Involvement of calcium and G proteins in the acute release of tissue- type plasminogen activator and von Willebrand factor from cultured human endothelial cells

Involvement of calcium and G proteins in the acute release of tissue- type plasminogen activator and von Willebrand factor from cultured human endothelial cells

van den Eijnden-Schrauwen, Y.
Atsma, D.E.
Lupu, F.
de Vries, R.E.M.
Kooistra, T.
Emeis, J.J.
TNO Preventie en Gezondheid

1997

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 yon 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 α-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. Halfmaximal 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-β-S, which inhibits heterotrimeric and small G proteins, significantly inhibited thrombin-induced vWF and TPA release from permeabilized cells. A1F4-, 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.

Calcium
Endothelial cells
G proteins
Tissue-type plasminogen activator
Von Willebrand factor
Calcium
Cells, Cultured
Eicosanoids
Endothelium, Vascular
GTP-Binding Proteins
Humans
Ionomycin
Pertussis Toxin
Protein Kinase C
Thrombin
Tissue Plasminogen Activator
Virulence Factors, Bordetella
von Willebrand Factor

http://resolver.tudelft.nl/uuid:f8c19eb1-5062-498c-babe-2b11da5e992d

234229

1079-5642

Arteriosclerosis, Thrombosis, and Vascular Biology, 17 (10), 2177-2187

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