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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2177-2187.)
© 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

Y. van den Eijnden-Schrauwen; D. E. Atsma; F. Lupu; R. E. M. de Vries; T. Kooistra; ; J. J. Emeis

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

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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

	Introduction

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The regulated secretion (acute release) of TPA from the endothelium, which can be induced by various stimuli, is considered important in fibrinolysis.^{1 2 3} Besides TPA, vWF is also acutely released from the endothelium on application of these stimuli.^{4 5 6 7 8 9 10 11} In contrast to TPA, vWF favors coagulation and promotes platelet aggregation and adhesion.

In this study, we investigated the role of second messenger systems in acute TPA and vWF release from cultured human endothelial cells. To this end, we used a system with HUVECs, which shows acute release characteristics comparable to the situation in vivo and which is suitable to study both TPA and vWF release.^{1 12} Thrombin was used as a stimulus to induce acute release, since it is a potent and physiologically important stimulus for acute TPA and vWF release.^{13 14}

It is known that the addition of thrombin rapidly increases the [Ca²⁺]_i in endothelial cells.¹⁵ Furthermore, thrombin and Ca²⁺ ionophore, which both increase [Ca²⁺]_i, stimulate vWF release from HUVECs in vitro,^{4 5 6 7 8 9} as does the introduction of Ca²⁺ into permeabilized cells.^{7 16} It is well established that next to Ca²⁺, G proteins are also involved in secretion.^{17 18 19} G proteins, including one or more PTX-sensitive G proteins, are involved in transducing (thrombin) receptor-mediated signals in endothelial cells^{20 21 22 23} ; the latter G proteins include one or more pertussis toxin–sensitive G proteins.²³ Since only little is known about the involvement of Ca²⁺ in TPA release^{24 25} or about the involvement of G proteins in TPA and vWF release,¹⁶ we investigated in this study the role of Ca²⁺ and G proteins in acute release (regulated secretion) of TPA and vWF from HUVECs.

We demonstrated that Ca²⁺ was essential for thrombin-induced TPA and vWF release and that it most likely acted via calmodulin. At comparable levels of [Ca²⁺]_i, thrombin induced release of TPA and of vWF to the same extent. On stimulation with Ca²⁺ ionophore, however, TPA release required higher levels of [Ca²⁺]_i than did vWF release. In experiments involving GDP-ß-S, AlF₄^- (AlCl₃ plus NaF), and PTX, PTX-sensitive G proteins were found to be differentially involved in the acute release of TPA and vWF.

	Methods

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Materials
Cell-culture reagents were from Flow Laboratories. Sterile, pyrogen-free, HSA (20% wt/vol) was from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service. Human -thrombin, sodium butyrate, cyclopiazonic acid, TFP, glutaraldehyde, indomethacin, PMA, LDH reagent kit, AlF₄^-, saponin, PTX, and GDP-ß-S were from Sigma Chemical Company. W-7, ETYA, and SK&F96365 were bought from Biomol. Calmidazolium came from Janssen Pharmaceutica. Fura 2-AM and BAPTA-AM were bought from Molecular Probes, ionomycin from Calbiochem, and EGTA from Aldrich. Materials used in the vWF and TPA ELISAs have been described.^{25 26} All other chemicals were from Merck. AACOCF₃ was a gift from Dr G. Nalbone (Laboratoire d'Hématologie, Marseille, France). Ro-31-8220 was a gift from Dr G. Lawton (Hoffmann La Roche, Welwyn Garden City, UK).

Cell Culture
HUVECs were isolated by using the method of Jaffe et al²⁷ and cultured as previously described²⁸ in total M199 (medium 199 supplemented with 10% [vol/vol] human serum, 10% [vol/vol] heat-inactivated newborn calf serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, 200 µg/mL endothelial cell growth factor, 2.5 U/mL heparin, and 2 mmol/L L-glutamine), under 5% CO₂ at 37°C. Cells were passaged once by trypsin/EDTA treatment at a split ratio of 1:3 and cultured to confluence in 2-cm² wells (final density 2x10⁵ cells per well) or on 1.44-cm² glass coverslips that had been coated with glutaraldehyde cross-linked gelatin.

Induction of Acute Release of TPA and vWF
Acute release of TPA and vWF was studied as previously described.¹² In short, first-passage confluent HUVECs were incubated for 24 hours before an experiment in total M199 containing 1 mmol/L butyrate.²⁹ Subsequently, HUVECs were incubated in medium 199 containing 20% (vol/vol) newborn calf serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine for 2 hours before an experiment, and the conditioned media were collected. The cells were then washed with sterile PBS, and 0.3 mL M199/HSA (medium 199 containing 0.03% [wt/vol] HSA, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine) was added to the cells. Inhibitors were diluted in M199/HSA and added during this time period, as indicated. Thirty minutes later, the appropriate stimulus in M199/HSA or vehicle was added to the cells and the media were collected after 3 minutes. In this study, only those cell cultures that released TPA on stimulation are reported (for unknown reasons, 10% to 15% of HUVEC cultures do not release detectable amounts of TPA on stimulation). When an experiment involving Ca²⁺ was performed, M199 was replaced by Tyrode's solution (composition in mmol/L: NaCl 132, KCl 4, MgCl₂ 1, NaHCO₃ 11.9, NaH₂PO₄ 0.36, D-glucose 10, HEPES 12.25, and CaCl₂ 1.6 or EGTA 0.1) supplemented with vitamins and amino acids, starting at the 2-hour preincubation period. Before the induction of acute release, cells were incubated with Tyrode's/HSA (Tyrode's solution containing 0.03% [wt/vol] HSA, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine) for 10 minutes. If required, 10 µmol/L BAPTA-AM was present during the last hour of the preincubation period. All incubations were done at 37°C. Assays for TPA and vWF were always done on the same conditioned media.

