Involvement of calcium and G proteins in the acute release of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells.
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 alpha-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-beta-S, which inhibits heterotrimeric and small G proteins, significantly inhibited thrombin-induced vWF and TPA release from permeabilized cells. AlF-4, 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.
van den Eijnden-Schrauwen
Y
Y
Gaubius Laboratory TNO-PG, Leiden, The Netherlands.
Atsma
D E
DE
Lupu
F
F
de Vries
R E
RE
Kooistra
T
T
Emeis
J J
JJ
D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D015777:Eicosanoids / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism*
D015777:Eicosanoids; D010566:Virulence Factors, Bordetella; D014841:von Willebrand Factor; D015759:Ionomycin; D037342:Pertussis Toxin; D011493:Protein Kinase C; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D019204:GTP-Binding Proteins; D002118:Calcium