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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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Gradient centrifugation of rat lung homogenate on a Nycodenz density  gradient. In this and in all  subsequent figures, percentages refer to percentage per  fraction of total activity (or  antigen) recovered in the 14  fractions of a gradient. The  numbers 1–14 of the fractions are displayed on the X  axis. (a) The percentage per  fraction of tPA antigen (•)  and the percentage per fraction of tPA activity (▴) are  shown as mean ± SD (n =  4). The mean density of the  fractions is also indicated  (♦). (b) The percentage per  fraction of vWf antigen (▪)  is shown as mean ± SD (n =  4). The mean density of the  fractions is given as (♦). (c)  The mean (n = 4) percentage  per fraction of cell protein  (•; dashed line), alkaline  phosphatase (○), angiotensin-converting enzyme (□), 5′-nucleotidase (▿), and cellular fibronectin (▪). (d) The mean (n = 4) percentage per fraction of cell protein (•; dashed line), LDH (▾), acid phosphatase (▵), neutral esterase (□), and glutamate dehydrogenase (▪; dashed line).
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Figure 1: Gradient centrifugation of rat lung homogenate on a Nycodenz density gradient. In this and in all subsequent figures, percentages refer to percentage per fraction of total activity (or antigen) recovered in the 14 fractions of a gradient. The numbers 1–14 of the fractions are displayed on the X axis. (a) The percentage per fraction of tPA antigen (•) and the percentage per fraction of tPA activity (▴) are shown as mean ± SD (n = 4). The mean density of the fractions is also indicated (♦). (b) The percentage per fraction of vWf antigen (▪) is shown as mean ± SD (n = 4). The mean density of the fractions is given as (♦). (c) The mean (n = 4) percentage per fraction of cell protein (•; dashed line), alkaline phosphatase (○), angiotensin-converting enzyme (□), 5′-nucleotidase (▿), and cellular fibronectin (▪). (d) The mean (n = 4) percentage per fraction of cell protein (•; dashed line), LDH (▾), acid phosphatase (▵), neutral esterase (□), and glutamate dehydrogenase (▪; dashed line).

Mentions: Rat lung was chosen for the first experiments, since it is, of all rat tissues, richest in tPA (Padró et al., 1990). In pilot experiments, using a Nydodenz gradient with density range 1.05–1.27 g/ml, all tPA and vWf was recovered at densities <1.18 g/ml. In subsequent experiments, therefore, a more shallow gradient (density range 1.03 to 1.17 g/ ml) was employed. In such a gradient (Fig. 1, a–d), tPA was found as a single symmetrical peak, with a maximum at density 1.11–1.12 g/ml. Both tPA activity and tPA antigen were found at the same position in the gradient (Fig. 1 a). The correlation between tPA antigen and activity in the 14 fractions was in all four experiments >0.97. In those two fractions that contained maximal amounts of tPA, ∼50% of tPA (Fig. 1 a) and 6–7% of protein (Fig. 1, c and d) was recovered. vWf antigen also peaked at density 1.11–1.12 g/ml, but in addition showed a second, smaller peak at density 1.06–1.07 g/ml (Fig. 1 b). Tissue protein, and most of the marker enzymes measured, showed peak values in the fractions with densities of 1.03 to 1.08 g/ml (Fig. 1, c and d). Typically, >70% of the protein was recovered in the these fractions. Maximal alkaline phosphatase and ACE activity (used as plasma membrane marker enzymes) were found around d = 1.05–1.08 g/ml; acid phosphatase (a marker for lysosomes), neutral esterase, and 5′-nucleotidase (markers for microsomes) around d = 1.05–1.07 g/ml, and LDH (a marker for the cytosol) ∼d = 1.03 g/ml. Only glutamate dehydrogenase activity, a marker enzyme for mitochondria, was recovered at d = 1.11–1.12 g/ml, the density of maximal tPA and vWf concentrations. Cellular fibronectin, a marker of connective tissue contamination, was found at density 1.06 g/ml.