Minimal Cell Permeabilization
First-passage HUVECs were cultured as described above and permeabilized essentially as described by Birch et al.⁷ The cells were washed with sterile PBS and then incubated for 10 minutes with permeabilization buffer (composition in mmol/L: NaCl 20, KCl 100, MgCl₂ 1, HEPES 30, EGTA 0.1, L-glutamine 2, gelatin 0.1% [wt/vol], HSA 0.03% [wt/vol], pH 7.0) containing 15 µg/mL saponin. The [Ca²⁺]_e in this buffer was 100 nmol/L (from impurities). A single stock solution of saponin (20 mg/mL in permeabilization buffer) was used in all experiments described. Saponin was diluted in a strictly standardized way and prewarmed to 37°C for 45 minutes before each experiment. Cells were stimulated for 3 minutes with I-thrombin after a 10-minute incubation with permeabilization buffer.

Intracellular Free Ca²⁺ Measurements
First-passage HUVECs cultured on coverslips were loaded with 4 µmol/L fura 2-AM in M199/HSA for 45 minutes at 37°C. After the loading period, the cells were washed twice with PBS and put immediately in a coverslip holder, which was then placed into a cuvette containing Tyrode's solution. The cuvette was introduced into a thermostatically controlled chamber at 37°C in a Perkin-Elmer LS-5B single-wavelength fluorescence spectrophotometer. The excitation wavelength was alternated between 338 and 380 nm, and emitted light was collected at 495 nm. Slits were set at the 10.0 setting for excitation and emission. In each experiment, background autofluorescence was determined and subtracted. Calibration and calculations were performed as described by Grynkiewicz et al.³⁰ The maximal fluorescence ratio was determined by addition of 1 or 2 µmol/L ionomycin, and the minimal fluorescence ratio was determined after subsequent incubation with 10 mmol/L EGTA in 1 mol/L Tris, pH 8.5, for 15 minutes.

Use of PTX, GDP-ß-S, and AlF₄^-
PTX, which inactivates G_i/o,^{31 32} was included in the culture medium during both the 2-hour and the 30-minute preincubation periods. A concentration of 1 µg/mL PTX was used, since that concentration fully inactivates G_i/o.³³ GDP-ß-S (2 mmol/L), an inhibitor of both small and heterotrimeric G proteins, was present during the 10 minutes of permeabilization and also during the 3 minutes of thrombin stimulation. AlF₄^- (20 µmol/L AlCl₃ plus 30 mmol/L NaF), which stimulates heterotrimeric G proteins,³⁴ was dissolved in permeabilization buffer to prevent formation of CaF₂ and Al₂(PO₄)₃.

Assays
Human TPA antigen was measured by ELISA as described.²⁶ Recombinant human one-chain TPA (activase) was used for calibration. The detection limit in this assay is 10 pg/mL. vWF antigen was measured by ELISA as described.³⁵ Human pooled plasma (0.078% to 1.25%) was used for calibration. The vWF concentration is given as units per milliliter, 100 U being defined as the amount of vWF antigen present in 1 mL of pooled human plasma. 6-Ketoprostaglandin F₁ was measured by radioimmunoassay by Dr F.J. Zijlstra (Erasmus University, Rotterdam, the Netherlands).

Data Presentation, Units Used, Definitions, and Statistics
Acute release is defined as the concentration of TPA (or vWF) in medium with stimulus minus the concentration in medium without stimulus at t=3 minutes. The amount of acute release is expressed as a percentage (±SD) of the acute release found in thrombin-treated cells (determined in at least three independent experiments performed in triplicate). Constitutive secretion is defined as the amount of protein secreted in Tyrode's/HSA or M199/HSA during the 10- or 30-minute preincubation period. Changes in the amount of protein secreted during this preincubation period as a result of using inhibitors may be due to altered constitutive secretion or to regulated secretion. Such changes will be indicated, but they were not further investigated. [Ca²⁺]_i was measured in duplicate, while TPA and vWF release were measured in parallel in triplicate cups.

All data shown are mean±SD (number of determinations), or median (95% CI). Statistical significance was determined by comparing treated cells with control cells by one-way ANOVA, followed by Bonferroni's modified t test. Experiments involving dose-response curves in the presence or absence of an inhibitor were analyzed by two-way ANOVA. Differences are considered significant at P<.05 (two sided).

	Results

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General
In this study, we used our previously described¹² system of first-passage HUVECs, in which the acute release of both TPA and vWF can be studied in vitro. Compared with our previous report, the system was modified in that HUVECs were preincubated with 1 mmol/L sodium butyrate for 24 hours to increase TPA synthesis. Without butyrate treatment, TPA synthesis is often so low that it makes the measurement of acute TPA release imprecise. Butyrate increased TPA in all cells, as shown by immunocytochemistry, but did not affect vWF synthesis. Control experiments demonstrated that the sensitivity of HUVECs to thrombin stimulation and the time course of acute TPA and vWF release were not affected by butyrate. The amounts of TPA and vWF constitutively released during the 30-minute preincubation period and the amounts acutely released by thrombin over 3 minutes were of a similar magnitude.

Increase in [Ca²⁺]_i and Release of TPA and vWF by Thrombin
The system described was then used in combination with the Ca²⁺ dye fura 2-AM to investigate the involvement of Ca²⁺ in the acute release of TPA and vWF (Fig 1A to 1C). Under basal conditions, the [Ca²⁺]_i was 76±36 nmol/L (n=10) in first-passage HUVECs. When cells were stimulated with 1 NIH U/mL of thrombin, [Ca²⁺]_i increased in less than 1 minute to a peak level of 792±187 nmol/L (n=10). Thereafter, [Ca²⁺]_i decreased to a plateau level of 224±218 nmol/L (n=10). The peak [Ca²⁺]_i induced by thrombin preceded in time the acute release of TPA and vWF: these latter processes are maximal at 1 and 3 minutes, respectively.¹²

