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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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(a) A rat lung homogenate was centrifuged on  a Nycodenz density gradient,  and fractions 6 and 7, containing the highest concentrations of tPA and vWf,  were recentrifuged after dilution on an identical Nycodenz gradient. Shown are the  percentage per fraction of  tPA antigen (▪) and vWf antigen (□) in the second density gradient. The fraction  numbers 1–14 are shown on the X axis. The position of the original fractions 6 and 7 of the first gradient is indicated by the line. (b) Human recombinant tPA was added to a rat lung homogenate to a final concentration of 50 ng/ml, followed by centrifugation on a Nycodenz density gradient. Shown on the Y axis are the percentage per fraction of endogenous rat tPA antigen (•) and of exogenously  added human tPA antigen (▾). The fraction numbers 1–14 are given on the X axis. Rat and human tPA antigen concentrations were determined by species-specific ELISA assays.
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Figure 2: (a) A rat lung homogenate was centrifuged on a Nycodenz density gradient, and fractions 6 and 7, containing the highest concentrations of tPA and vWf, were recentrifuged after dilution on an identical Nycodenz gradient. Shown are the percentage per fraction of tPA antigen (▪) and vWf antigen (□) in the second density gradient. The fraction numbers 1–14 are shown on the X axis. The position of the original fractions 6 and 7 of the first gradient is indicated by the line. (b) Human recombinant tPA was added to a rat lung homogenate to a final concentration of 50 ng/ml, followed by centrifugation on a Nycodenz density gradient. Shown on the Y axis are the percentage per fraction of endogenous rat tPA antigen (•) and of exogenously added human tPA antigen (▾). The fraction numbers 1–14 are given on the X axis. Rat and human tPA antigen concentrations were determined by species-specific ELISA assays.

Mentions: Recentrifugation of the two-peak fractions (d = 1.11– 1.12 g/ml, diluted 1:1 with homogenization buffer to reduce density) on an identical Nycodenz gradient resulted in recovery of tPA and vWf at the density of the original fractions (Fig. 2 a). To decide whether the distribution of tPA in the gradient might have been due to binding of particle-free tPA to a particle of d = 1.11–1.12 g/ml, recombinant human tPA (Activase; final concentration 50 ng/ml) was added to a rat lung homogenate before gradient centrifugation. Subsequently, we separately measured the exogenously added human tPA antigen and the endogenous rat tPA antigen by species-specific tPA ELISA assays. Rat tPA was recovered as a single peak at its normal density, while the human tPA was found distributed diffusely through the gradient (Fig. 2 b). Upon ultracentrifugation of a lung homogenate in homogenization buffer without Nycodenz (d = 1.03 g/ml), >90% of tPA and vWf was recovered at the bottom of the tube. After ultracentrifugation in homogenization buffer without Nycodenz but now containing 1% Triton X-100 to lyse organelles, tPA and vWf were diffusely distributed through the gradient (not shown). From these data it was concluded that almost all tPA was present in rat lung homogenates in sedimentable particles of d = 1.11–1.12 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)

(a) A rat lung homogenate was centrifuged on  a Nycodenz density gradient,  and fractions 6 and 7, containing the highest concentrations of tPA and vWf,  were recentrifuged after dilution on an identical Nycodenz gradient. Shown are the  percentage per fraction of  tPA antigen (▪) and vWf antigen (□) in the second density gradient. The fraction  numbers 1–14 are shown on the X axis. The position of the original fractions 6 and 7 of the first gradient is indicated by the line. (b) Human recombinant tPA was added to a rat lung homogenate to a final concentration of 50 ng/ml, followed by centrifugation on a Nycodenz density gradient. Shown on the Y axis are the percentage per fraction of endogenous rat tPA antigen (•) and of exogenously  added human tPA antigen (▾). The fraction numbers 1–14 are given on the X axis. Rat and human tPA antigen concentrations were determined by species-specific ELISA assays.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: (a) A rat lung homogenate was centrifuged on a Nycodenz density gradient, and fractions 6 and 7, containing the highest concentrations of tPA and vWf, were recentrifuged after dilution on an identical Nycodenz gradient. Shown are the percentage per fraction of tPA antigen (▪) and vWf antigen (□) in the second density gradient. The fraction numbers 1–14 are shown on the X axis. The position of the original fractions 6 and 7 of the first gradient is indicated by the line. (b) Human recombinant tPA was added to a rat lung homogenate to a final concentration of 50 ng/ml, followed by centrifugation on a Nycodenz density gradient. Shown on the Y axis are the percentage per fraction of endogenous rat tPA antigen (•) and of exogenously added human tPA antigen (▾). The fraction numbers 1–14 are given on the X axis. Rat and human tPA antigen concentrations were determined by species-specific ELISA assays.
Mentions: Recentrifugation of the two-peak fractions (d = 1.11– 1.12 g/ml, diluted 1:1 with homogenization buffer to reduce density) on an identical Nycodenz gradient resulted in recovery of tPA and vWf at the density of the original fractions (Fig. 2 a). To decide whether the distribution of tPA in the gradient might have been due to binding of particle-free tPA to a particle of d = 1.11–1.12 g/ml, recombinant human tPA (Activase; final concentration 50 ng/ml) was added to a rat lung homogenate before gradient centrifugation. Subsequently, we separately measured the exogenously added human tPA antigen and the endogenous rat tPA antigen by species-specific tPA ELISA assays. Rat tPA was recovered as a single peak at its normal density, while the human tPA was found distributed diffusely through the gradient (Fig. 2 b). Upon ultracentrifugation of a lung homogenate in homogenization buffer without Nycodenz (d = 1.03 g/ml), >90% of tPA and vWf was recovered at the bottom of the tube. After ultracentrifugation in homogenization buffer without Nycodenz but now containing 1% Triton X-100 to lyse organelles, tPA and vWf were diffusely distributed through the gradient (not shown). From these data it was concluded that almost all tPA was present in rat lung homogenates in sedimentable particles of d = 1.11–1.12 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