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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1315.)
© 2004 American Heart Association, Inc.

Thrombosis

Small GTP-Binding Protein Ral Is Involved in cAMP-Mediated Release of von Willebrand Factor From Endothelial Cells

Mariska G. Rondaij; Erica Sellink; Karina A. Gijzen; Jean Paul ten Klooster; Peter L. Hordijk; Jan A. van Mourik; Jan Voorberg

From the Departments of Plasma Proteins (M.G.R., E.S., K.A.G., J.A.v.M., J.V.) and Experimental Immunohematology (J.P.t.K., P.L.H.), Sanquin Research at CLB, Amsterdam, the Netherlands; and the Department of Vascular Medicine (J.A.v.M.), Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.

Correspondence to Dr J. Voorberg, Department of Plasma Proteins, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands. E-mail J.Voorberg{at}sanquin.nl

	Abstract

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Objective— von Willebrand factor (vWF) is synthesized by endothelial cells and stored in specialized vesicles called Weibel-Palade bodies (WPBs). Recently, we have shown that the small GTP-binding protein Ral is involved in thrombin-induced exocytosis of WPBs. In addition to Ca²⁺-elevating secretagogues such as histamine and thrombin, release of WPB is also observed after administration of cAMP-raising substances such as epinephrine and vasopressin. In the present study, we investigated whether Ral is also involved in cAMP-mediated vWF release.

Methods and Results— Activation of Ral was observed 15 to 20 minutes after stimulation of endothelial cells with epinephrine, forskolin, or dibutyryl-cAMP. A cell-permeable peptide comprising the carboxy-terminal part of the Ral protein reduced both thrombin-induced and epinephrine-induced vWF secretion supporting a crucial role for Ral in this process. Furthermore, inhibition of protein kinase A by H-89 resulted in a marked reduction of vWF release and greatly diminished levels of GTP-Ral on stimulation with epinephrine. Activation of Ral was independent of the activation of Epac, a cAMP-regulated exchange factor for the small GTPases Rap1 and Rap2.

Conclusions— These results suggest that protein kinase A-dependent activation of Ral regulates cAMP-mediated exocytosis of WPB in endothelial cells.

Epinephrine, a cAMP-raising agonist of WPB exocytosis, activates the small GTPase Ral in a PKA-dependent manner. Furthermore, a cell-permeable, Ral-derived peptide inhibited epinephrine-induced and thrombin-induced vWF secretion. These results suggest that Ral is a crucial component of cAMP-dependent and Ca²⁺-dependent signaling pathways that mediate WPB exocytosis.

Key Words: Weibel-Palade bodies • von Willebrand factor • Ral • cAMP • endothelial cells

	Introduction

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Endothelial cells of virtually all blood vessels contain specialized storage organelles known as Weibel-Palade bodies (WPBs).¹ Twenty years after their initial description, it was shown by several groups that this subcellular compartment is the residence of von Willebrand factor (vWF).^2–4 Since then, a variety of bioactive substances have been localized to WPBs (reviewed in^5,6). Several lines of evidence support the notion that vWF is the driving force for the biogenesis of WPBs. Ectopic expression of vWF cDNA in nonendothelial cells results in the formation of rod-shaped organelles that closely resemble WPBs.^7–9 Importantly, sorting of P-selectin, CD63, and IL-8 to WPBs is dependent on the presence of vWF.^10–12 In the absence of vWF, these inflammatory mediators are not subject to regulated exocytosis. Elegant in vivo studies have shown that P-selectin-mediated leukocyte recruitment to endothelial cells is severely compromised in vWF-deficient mice.¹³ Moreover, atherosclerosis was also significantly reduced in these mice, suggesting an important contribution of WPB exocytosis to the pathogenesis of this vascular abnormality.¹⁴

The exocytosis of WPBs can be stimulated by a large number of different agonists that can be separated into 2 distinct groups, those that act by elevating intracellular Ca²⁺ levels and those that act by raising cAMP levels in the cell.^15–25 Thrombin, one of the best studied agonists of vWF secretion, acts on proteinase activated receptors (PARs) to induce an increase in intracellular Ca²⁺ levels.^26,27 The cellular responses to these elevated Ca²⁺ levels are most likely mediated by calmodulin (CaM).^26,27