View larger version (17K): [in this window] [in a new window]   Figure 1. Modulation of calcium fluxes affects thrombin-stimulated Ca2+i. Ca2+i was measured in HUVECs as described in "Methods," and representative traces (one of six experiments) show the fluorescence ratio (340 nm/380 nm) in time. After 10 minutes' preincubation with the described agents and buffers, 1 NIH U/mL -thrombin was added (at arrow) and the fluorescence ratio recorded for 3 minutes. A, Intracellular chelation of Ca2+. HUVECs were preincubated with 10 µmol/L BAPTA-AM in M199/HSA for 1 hour at 37°C and subsequently incubated with Tyrode's solution containing 100 µmol/L EGTA without Ca2+ (dashed line). Control cells were incubated with Tyrode's solution containing 1.6 mmol/L Ca2+(solid line). B, Inhibition of Ca2+ influx. HUVECs were incubated with Tyrode's solution containing 1.6 mmol/L Ca2+ plus 50 µmol/L SK&F96365 (dotted line), Tyrode's solution containing 100 µmol/L EGTA without Ca2+(dashed line), or Tyrode's solution containing 1.6 mmol/L Ca2+ (solid line,) for 10 minutes at 37°C. C, Depletion of the Ca2+i pool. After a 2-minute equilibration period, HUVECs were stimulated with 10 µmol/L cyclopiazonic acid (first arrows) in Tyrode's solution containing 100 µmol/L EGTA without Ca2+(dashed line). The effect of thrombin on control cells is also shown (solid line).

Role of Ca²⁺-Calmodulin in Thrombin-Induced TPA and vWF Release
To study whether the increase in [Ca²⁺]_i is essential in thrombin-induced TPA and vWF release, [Ca²⁺]_i was minimized by incubating the cells with the cell-permeable Ca²⁺ chelator BAPTA-AM in the absence of Ca²⁺_e. Under these conditions, no increase in [Ca²⁺]_i was found on addition of thrombin (Fig 1A). The acute release of TPA and vWF, measured in parallel, was almost zero (Table 1).

View this table: [in this window] [in a new window]   Table 1. Intracellular and Extracellular Ca2+ Effects on Thrombin-Stimulated TPA and vWF Release

To study whether Ca²⁺ influx contributed to acute release of TPA and vWF, HUVECs were incubated for 10 minutes before thrombin treatment with either 50 µmol/L SK&F96365 (an inhibitor of Ca²⁺ influx)³⁶ or Ca²⁺-free Tyrode's solution containing 100 µmol/L EGTA. The peak level of [Ca²⁺]_i was slightly reduced, and [Ca²⁺]_i rapidly returned to basal levels in Tyrode's solution containing SK&F96365, as well as in Ca²⁺-free Tyrode's solution containing 100 µmol/L EGTA (Fig 1B). However, TPA and vWF release were only partially inhibited under these conditions (Table 1).

The contribution of Ca²⁺_i pools was studied as follows: 10 µmol/L cyclopiazonic acid³⁷ was used to empty the Ca²⁺_i pools, in the absence of Ca²⁺_e. On addition of cyclopiazonic acid in Ca²⁺-free Tyrode's solution, a small transient increase in [Ca²⁺]_i was seen. At 10 minutes after cyclopiazonic acid addition, thrombin induced no further increase in [Ca²⁺]_i (Fig 1C). TPA and vWF release were diminished to 41% and 60%, respectively, under these conditions (Table 1; see also "Discussion"). Cyclopiazonic acid did not change the constitutive secretion of TPA or vWF in the absence of Ca²⁺_e.

Together, these experiments suggested that an increase in [Ca²⁺]_i was necessary for thrombin-induced acute release of TPA and vWF but that it was not important from which source the Ca²⁺ was derived.

To answer the question as to whether Ca²⁺ acted via binding to calmodulin, HUVECs were preincubated for 30 minutes with various concentrations of the calmodulin inhibitor TFP, calmidazolium, or W-7 before stimulation with -thrombin. All inhibitors inhibited TPA and vWF release dose dependently (not shown). At optimal concentrations, all three inhibitors significantly inhibited the release of TPA and vWF (Table 2), suggesting that the increase in [Ca²⁺]_i is transduced to calmodulin. TFP and W-7 did not significantly affect constitutive secretion during the preincubation period. Calmidazolium, however, significantly enhanced vWF secretion during the preincubation period; a similar effect has been observed in vivo in a perfused rat hindleg system.²⁵

View this table: [in this window] [in a new window]   Table 2. Effect of Calmodulin Inhibitors on Thrombin-Stimulated TPA and vWF Release

