© 1997 American Heart Association, Inc.
Articles |
Involvement of Calcium and G Proteins in the Acute Release of Tissue-Type Plasminogen Activator and von Willebrand Factor From Cultured Human Endothelial Cells
From the Gaubius Laboratory TNO-PG (Y. van den E.-S., R.E.M. de V., T.K., J.J.E.) and the Department of Cardiology, University Hospital (D.E.A.), Leiden, the Netherlands; and the Thrombosis Research Institute, London, UK (F.L.).
Correspondence to J.J. Emeis, Gaubius Laboratory TNO-PG, Zernikedreef 9, 2333 CK Leiden, the Netherlands. E-mail jj.emeis{at}pg.tno.nl
Abstract In this study, we investigated the role of
Ca2+ and G proteins in thrombin-induced acute release
(regulated secretion) of tissue-type plasminogen
activator (TPA) and von Willebrand factor (vWF),
using a previously described system of primary human umbilical vein
endothelial cells (HUVECs). The acute release of TPA
and vWF, as induced by 
-thrombin, was almost zero after chelation of
Ca2+i, showing that an increase in
[Ca2+]i was required. It did not matter
whether the increase in [Ca2+]i came from an
intracellular or extracellular Ca2+ source.
Thrombin-induced release of TPA and vWF already started at low
[Ca2+]i, around 100 nmol/L. Half-maximal
release was found at a [Ca2+]i of 261 nmol/L
for TPA and at 222 nmol/L for vWF. The
Ca2+ signal was transduced to
calmodulin, as calmodulin
inhibitors inhibited TPA and vWF release. The
Ca2+ ionophore ionomycin dose dependently
released vWF; half-maximal vWF release occurred at a
[Ca2+]i of 311 nmol/L. In contrast, no TPA
release was found at all below a [Ca2+]i of
500 nmol/L. Thus, below 500 nmol/L [Ca2+]i,
an increase in [Ca2+]i alone was sufficient
to induce vWF release but not sufficient to induce TPA release. Protein
kinase C did not appear to be involved in TPA or vWF release, as
neither an activator nor an inhibitor of
protein kinase C significantly influenced release. Inhibition of
phospholipase A2 also did not reduce thrombin-induced TPA
and vWF release. The involvement of G proteins was studied by using
both saponin-permeabilized and intact cells. GDP-ß-S,
which inhibits heterotrimeric and small G proteins, significantly
inhibited thrombin-induced vWF and TPA release from
permeabilized cells. AlF4-,
which activates heterotrimeric G proteins, induced TPA and vWF
release in both intact and permeabilized
HUVECs. Preincubation of HUVECs with pertussis toxin
significantly inhibited thrombin-induced vWF release, due to inhibition
of thrombin-induced Ca2+ influx. Pertussis
toxin did not affect ionomycin-induced release. The
inhibitory effect of pertussis toxin was less obvious in
thrombin-induced TPA release, because it was counterbalanced by a
positive effect of the toxin on TPA release. Thus, both
inhibitory and stimulatory (pertussis toxin–sensitive) G
proteins were involved in TPA release. Therefore, thrombin-induced
acute release of TPA and vWF differed in two respects. First, below a
[Ca2+]i of 500 nmol/L, an increase in
Ca2+ was sufficient for vWF release but
not for TPA release. Second, pertussis toxin–sensitive G proteins were
differentially involved in acute TPA and vWF release.
Key Words: endothelial cells • tissue-type plasminogen activator • von Willebrand factor • calcium • G proteins
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