUVEC culture. On the X axis the fraction numbers are indicated. The difference in density between corresponding fractions from the thrombin-treated and control Nycodenz gradient fractionations was always <0.004 g/ml. Note that in Fig. 6 a, tPA is lost mainly from fractions 6–10, while in Fig. 6 b, tPA is lost from fractions 4–8. See text for details.
Mentions: For experiments involving thrombin stimulation of regulated secretion (acute release) of tPA, 300-cm2 HUVEC cultures were used that had been preincubated for 24 h with 1 mM sodiumbutyrate to increase tPA synthesis and storage (Kooistra et al., 1987). HUVEC were stimulated with 1 NIH U/ml of human α-thrombin for 3 min (van den Eijnden-Schrauwen et al., 1995), scraped into cold buffer, and homogenized. Control cultures, not treated with thrombin, were run in parallel. After thrombin, a loss of tPA antigen from the high density range of the gradient (fractions 5–8) was observed, compared to the parallel control cultures. Two typical cultures (out of five) are shown in Fig. 5, a and b. In the experiment shown in Fig. 5 a, tPA disappeared mainly from density range 1.11–1.12 g/ml (fractions 6–8), while in the experiment shown in Fig. 5 b, tPA was reduced in fractions 4–6 (density 1.14 g/ml). The differences between cultures possibly reflect greater experimental variation in cultured HUVEC than in experiments involving rat lungs (note the small SDs in Fig. 1, a and b).

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