Increase in [Ca²⁺]_i Necessary for Thrombin-Induced TPA and vWF Release
The Ca²⁺_i levels and the acute release of TPA and vWF were measured in parallel after stimulation with increasing concentrations of thrombin. In Fig 2, the peak concentration [Ca²⁺]_i is plotted against TPA or vWF release (expressed as percentage of the release induced by 1 NIH U/mL of thrombin). Acute release of TPA and vWF started around 100 nmol/L [Ca²⁺]_i and increased very rapidly when [Ca²⁺]_i increased further. By linear regression analysis, it was calculated that 50% TPA release occurred at 269 nmol/L Ca²⁺ (95% CI, 211 to 338 nmol/L) and 50% vWF release at 222 nmol/L (95% CI, 182 to 279 nmol/L; see legend to Fig 2). In combination with the fact that complete removal of [Ca²⁺]_i in HUVECs with BAPTA-AM in the absence of Ca²⁺_e inhibited thrombin-induced TPA and vWF release (see Table 1), these data suggested that an increase in [Ca²⁺]_i above 100 nmol/L was necessary for thrombin-induced acute release of TPA and vWF to occur.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of thrombin on [Ca2+]i and TPA and vWF release. Various concentrations of -thrombin (0 to 1 NIH U/mL) were added to HUVECs for 3 minutes and the peak [Ca2+]i was measured in duplicate by using the fura 2-AM technique. In parallel, TPA and vWF release were measured in triplicate and expressed as percentage of the release induced by 1 NIH U/mL, as described in "Methods." When [Ca2+]i exceeded 1 µmol/L, data are shown as 1 µmol/L, because Ca2+ measurements are unreliable in that range.30 A and C, Relationship between [Ca2+]i induced by thrombin and TPA release. The increase in [Ca2+]i induced by thrombin is plotted against the percentage TPA release (mean±SD). The absolute TPA release induced by 1 NIH U/mL -thrombin was 0.24, 0.15, and 0.18 ng/2.105 cells in the three experiments shown. In C, the same data as in A are shown, with [Ca2+]i plotted on a log scale. After linear regression analysis, a significant correlation (r=.90) was found between log [Ca2+]i and TPA release. At 50% TPA release, the Ca2+ concentration was calculated to be 261 nmol/L (95% CI, 211 to 338 nmol/L). B and D, Relationship between [Ca2+]i induced by thrombin and vWF release. The increase in [Ca2+]i induced by thrombin is plotted against the percentage vWF release (mean±SD). The absolute vWF release induced by 1 NIH U/mL -thrombin was 0.43, 0.21, and 0.24 U/2.105 cells in the three experiments shown. In D, the same data as in B are shown, with [Ca2+]i plotted on a log scale. After linear regression analysis, a significant correlation (r=.89) was found between log [Ca2+]i and vWF release. At 50% vWF release, the Ca2+ concentration was calculated to be 222 nmol/L (95% CI, 182 to 279 nmol/L).

Effect of Ionomycin on TPA and vWF Release
To study whether an increase in [Ca²⁺]_i was sufficient to induce acute release of TPA and vWF, we used increasing doses (up to 0.9 µmol/L) of the Ca²⁺ ionophore ionomycin to increase [Ca²⁺]_i stepwise. The amount of TPA and vWF acutely released by ionomycin (expressed as percentage of the release induced by 1 NIH U/mL of -thrombin) is plotted against [Ca²⁺]_i in Fig 3A for TPA and 3B for vWF. The results showed that increases in [Ca²⁺]_i induced by ionomycin dose dependently induced acute release of vWF (Fig 3B). The [Ca²⁺]_i needed for 50% vWF release by ionomycin was 311 nmol/L (95% CI, 233 to 424 nmol/L), which is slightly higher than the [Ca²⁺]_i needed for 50% vWF release induced by thrombin (222 nmol/L). In contrast, ionomycin did not induce any TPA release below 500 nmol/L [Ca²⁺]_i (Fig 3A). Together, these data showed that ionomycin did release vWF but was unable to release TPA as long as [Ca²⁺]_i remained below 500 nmol/L. Thus, thrombin may activate, besides Ca²⁺, other regulatory mechanisms required for TPA release but not for vWF release.

View larger version (19K): [in this window] [in a new window]   Figure 3. Relationship between [Ca2+]i induced by ionomycin and TPA and vWF release. Various concentrations of ionomycin (0 to 0.9 µmol/L) or 1 NIH U/mL -thrombin were added to HUVECs for 3 minutes and the maximum [Ca2+i] was measured in duplicate. In parallel, TPA and vWF release were measured in triplicate and expressed as percentage of the release induced by 1 NIH U/mL -thrombin. The peak [Ca2+]i is plotted against release (mean±SD; n=3). When the concentration of [Ca2+]i exceeded 1 µmol/L, data are shown as 1 µmol/L, because Ca2+ measurements are unreliable in that range.30 A and C, Relationship between [Ca2+]i induced by ionomycin and TPA release. The increase in [Ca2+]i induced by ionomycin is plotted against TPA release (mean±SD; n=3). The absolute TPA release induced by 1 NIH U/mL -thrombin was 0.05, 0.58, and 0.07 ng/2.105 cells. In C, the same data as in A are shown, with [Ca2+]i plotted on a log scale. After linear regression analysis, no significant correlation was found between log [Ca2+]i and TPA release. B and D, Relationship between [Ca2+]i induced by ionomycin and vWF release. The increase in [Ca2+]i induced by ionomycin is plotted against vWF release (mean±SD; n=3). The absolute vWF release induced by 1 NIH U/mL -thrombin was 0.37, 0.21, and 0.33 U/2.105 cells. In D, the same data as in B are shown, with [Ca2+]i plotted on a log scale. After linear regression analysis, a significant correlation (r=.91) was found between log [Ca2+]i and vWF release. At 50% vWF release, the Ca2+ concentration was calculated to be 311 nmol/L (95% CI, 233 to 424 nmol/L).

Involvement of PKC in Thrombin-Induced TPA Release
Because thrombin activates PKC in HUVECs,³⁸ we investigated whether PKC was involved in thrombin-stimulated TPA release. To this end, the cells were preincubated for 30 minutes with the PKC inhibitor Ro-31-8220, followed by induction of acute release with 1 NIH U/mL -thrombin. Ro-31-8220 increased thrombin-induced TPA and vWF release slightly, but not significantly (not shown).

It proved not possible to study the role of PKC by downregulating PKC activity by PMA, since no TPA storage pool was left after 24 hours of treatment with 1 µmol/L PMA. This finding is comparable to that of the vWF storage pool, which is also absent after 24 hours of PMA treatment.³⁹ Next, we investigated whether activation of PKC by PMA could improve the poor TPA release induced by ionomycin at below 500 nmol/L [Ca²⁺]_i. HUVECs were stimulated with increasing doses of the Ca²⁺ ionophore ionomycin in the presence or absence of 100 nmol/L PMA (which itself did not induce any TPA release during the 3-minute stimulation period). PMA had no effect on ionomycin-induced TPA release below 500 nmol/L [Ca²⁺]_i, whereas it slightly, but not significantly, enhanced the ionomycin-induced TPA release above 500 nmol/L [Ca²⁺]_i. This slight effect of PMA on ionomycin-induced TPA release is not mediated by an effect of PMA on [Ca²⁺]_i in HUVECs (see Reference 38³⁸ and personal communication from R. Draaijer, July 1996). By Western blot analysis (not shown), we found only the PMA-sensitive PKC isotypes , ß, and in our first-passage endothelial cells, but not the isotypes , , and I (in agreement with published data⁴⁰ ), which makes it unlikely that thrombin would influence release via PMA-insensitive PKC isotypes.

