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 thro mbin-induced acute release (regulated secretion) of tissue-type plasmi nogen activator (TPA) and von Willebrand factor (vWF), using a previou sly described system of primary human umbilical vein endothelial cells (HUVECs). The acute release of TPA and vWF, as induced by alpha-throm bin, was almost zero after chelation of Ca-i(2+), showing that an incr ease 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 t ransduced to calmodulin, as calmodulin inhibitors inhibited TPA and VW F release. The Ca2+ ionophore ionomycin dose dependently released VWF; half-maximal vWF release occurred at a [Ca2+](i), of 311 nmol/L. In c ontrast, 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 w as sufficient to induce vWF release but not sufficient to induce TPA r elease. Protein kinase C did not appear to be involved in TPA or VWF r elease, as neither an activator nor an inhibitor of protein kinase C s ignificantly influenced release. Inhibition of phospholipase A(2) also did not reduce thrombin-induced TPA and vWF release. The involvement of G proteins was studied by-using both saponin-permeabilized and inta ct cells. GDP-beta-S, which inhibits heterotrimeric and small G protei ns, 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 t hrombin-induced vWF release, due to inhibition of thrombin-induced Ca2 + influx. Pertussis toxin did not affect ionomycin-induced release. Th e inhibitory effect of pertussis toxin was less obvious in thrombin-in duced TPA release, because it was counterbalanced by a positive effect of the toxin on TPA release. Thus, both inhibitory and stimulatory (p ertussis toxin-sensitive) G proteins were involved in TPA release. The refore, thrombin-induced acute release of TPA and VWF differed in two respects. First, below a [Ca2+](i) of 500 nmol/L, an increase in Ca2was sufficient for VWF release but not for TPA release. Second, pertus sis toxin-sensitive G proteins were differentially involved in acute T PA and VWF release.