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)

Gradient centrifugation of rat lung homogenate on a Nycodenz density  gradient. In this and in all  subsequent figures, percentages refer to percentage per  fraction of total activity (or  antigen) recovered in the 14  fractions of a gradient. The  numbers 1–14 of the fractions are displayed on the X  axis. (a) The percentage per  fraction of tPA antigen (•)  and the percentage per fraction of tPA activity (▴) are  shown as mean ± SD (n =  4). The mean density of the  fractions is also indicated  (♦). (b) The percentage per  fraction of vWf antigen (▪)  is shown as mean ± SD (n =  4). The mean density of the  fractions is given as (♦). (c)  The mean (n = 4) percentage  per fraction of cell protein  (•; dashed line), alkaline  phosphatase (○), angiotensin-converting enzyme (□), 5′-nucleotidase (▿), and cellular fibronectin (▪). (d) The mean (n = 4) percentage per fraction of cell protein (•; dashed line), LDH (▾), acid phosphatase (▵), neutral esterase (□), and glutamate dehydrogenase (▪; dashed line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139817&req=5

Figure 1: Gradient centrifugation of rat lung homogenate on a Nycodenz density gradient. In this and in all subsequent figures, percentages refer to percentage per fraction of total activity (or antigen) recovered in the 14 fractions of a gradient. The numbers 1–14 of the fractions are displayed on the X axis. (a) The percentage per fraction of tPA antigen (•) and the percentage per fraction of tPA activity (▴) are shown as mean ± SD (n = 4). The mean density of the fractions is also indicated (♦). (b) The percentage per fraction of vWf antigen (▪) is shown as mean ± SD (n = 4). The mean density of the fractions is given as (♦). (c) The mean (n = 4) percentage per fraction of cell protein (•; dashed line), alkaline phosphatase (○), angiotensin-converting enzyme (□), 5′-nucleotidase (▿), and cellular fibronectin (▪). (d) The mean (n = 4) percentage per fraction of cell protein (•; dashed line), LDH (▾), acid phosphatase (▵), neutral esterase (□), and glutamate dehydrogenase (▪; dashed line).
Mentions: Rat lung was chosen for the first experiments, since it is, of all rat tissues, richest in tPA (Padró et al., 1990). In pilot experiments, using a Nydodenz gradient with density range 1.05–1.27 g/ml, all tPA and vWf was recovered at densities <1.18 g/ml. In subsequent experiments, therefore, a more shallow gradient (density range 1.03 to 1.17 g/ ml) was employed. In such a gradient (Fig. 1, a–d), tPA was found as a single symmetrical peak, with a maximum at density 1.11–1.12 g/ml. Both tPA activity and tPA antigen were found at the same position in the gradient (Fig. 1 a). The correlation between tPA antigen and activity in the 14 fractions was in all four experiments >0.97. In those two fractions that contained maximal amounts of tPA, ∼50% of tPA (Fig. 1 a) and 6–7% of protein (Fig. 1, c and d) was recovered. vWf antigen also peaked at density 1.11–1.12 g/ml, but in addition showed a second, smaller peak at density 1.06–1.07 g/ml (Fig. 1 b). Tissue protein, and most of the marker enzymes measured, showed peak values in the fractions with densities of 1.03 to 1.08 g/ml (Fig. 1, c and d). Typically, >70% of the protein was recovered in the these fractions. Maximal alkaline phosphatase and ACE activity (used as plasma membrane marker enzymes) were found around d = 1.05–1.08 g/ml; acid phosphatase (a marker for lysosomes), neutral esterase, and 5′-nucleotidase (markers for microsomes) around d = 1.05–1.07 g/ml, and LDH (a marker for the cytosol) ∼d = 1.03 g/ml. Only glutamate dehydrogenase activity, a marker enzyme for mitochondria, was recovered at d = 1.11–1.12 g/ml, the density of maximal tPA and vWf concentrations. Cellular fibronectin, a marker of connective tissue contamination, was found at density 1.06 g/ml.

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