Involvement of Eicosanoids in Thrombin-Induced TPA Release
Thrombin will also activate PLA₂ in HUVECs.^{16 19 23} However, neither the combined lipoxygenase/cyclooxygenase inhibitor ETYA⁴¹ (25 µmol/L), the cyclooxygenase inhibitor indomethacin⁴¹ (25 µmol/L), nor the PLA₂ inhibitor AACOCF3⁴² (10 µmol/L) caused any inhibition of TPA or vWF release (not shown). As the inhibitors fully (ETYA, indomethacin) or significantly (for 70%, AACOC3) inhibited the thrombin-induced production of 6-ketoprostaglandin F₁, these experiments indicated that eicosanoids did not play a role in thrombin-induced release.

Involvement of G Proteins in Thrombin-Induced TPA Release
Since thrombin activates G proteins in HUVECs^{20 21 22 23} and because G proteins play a role in exocytosis,^{17 18 19} we studied the involvement of G proteins in thrombin-stimulated acute release. To be able to use cell-impermeable or poorly cell-permeable compounds such as GDP-ß-S and AlF₄^-, which are necessary to be able to study G protein involvement, we used permeabilized HUVECs. To this end, we chose the system described by Birch et al,⁷ in which HUVECs are minimally permeabilized by saponin, because in this system, exocytosis of at least vWF was shown to be maintained.⁷

Release From Minimally Permeabilized Cells
In HUVECs minimally permeabilized by using saponin,⁷ the amounts of vWF and TPA released into the permeabilization medium during the 10-minute permeabilization period were increased by about 50% compared with cells incubated without saponin (54±25% for vWF and 54±36% for TPA, respectively; n=6). Thrombin dose dependently released vWF (Fig 4A) and TPA (Fig 4B) from saponin-permeabilized cells. The release induced by 1 NIH U/mL human -thrombin was, in permeabilized cells, very similar to the release induced by thrombin in nonpermeabilized cells (114±12% for vWF and 89±16% for TPA, respectively; n=6). The release was not due to cell lysis, as the amount of LDH in the medium was only 5±3% of that present in the cell extracts. To further check the integrity of our cells, permeabilized cells were preincubated with the calmodulin inhibitor TFP to verify whether the acute release of vWF and TPA was still mediated by calmodulin. Acute thrombin-induced release from permeabilized cells was inhibited by TFP by 83±9% for vWF and 100±0% for TPA (both n=3). Thus, thrombin-induced release from minimally saponin-permeabilized HUVECs was very similar to thrombin-induced release from intact cells.

View larger version (16K): [in this window] [in a new window]   Figure 4. G proteins in thrombin-induced vWF and TPA release: effect of GDP-ß-S. HUVECs were treated for 10 minutes with permeabilization buffer containing 15 µg/mL saponin and 100 nmol/L Ca2+e in the absence () or presence () of 2 mmol/L GDP-ß-S. The cells were then stimulated for 3 minutes with various concentrations of human -thrombin (0 to 1 NIH U/mL) in the same buffers. A, Effect of GDP-ß-S on vWF release. vWF release is expressed as a percentage (mean±SEM; n=3) of the release induced by 1 NIH U/mL -thrombin in the presence of 100 nmol/L Ca2+e. The absolute values of vWF release were 0.20, 0.31, and 0.36 U/2.105 cells. The effect of GDP-ß-S was significant by two-way ANOVA (F1,30=17.20, P<.005). B, Effect of GDP-ß-S on TPA release. TPA release is expressed as a percentage (mean±SEM; n=3) of the release induced by 1 NIH U/mL -thrombin in the presence of 100 nmol/L Ca2+e. The absolute values of TPA release were 0.40, 0.31, and 0.34 ng/2.105 cells. The effect of GDP-ß-S was significant by two-way ANOVA (F1,30=15.05, P<.005).

G Proteins in Thrombin-Induced vWF and TPA Release
Since both heterotrimeric and small G proteins might be involved in thrombin-induced vWF and TPA release, the effect of GDP-ß-S (a stable GDP analogue which inactivates both types of G proteins) on thrombin-induced vWF and TPA release was studied first. When HUVECs had been preincubated for 10 minutes with 2 mmol/L GDP-ß-S, the thrombin-induced vWF and TPA release was inhibited completely up to 0.03 NIH U/mL -thrombin and significantly by about 30% above this concentration (Fig 4A and 4B).

To discriminate between the role of heterotrimeric G proteins and small G proteins, HUVECs were stimulated with AlF₄^-, which activates heterotrimeric but not small G proteins.³⁴ AlF₄^- induced vWF and TPA release dose dependently (not shown). At 30 mmol/L fluoride, AlF₄^- induced (compared with thrombin treatment) 21±19% vWF release in intact cells and 79±43% in permeabilized cells. Release of TPA induced by AlF₄^- was 81±31% in intact cells and 121±22% in permeabilized cells, respectively, compared with thrombin treatment (n=3). Together, these data suggested that at least heterotrimeric G proteins were involved in vWF and TPA release.

Effect of PTX on vWF Release
To further define the classes of heterotrimeric G proteins involved in thrombin-induced release, HUVECs were incubated for 2.5 hours with 1 µg/mL PTX, which inactivates heterotrimeric G proteins of the classes G_i/o.³³ Pretreatment with PTX did not change the constitutive secretion of vWF, but thrombin-induced vWF release was significantly inhibited by PTX at -thrombin concentrations 0.03 NIH U/mL (Fig 5A) and maximally by 29% at 0.3 NIH U/mL of -thrombin. Pretreatment with PTX reduced the fluorescence ratio of the peak Ca²⁺ phase and (significantly) the fluorescence ratio of the plateau Ca²⁺ phase (Table 3), supporting the hypothesis that PTX inhibited thrombin-induced Ca²⁺ influx into HUVECs.