Secretion of vWF, however, is also induced by cAMP-raising substances like epinephrine²⁴ and vasopressin.²⁵ In a subset of patients with von Willebrand disease, vWF levels in plasma are increased by the administration of 8-arginine vasopressin (AVP) or the vasopressin analogue desmopressin (DDAVP).²⁸ Both epinephrine and vasopressin act on G-protein-coupled receptors of the G_s subtype, the ß₂-adrenergic receptor and the vasopressin V2 receptor, respectively.^24,25 Stimulation of these G-protein-coupled receptors results in activation of adenylate cyclase, which catalyzes the formation of the second messenger cAMP. Incubation with the cAMP analogue Rp-8CPT-cAMPS blocked forskolin-induced and DDAVP-induced but not thrombin-induced vWF secretion.^22,25 These findings show that thrombin and epinephrine induce vWF secretion through different signaling pathways.

Recently, the small GTPase Ral has been implicated in thrombin-induced vWF secretion. In endothelial cells, Ral was found in subcellular fractions containing WPBs.²⁹ Moreover, activation of Ral coincided with thrombin-induced vWF secretion.³⁰ In addition, expression of an active, GTP-bound Ral variant in human umbilical vein endothelial cells (HUVECs) resulted in the disappearance of WPBs.³⁰ Independent of our observations, it has been shown that Ral interacts with Sec5³¹ and Exo84,³² both members of the exocyst, a multiprotein complex that mediates targeting of secretory vesicles to the plasma membrane. Ral is also involved in cytoskeletal rearrangements via its effectors filamin³³ and RalBP1,³⁴ a GTPase-activating protein (GAP) for Cdc42. In this study, we investigated whether Ral is also activated in response to cAMP-raising compounds like epinephrine and forskolin. Our results show that epinephrine as well as forskolin and the cAMP-analogue dibutyryl-cAMP are able to induce Ral activation. Further substantiation that Ral also mediates cAMP-induced vWF secretion comes from the finding that a Ral-derived, cell-permeable peptide inhibits both thrombin-induced and epinephrine-induced vWF secretion. In addition, we found that epinephrine-induced Ral activation is dependent on protein kinase A (PKA) and proceeds independently of the cAMP-regulated exchange protein Epac. These results suggest that Ral is a crucial component of cAMP-dependent and Ca²⁺-dependent signaling pathways that mediate exocytosis of WPBs.

	Methods

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Please see online Methods (available at http://atvb.ahajournals.org) for details of the methods used in this study.

	Results

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Activation of Ral by cAMP-Dependent Agonists
To investigate whether secretion of vWF induced by cAMP-raising substances is also mediated by Ral, HUVECs were stimulated with epinephrine for various time periods in the presence of the phosphodiesterase inhibitor IBMX. In accordance with previous observations, vWF secretion in response to epinephrine was more gradual compared with the rapid thrombin-induced vWF release²⁴ (Figure 1A). In contrast to thrombin-induced vWF secretion, which reaches a maximum after 10 minutes of stimulation, epinephrine-induced vWF release was previously found to reach a plateau between 45 and 60 minutes of stimulation, with a maximal response of twice the maximal level of thrombin-induced vWF secretion.²⁴ Activation of Ral was measured using a Ral-GTP specific pull-down assay.³⁵ Stimulation of HUVECs with epinephrine resulted in a gradual increase in the amount of activated Ral, reaching a maximum after 20 minutes of stimulation. Ral activation induced by epinephrine was clearly delayed compared with thrombin-induced Ral activation, which is maximal after 2 minutes of stimulation (Figure 1A) and returns to baseline levels after 10 minutes (not shown). The gradual increase in activated Ral in response to epinephrine is compatible with the slow onset of vWF secretion observed on stimulation with this agonist (Figure 1A). To verify whether the Ral activation we observed on epinephrine stimulation was dependent on the formation of the second messenger cAMP, we incubated cells with the adenylate cyclase activator forskolin and the cAMP analogue dibutyryl cAMP (db-cAMP). Stimulation with forskolin (Figure 1B) and db-cAMP (Figure I, available online at http://atvb.ahajournals.org) resulted in Ral activation with similar kinetics as epinephrine-induced activation of Ral. These results indicate that increases in intracellular cAMP result in the activation of the small GTPase Ral.

