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© The Rockefeller University Press, 0021-9525/1997//245 $5.00
The Journal of Cell Biology, Volume 139, Number 1, , 1997 245-256

Article

An Endothelial Storage Granule for Tissue-Type Plasminogen Activator



J.J. Emeis*, Y. van den Eijnden-Schrauwen*, C.M. van den Hoogen*, W. de Priester{ddagger}, A. Westmuckett§, and F. Lupu§

* Gaubius Laboratory TNO-PG, 2301 CE Leiden, The Netherlands; {ddagger} Electron Microscopy Unit, Clusius Laboratory, University of Leiden, 2333 AL Leiden, The Netherlands; and § Thrombosis Research Institute, SW3 6LR London, United Kingdom

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.

Abbreviations used in this paper: HUVEC, human umbilical vein endothelial cells; TFPI, tissue factor pathway inhibitor; tPA, tissue-type plasminogen activator; vWf, von Willebrand factor.

Address all correspondence to J.J. Emeis, Gaubius Laboratory TNO-PG, P.O. Box 2215, 2301 CE Leiden, The Netherlands. Tel.: (31) 71-518-1451. Fax: (31) 71-518-1904. e-mail: JJ.Emeis{at}pg.tno.nl

The assistance, during various stages of this study, of Sachin Apte, Alette van Binsbergen, René Hegeman, Marielle Kroon, and Linda Oudsen is gratefully acknowledged. The authors are indebted to Dr M. Bijsterbosch for introducing them to subcellular fractionation techniques.


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