View larger version (12K): [in this window] [in a new window]   Figure 5. Effects of PTX on vWF release. HUVECs () were pretreated for 2 hours with 1 µg/mL PTX, and control cells () were not. The data are expressed as percentage (mean±SEM) of release, relative to the release induced by 1 NIH U/mL -thrombin in control cells. A, PTX and thrombin. HUVECs were stimulated with various concentrations of -thrombin (0 to 1 NIH U/mL) for 3 minutes in M199/HSA. vWF release induced by 1 NIH U/mL -thrombin was 0.25 U/2.105 cells (median; 95% CI, 0.11 to 0.38; n=5). The effect of PTX on vWF release was significant by two-way ANOVA (F1,40=14.73, P<.005). B, PTX and thrombin in Ca2+-free Tyrode's solution. HUVECs were stimulated with various concentrations of -thrombin (0 to 1 NIH U/mL) for 3 minutes in Ca2+-free Tyrode's solution (n=3). The absolute vWF release values induced by 1 NIH U/mL -thrombin were 0.41, 0.14, and 0.31 U/2.105 cells. PTX did not significantly affect vWF release in Ca2+-free Tyrode's solution. C, PTX and ionomycin. HUVECs were stimulated with various concentrations of ionomycin (0 to 1 µmol/L) or 1 NIH U/mL -thrombin. vWF release induced by 1 NIH U/mL -thrombin was 0.25 U/2.105 cells (median; 95% CI, 0.11 to 0.44; n=5). The effect of PTX on ionomycin-induced vWF release was not significant.

View this table: [in this window] [in a new window]   Table 3. Effect of PTX on Thrombin-Induced Increase in [Ca2+]i

To test the hypothesis that PTX exerted its action on vWF release via Ca²⁺ influx, we induced acute release of vWF with various concentrations of -thrombin in Ca²⁺-free Tyrode's solution. No effect of PTX on release was seen under these conditions (Fig 5B). Pretreatment with PTX did not affect vWF release induced by ionomycin either (Fig 5C). The effect of PTX on thrombin-induced vWF thus was not seen when [Ca²⁺]_i was increased receptor independently or when there was no Ca²⁺ influx.

Effects of PTX on TPA Release
PTX did not affect constitutive TPA secretion but inhibited thrombin-induced TPA release (maximally 20% at 0.1 NIH U/mL; Fig 6A). The effect of PTX on thrombin-induced TPA release was, however, not significant (by two-way ANOVA), possibly because the inhibition by PTX was less at lower and higher concentrations of -thrombin, and no inhibition was found at 1 NIH U/mL -thrombin. These data showed that the effects of PTX on TPA release were more complex than on vWF release. In contrast to vWF release (Fig 5B), PTX significantly enhanced TPA release in Ca²⁺-free Tyrode's solution (Fig 6B). Pretreatment with PTX also slightly, but not significantly, enhanced TPA release by ionomycin (Fig 6C), maximally to 132±12% (n=3) at 1 µmol/L ionomycin.

View larger version (13K): [in this window] [in a new window]   Figure 6. Effects of PTX on TPA release. HUVECs () were pretreated for 2 hours with 1 µg/mL PTX, and control cells () were not. Data are expressed as percentage (mean±SEM) of release, relative to the release induced by 1 NIH U/mL -thrombin in control cells. A, PTX and thrombin. HUVECs were stimulated with various concentrations of -thrombin (0 to 1 NIH U/mL) for 3 minutes in M199/HSA. TPA release induced by 1 NIH U/mL -thrombin was 0.34 ng/2.105 cells (mean; 95% CI, 0.18 to 0.61; n=5). By two-way ANOVA, the effect of PTX was not significant (F1,40=1.32). B, PTX and thrombin in Ca2+-free Tyrode's solution. HUVECs were stimulated with various concentrations of -thrombin (0 to 1 NIH U/mL) for 3 minutes in Ca2+-free Tyrode's solution (n=3). The absolute values of TPA release induced by 1 NIH U/mL -thrombin were 0.49, 0.74, and 0.64 ng/2.105 cells. PTX significantly stimulated TPA release under these conditions (F1,30=18.22, P<.005). C, PTX and ionomycin. HUVECs were stimulated with various concentrations of ionomycin (0 to 1 µmol/L) or 1 NIH U/mL -thrombin (n=3). The absolute values of TPA release induced by 1 NIH U/mL -thrombin were 0.29, 0.21, and 0.49 ng/2.105 cells in three experiments. PTX did not significantly affect ionomycin-induced TPA release.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In vivo, TPA and vWF are often secreted simultaneously from the endothelium on the addition of stimuli.^{12 35} In a previous report, we described the development of a system in which acute release (regulated secretion) of TPA and vWF from human endothelial cells can be studied in vitro.¹² In this system, we used thrombin as the stimulus to induce acute release. Thrombin presumably acted via its (G protein–coupled) seven-transmembrane-domain receptor, since release of TPA and vWF could also be induced by the thrombin receptor–activating peptide SFLLRN (our unpublished data, 1996).