View larger version (25K): [in this window] [in a new window]   Figure 1. cAMP-raising agonists of vWF secretion induce activation of Ral. HUVECs were grown to confluency in 6-wells plates. Cells were washed twice with SF medium, incubated with SF medium for 1 hour, and then incubated with thrombin (1 U/mL), epinephrine (10 µmol/L +100 µmol/L IBMX), forskolin (10 µmol/L +100 µmol/L IBMX), or SF medium alone (control). Results are given as the average of 4 independent experiments. Error bars represent SEM. A, vWF secretion on stimulation was determined measuring the amount of vWF released in the medium by enzyme-linked immunosorbent assay (ELISA). vWF released by control cells after 30 minutes incubation with SF medium is taken as 100%. Ral activation in response to stimulation with epinephrine, thrombin, or SF medium alone was measured using a Ral-GTP-specific pull-down assay as described in the Methods. Lower panels show total Ral levels as loading control. B, vWF release and Ral activation induced by the adenylate cyclase activator forskolin.

Both Thrombin-Mediated and cAMP-Mediated Release of vWF Is Inhibited by a Cell-Permeable Peptide Corresponding to the Carboxy-Terminus of Ral
To further establish the involvement of Ral in cAMP-dependent release of vWF, we designed a cell permeable, Ral-derived peptide corresponding to the carboxy-terminal part of this small GTPase. The C-terminal polybasic region of small GTPases is hypervariable and is thought to play a role in the targeting and signaling specificity.³⁶ This peptide contains the protein transduction domain (TAT) of human immunodeficiency virus, which enables the peptide to be transduced directly into the cell,³⁷ and the last 26 amino acids of Ral, excluding the CAAX box. In the presence of 200 µg/mL of TATRal-c, vWF secretion of both thrombin-stimulated and epinephrine-stimulated cells was significantly reduced (Figure 2), supporting the involvement of Ral in vWF secretion from endothelial cells. In contrast, 2 control peptides derived from the carboxy-terminus of H-Ras and K-Ras did not inhibit vWF release, suggesting that the reduction in vWF secretion on stimulation in the presence of TATRal-c is a Ral-specific effect (Figure II, available online at http://atvb.ahajournals.org). To assess whether this inhibitory effect on vWF secretion is caused by an effect on the exocytosis of WPBs, corresponding to regulated vWF secretion, HUVECs were incubated with serum-free medium, thrombin, epinephrine, or forskolin in the absence or presence of TATRal-c. Stimulation of endothelial cells in the presence of the TATRal-c peptide resulted in higher residual numbers of WPBs in the cell compared with cells that were stimulated in the absence of the TATRal-c peptide (Figure III, available online at http://atvb.ahajournals.org). The TATRal-c peptide did not affect perinuclear clustering of WPBs on epinephrine and forskolin stimulation³⁸ (compare Figure IIIB c and d with g and h), suggesting that TATRal-c affects exocytosis rather than vesicle transport in general. These findings provide a direct link between Ral and the secretion of vWF from endothelial cells.

View larger version (21K): [in this window] [in a new window]   Figure 2. A, Ral-derived peptide inhibits thrombin-induced and epinephrine-induced vWF secretion. HUVECs were grown in 6-wells plates to confluency. Cells were incubated with SF medium for 1 hour and then stimulated with thrombin (1 U/mL), epinephrine (10 µmol/L +100 µmol/L IBMX), or SF medium alone (control) in the absence or presence of 200 µg/mL of the TATRal-c peptide. vWF secretion on stimulation in the absence or presence of 200 µg/mL of TATRal-c was determined and given as the average of 4 independent experiments. vWF released by control cells after 30 minutes incubation with SF medium is taken as 100%. Error bars represent SEM. *P<0.05; **P<0.005 by Student t test.

