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.

The process of acute release of tissue-type plasminogen activator (tPA) is important in locally speeding up fibrinolysis. Using a sensitive enzyme-linked immunosorbent assay for tPA, we investigated the acute release of tPA from cultured human umbilical vein endothelial cells. The addition of thrombin (0.003 to 3 NIH U/mL) caused the dose-dependent release of noncomplexed, enzymatically active tPA into the medium. The amount of tPA released into the medium by thrombin was similar to the difference in the amounts of tPA present in extracts from thrombin-treated cells and control cells. The process of acute release of tPA was complete in 1 minute, whereas the concomitant release of von Willebrand factor into the medium was slightly slower (maximum after 3 minutes). By increasing (c.q. decreasing) tPA synthesis, it was found that the amount of tPA constitutively secreted, the amount acutely released, and the amount in cell extracts were increased (c.q. decreased) to the same extent. The same relation was found in vivo. When rats were pretreated with cholera toxin or retinoic acid to increase tPA synthesis, plasma levels of tPA were increased, whereas acute release of tPA, as induced by bradykinin, was increased to the same extent. Acutely released tPA and constitutively secreted tPA were liberated from different pathways in human umbilical vein endothelial cells; tPA had, relative to the in vivo situation, a short residence time in the acutely releasable pathway.

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.

SummaryThe effect of compounds increasing intracellular adenosine 3’:5’-cyclic monophosphate [cAMP]i levels (prostacyclin, isoproterenol, forskolin, cholera toxin), and of the cAMP analogs 8-bromo-cAMP and dibutyryl-cAMP, on the regulated secretion (acute release) of tissue-type plasminogen activator (tPA) and von Willebrand factor (vWF) was studied in cultured human umbilical vein endothelial cells (HUVEC).Prostacyclin, isoproterenol and forskolin, which increased [cAMP]i in HUVEC, and the cell-permeant cAMP analog 8-bromo-cAMP induced dose- and time-dependent secretion of tPA and vWF. The extent of vWF and tPA release correlated with [cAMP]i, and was increased by the phosphodiesterase inhibitor isobutylmethylxanthine.In contrast to thrombin, the cAMP-elevating agents did not increase the intracellular calcium concentration [Ca2+]i in HUVEC. At sub-maximal concentrations, the effects of thrombin and prostacyclin were additive.Our results show that an increase in [cAMP]i resulted in regulated secretion (acute release) of tPA and vWF from HUVEC, without the concomitant increase in [Ca2+]i which is, in HUVEC, essential for thrombin-induced regulated secretion to occur. cAMP-induced secretion represents a novel mechanism for causing regulated secretion of tPA and vWF from endothelial cells.

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