In the present paper, we studied the role of Ca²⁺ and G proteins in thrombin-stimulated TPA and vWF release. An increase in [Ca²⁺]_i was essential for TPA and vWF release, as complete chelation of [Ca²⁺]_i with BAPTA-AM in Ca²⁺-free Tyrode's solution almost completely inhibited both TPA and vWF release. Since preincubation with three different calmodulin inhibitors (TFP, W-7, and calmidazolium) significantly inhibited both TPA and vWF release, Ca²⁺ most probably transduced to the Ca²⁺-binding protein calmodulin. These data are in agreement with data from Birch et al,⁹ who showed that vWF release induced by thrombin was almost nil after incubation with EGTA and MAPTA-AM (a Ca²⁺-chelating agent similar to BAPTA-AM) and that introduction into permeabilized HUVECs of a calmodulin-inhibitory peptide inhibited vWF release.⁹ Only the observation that despite the absence of an increase in [Ca²⁺]_i on thrombin stimulation, TPA and vWF release still occurred in cells treated with cyclopiazonic acid in the absence of Ca²⁺_e was unexpected. However, under the latter conditions, [Ca²⁺]_i was not chelated as when BAPTA-AM was used. Possibly, the early transient increase in [Ca²⁺]_i induced by cyclopiazonic acid might have contributed later to TPA and vWF release. Another possibility is that local increases in [Ca²⁺]_i (not detectable in our system) might still have occurred and induced TPA and vWF release.

Furthermore, our data showed that inhibition of Ca²⁺ influx or depletion of the Ca²⁺_i pools inhibited TPA and vWF release only slightly (Table 1). Under these conditions, a reduced [Ca²⁺]_i peak was seen on the addition of thrombin (Fig 1). Because the peak [Ca²⁺]_i strongly correlated with the extent of TPA and vWF release (Fig 2), it is likely that depletion of the Ca²⁺_i pool or inhibition of Ca²⁺ influx affected TPA and vWF release via an effect on peak [Ca²⁺]_i. Apparently, the source from which the increase in [Ca²⁺]_i was derived was irrelevant (Table 1). These data are in contrast to experiments in a rat perfused hindleg system, in which depletion of Ca²⁺_e with EDTA after a few minutes fully inhibited TPA and vWF release.^{25 43} This difference might be due to the fact that the Ca²⁺_i pool is, in vivo, smaller than in vitro⁴⁴ and more rapidly depleted. We therefore suggest that Ca²⁺ influx plays a more prominent role in increasing [Ca²⁺]_i and hence also in TPA and vWF release in vivo than in vitro. The same may be true for other endothelial cell systems in which activation depends on an increase in [Ca²⁺]_i.

Discrepancies exist in the literature regarding the role of Ca²⁺ influx in vWF release from HUVECs. In some studies,^{4 5 7} chelation of Ca²⁺_e with an excess of EDTA or EGTA for at least 10 minutes inhibited vWF release. However, Birch et al,⁹ who used Ca²⁺-free buffers (not containing large amounts of EDTA or EGTA), did not find Ca²⁺ influx to be essential for thrombin-stimulated vWF release. In our experiments, Ca²⁺-free Tyrode's solution with a low concentration (0.1 mmol/L) of EGTA was used. We suggest that incubation of HUVECs with large amounts of EDTA and/or EGTA for a prolonged period of time not only removes Ca²⁺_e but also decreases the amount of Ca²⁺ in the intracellular storage pools sufficiently to reduce the peak [Ca²⁺]_i after stimulation and thus to reduce release.

The amount of [Ca²⁺]_i needed for 50% vWF release induced by ionomycin was 311 nmol/L, comparable to a [Ca²⁺]_i of 222 nmol/L needed for 50% vWF release induced by thrombin and also comparable to the [Ca²⁺]_i of 350 nmol/L needed for 50% vWF release induced by thrombin, as found by Birch et al.⁹ Because thrombin and ionomycin induced 50% vWF release at comparable levels of [Ca²⁺]_i, we suggest that Ca²⁺ alone is sufficient to induce acute vWF release, a conclusion also reached by Frearson et al.¹⁶ Carew et al⁸ noted that ATP increased [Ca²⁺]_i in HUVECs but did not induce vWF secretion. This result might have occurred because these authors measured vWF release only after 60 minutes, while, due to ATP breakdown by extracellular ATPases, the increase in [Ca²⁺]_i may not have been sustained long enough to have a measurable effect on the final vWF level over this long period. Another explanation might be that in contrast to most other Ca²⁺-increasing agents, ATP also increases cAMP in HUVECs.⁴⁵

Our ionomycin experiments showed different results for TPA release than for vWF release. While thrombin-induced TPA release occurred at all [Ca²⁺]_i >100 nmol/L, ionomycin-induced release of TPA was only seen if Ca²⁺ levels increased to >500 nmol/L. In contrast, vWF release occurred in all instances with [Ca²⁺]_i >100 nmol/L. We have no explanation for the sudden occurrence of ionomycin-induced TPA release above 500 nmol/L [Ca²⁺]_i.

Calmidazolium significantly increased vWF secretion during the preincubation period. Calmidazolium has been reported⁴⁶ to increase [Ca²⁺]_i in bovine aortic endothelial cells, an observation confirmed by us for HUVECs (our unpublished data, 1996). This increase in [Ca²⁺]_i likely induced acute vWF release during the preincubation period, which would explain the reduced subsequent thrombin-induced release of vWF (Table 2). Interestingly, calmidazolium did not increase TPA secretion during the preincubation period and in this respect resembled ionomycin. This finding suggests again that at low Ca²⁺ levels, an increase in [Ca²⁺]_i is not sufficient to induce TPA secretion.