cAMP-Mediated Activation of RalA Involves Protein Kinase A
An increase in cAMP levels commonly leads to the activation of the cAMP-dependent protein kinase A (PKA), which subsequently results in the phosphorylation of target proteins. Previous studies have shown that the PKA-inhibitory cAMP analogue Rp-8-CPT-cAMPS interferes with cAMP-mediated vWF secretion.^22,25 Here, we investigated whether epinephrine-induced Ral activation was PKA-dependent. We incubated HUVECs with epinephrine in the absence or presence of 100 µmol/L H-89. In the presence of H-89, epinephrine-induced vWF secretion was significantly reduced, whereas thrombin-induced vWF secretion was not affected by H-89 (Figure 3A). These results show that PKA activity is crucial for release of vWF in epinephrine-stimulated, but not in thrombin-stimulated, endothelial cells. Moreover, Ral activation in response to epinephrine was severely reduced in the presence of H-89. Ral-GTP levels in cells that were stimulated in the presence of H-89 were similar to those in unstimulated cells (Figure 3A). To verify whether cAMP-induced vWF release and Ral activation is, in addition to PKA, also partly mediated by the activation of Epac, a cAMP-regulated guanine exchange factor for Rap1 and Rap2, we incubated cells with 100 µmol/L 8-pCPT-2'-O-Me-cAMP (Me-cAMP), a newly characterized cAMP analogue that specifically activates Epac.³⁹ Stimulation with Me-cAMP induced a significant increase in vWF secretion and WPB exocytosis; however, in contrast to epinephrine, it did not result in Ral activation at the indicated time points (Figure 3B and Figure IV, available online at http://atvb.ahajournals.org). Together, these results suggest that vWF secretion induced by epinephrine is mediated by cAMP/PKA-dependent activation of Ral.

View larger version (21K): [in this window] [in a new window]   Figure 3. Epinephrine-induced Ral activation is PKA-dependent. HUVECs were cultured in 6-wells plates to confluency. Cells were incubated for 1 hour with SF medium before stimulation with thrombin (1 U/mL), epinephrine (10 µmol/L +100 µmol/L IBMX), SF medium alone (control) in the absence or presence of 100 µmol/L of the PKA inhibitor H-89, or the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP (Me-cAMP; 100 µmol/L). **P<0.005; *P<0.05 by Student t test. A, vWF secretion on 30 minutes of stimulation in the absence or presence of 100 µmol/L of H-89 was determined and given as the average of 3 independent experiments. vWF released by control cells after 30 minutes incubation with SF medium is taken as 100%. Activation of Ral on stimulation with epinephrine in absence and presence of H-89 was measured; lower panels show total Ral loading control. B, vWF secretion and Ral activation in response to Me-cAMP given as the average of 7 independent experiments. vWF released by control cells after 20-minute incubation with SF medium is taken as 100%.

	Discussion

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Administration of vasopressin or its analogue DDAVP is commonly used to increase plasma levels of vWF in a subset of patients with von Willebrand disease.²⁸ Binding of AVP or DDAVP to the vasopressin V2 receptor expressed in HUVECs results in an increase in intracellular cAMP levels, which promotes exocytosis of WPBs.²⁵ Similarly, epinephrine has been shown to induce secretion of vWF via the ß2-adrenergic receptor.²⁴ In this article, we show that cAMP-mediated secretion of vWF coincides with the activation of the small GTPase Ral. Ral activation in response to epinephrine was more gradual than in response to thrombin. Interestingly, this is in agreement with the slow release of vWF observed on stimulation with epinephrine compared with the rapid secretion of vWF induced by thrombin. The temporal difference between epinephrine-induced and thrombin-induced Ral activation and vWF secretion could be indicative for the difference in physiological function between these secretagogues. Thrombin functions as a local emergency signal for vascular injury, whereas epinephrine and AVP act more systemically, regulating vWF plasma levels.

To further explore the role of Ral in vWF secretion, we made use of a cell-permeable peptide, TATRal-c, comprising the carboxy terminus of Ral, including the putative CaM-binding domain of this protein.⁴⁰ It has been described that binding of CaM to Ral in the presence of Ca²⁺ stimulates GTP binding to Ral.⁴¹ Competition for CaM binding between TATRal-c and endogenous Ral would result in decreased vWF release on thrombin stimulation. In contrast, this peptide severely reduced epinephrine-induced as well as thrombin-induced vWF release, suggesting an alternative mechanism of action. Although Ca²⁺/CaM binding is found to increase the GTP binding to Ral, Ral activation is more likely the result of RalGEFs that may act in conjunction with other factors. Interestingly, a new RalGEF was recently identified that in contrast to the other known RalGEFs, activates Ral in a Ras-independent manner.⁴² This RalGPS (RalGEFs with PH domain and SH3 binding motif) or RalGEF2 was unable to bind to and induce nucleotide exchange of a C-terminally truncated Ral mutant, suggesting that the C-terminus of Ral is required for RalGEF2 binding.⁴³ However, we have not been able to show an inhibitory effect of TATRal-c on the activation of Ral on stimulation with either thrombin or epinephrine (data not shown). Alternatively, TATRal-c can interfere with binding of Ral to effector molecules like RalBP1,³⁴ filamin,³³ Sec5,³¹ or Exo84,³² which would be in agreement with the finding that a TATRac-c peptide interfered with localization and effector-binding rather than activation of Rac.³⁶ Sec5 and Exo84 are particularly interesting candidates because these Ral effectors are both members of the mammalian exocyst complex, which has been implicated in targeting secretory vesicles to specific membrane-fusion sites.⁴⁴ The binding of Ral to these effectors, however, is thought to involve the effector loop of Ral rather than the carboxy-terminus. This raises the possibility that the TATRal-c peptide interacts with an as yet unidentified effector molecule of Ral. The mechanisms by which epinephrine and thrombin activate Ral appear to be distinct (Figure 4). For thrombin, it has been found that stimulation of HUVECs in the presence of the CaM inhibitor trifluoroperazin results in a reduction in both Ral activation and vWF secretion.³⁰ Our results show that the PKA inhibitor H-89 significantly reduces epinephrine-induced but not thrombin-induced vWF secretion. In addition, Ral activation on stimulation with epinephrine is severely reduced in the presence of H-89. It should be noted that relatively large concentrations of H-89 were used to inhibit PKA. We cannot exclude that other kinases might be affected as well under these experimental conditions.⁴⁵ Previous reports used, respectively, 500 and 200 µmol/L of the cAMP analogue Rp-8-CPT-cAMPS to block cAMP-mediated vWF release from HUVECs.^22,25 Apparently, high concentrations of antagonists are required to completely block PKA activity in endothelial cells. Independent of H-89, the observed stimulatory effect of db-cAMP on Ral activation and vWF secretion (Figure I) suggests that PKA is involved in both Ral activation and vWF secretion. Recently, it has been reported that benzoyladenosine-cAMP (Bz-cAMP) selectively activates PKA.⁴⁶ Incubation of endothelial cells with 100 µmol/L Bz-cAMP induced vWF release from HUVECs, providing additional evidence that PKA mediates vWF secretion (data not shown). Recently, 2 other proteins that are directly regulated by cAMP have been described. These proteins, Epac1 and Epac2, display guanine exchange activity for 2 other small GTPases, Rap1 and Rap2.^47,48 In HEK-293 cells, cAMP formation was found to result in activation of Epac, which subsequently acts via Rap2B on IP₃-sensitive Ca²⁺ stores, consequently increasing intracellular Ca²⁺ levels.⁴⁹ Stimulation of endothelial cells with forskolin, however, failed to increase intracellular Ca²⁺ levels.²⁴ In agreement with these findings, we observed that the intracellular Ca²⁺-chelator BAPTA-AM did not inhibit epinephrine-induced secretion of vWF, whereas it significantly reduced both thrombin-induced and Me-cAMP-induced vWF secretion (data not shown). Furthermore, we did not observe an effect on Ral activation in response to the Epac-specific cAMP analogue Me-cAMP. These results suggest that both epinephrine-induced vWF release and Ral activation are not dependent on cAMP-induced activation of Epac, but require the activation of PKA. Further studies are needed to address the physiological significance of Epac-mediated release of vWF. As yet, it is not clear how PKA contributes to the activation of Ral. PKA-dependent phosphorylation of RasGEF CDC25^Mm/GFR1 has been shown,⁵⁰ and PKA activity was found to enhance Ras activation.⁵¹ Similarly, PKA-mediated phosphorylation of a RalGEF could account for the enhanced activation of Ral on stimulation with epinephrine. Further study, however, is required to elucidate the mechanism behind the current finding that epinephrine-induced vWF secretion is mediated by cAMP/PKA-dependent activation of the small GTPase Ral.

View larger version (23K): [in this window] [in a new window]   Figure 4. Model of epinephrine-mediated and thrombin-mediated signaling pathways leading to Ral activation and WPB exocytosis.

	Acknowledgments

 
Acknowledgments

This work was supported by grants from the Netherlands Heart Foundation (grant 2000.097) and the Netherlands Thrombosis Foundation (grant 20011). We thank Dr M. Fernadez-Borja and R. Bierings for critically reading the manuscript.

Received December 21, 2003; accepted March 15, 2004.

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