Since activation of the thrombin receptor also activates (in HUVECs) PLA₂ and PKC,^{16 19 23 38} we studied their role in TPA and vWF release. The PKC inhibitor Ro-31-8220 did not significantly affect thrombin-induced TPA and vWF release, while PMA did not induce acute TPA or vWF release (over 3 minutes). Our data concerning vWF release agree with results from other groups, who found that thrombin-induced vWF release is independent of PKC activation.^{7 8} Carew et al⁸ also found a small increase in thrombin-induced vWF release after preincubation with 0.1 µmol/L Ro-31-8220. Activation of PKC by PMA modulated ionomycin-induced TPA release but slightly, and only at high levels of [Ca²⁺]_i. We therefore conclude that PKC is not essential for acute (eg, within 3 minutes) thrombin-induced TPA and vWF release. However, PMA is known to induce delayed, low-level, and long-lasting vWF release, which is maximal in HUVECs after 30 minutes.⁴⁷ This "slow" vWF release has also been found in the rat perfused hindleg model.²⁵ This type of slow release might explain the effect of PMA on Ca²⁺ ionophore–induced vWF release found by Carew et al.⁸

Indomethacin, ETYA, and AACOCF₃ significantly inhibited prostacyclin synthesis without affecting TPA or vWF release. We thus consider it highly unlikely that metabolites of the eicosanoid pathway are involved in thrombin-mediated TPA or vWF release.

An important role has been ascribed to G proteins in exocytotic processes as such¹⁸ and in transducing (thrombin) receptor-mediated signals in the endothelial cell.^{18 19 20 21 22 23 48} An involvement of one or more heterotrimeric G proteins in acute release from HUVECs was suggested by the experiments with AlF₄^-, which released TPA and vWF from intact cells, and to an even greater extent from permeabilized cells. It is likely that AlF₄^- stimulated the heterotrimeric G proteins involved in release more easily in permeabilized cells than in intact cells because the latter are more accessible. Such findings have, for instance, been reported by Inoue et al,⁴⁹ who showed that AlF₄^- activated G_i but not G_s in intact cells, but that AlF₄^- activated both G proteins in cell membrane preparations.⁴⁹ The 30% inhibition of thrombin-induced release by GDP-ß-S (Fig 4) also suggested that G proteins were involved in TPA and vWF secretion.

Pretreatment of HUVECs with PTX inhibited vWF release by about 30% at all concentrations of -thrombin tested, most likely due to inhibition of Ca²⁺ influx (Fig 5 and Table 3). The mechanism by which a PTX-sensitive G protein mediates Ca²⁺ influx in HUVECs is still unknown. Possibly, a PTX-sensitive G protein that is coupled to the thrombin receptor activates, either directly or indirectly, a Ca²⁺ influx channel. Such a mechanism has been described by Graier et al,⁵⁰ who showed that an increase in 5,6-epoxyeicosatrienoic acid induced Ca²⁺ influx into HUVECs. 5,6-Epoxyeicosatrienoic acid is formed out of arachidonic acid, liberated from membrane lipids by activated PLA₂, by monooxygenation. However, in our system, neither inhibition of PLA₂ by AACOCF₃ nor inhibition of monooxygenation by ETYA inhibited thrombin-induced Ca²⁺ influx, making this explanation unlikely. The initial rise in [Ca²⁺]_i due to activation of phospholipase C and the formation of IP₃ was slightly, though not significantly, inhibited by PTX (Table 3). This observation is in agreement with results from Garcia et al,³³ who also showed that the thrombin-induced initial increase in [Ca²⁺]_i is PTX insensitive. Other effects of thrombin, eg, the activation of c-fos,²³ may, however, be PTX sensitive. Also, other receptors may be coupled in HUVECs to phospholipase C via PTX-sensitive G proteins, as shown for ATP⁵¹ and histamine.²¹

Our data suggest that a second PTX-sensitive G protein may additionally be involved in acute release of TPA specifically. The effect of this G protein, which reduces TPA release, is usually masked by the effect of the G protein modulating Ca²⁺ influx. Its effect could be observed when HUVECs were stimulated with thrombin under Ca²⁺-free conditions (Fig 6B) or when [Ca²⁺]_i was increased receptor independently by using ionomycin (Fig 6C). Although the effects on thrombin- and ionomycin-induced secretion were not significant, but only suggestive, these data (Fig 6A through 6C) may suggest that the inhibitory G protein acted distally to the increase in [Ca²⁺]_i.⁵² This observation is comparable to those in chromaffin cells, in which the regulated secretion of catecholamines induced by various concentrations of Ca²⁺ is also enhanced by pretreatment with PTX.⁵³ The inhibitory G protein in chromaffin cells is characterized as a G_o protein and is located on the chromaffin granule membrane.⁵⁴ The activation of this G_o is mediated via GAP43, a cytoplasmic pseudoreceptor, which is sensitive to PKC and calmodulin.⁵⁵ It has been proposed that this G_o is continuously activated by basal levels of Ca²⁺ and thus prevents regulated secretion under nonstimulated conditions. It is possible that the inhibitory G protein in our cells is also continuously activated by basal levels of Ca²⁺ and thus prevents the acute release of TPA under nonstimulated conditions.

In summary, our results showed that acute release of both TPA and vWF requires an increase in [Ca²⁺]_i above 100 nmol/L. Such an increase is sufficient to cause the acute release of vWF but (below 500 nmol/L) not sufficient to cause release of TPA. A PTX-sensitive G protein was involved in thrombin-induced Ca²⁺ influx. A second PTX-sensitive G protein is likely involved in increasing the acute release of TPA but not vWF.

	Selected Abbreviations and Acronyms

 

AACOCF3 = trifluoromethyl ketone arachidonic acid CI = confidence interval ELISA = enzyme-linked immunosorbent assay ETYA = 5,8,11,14-eicosatetranoic acid HSA = human serum albumin HUVEC = human umbilical vein endothelial cell PKC = protein kinase C PLA2 = phospholipase A2 PMA = phorbol 12-myristate 13-acetate PTX = pertussis toxin TFP = trifluoperazine TPA = tissue-type plasminogen activator vWF = von Willebrand factor

	Acknowledgments

 
This study was supported by grants 90.075 and 93.126 from the Netherlands Heart Foundation. AACOCF₃ was a gift from Dr G. Nalbone (Laboratoire d'Hématologie, Marseille, France). Ro-31-8220 was a gift from Dr G. Lawton (Hoffmann La Roche, Welwyn Garden City, UK).

Received June 30, 1996; accepted December 19, 1996.

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