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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:899.)
© 2001 American Heart Association, Inc.

Vascular Biology

Small GTP-Binding Protein Ral Modulates Regulated Exocytosis of von Willebrand Factor by Endothelial Cells

Hubert P. J. C. de Leeuw; Mar Fernandez-Borja; Eric A. J. Reits; Thalia Romani de Wit; Pauline M. Wijers-Koster; Peter L. Hordijk; Jacques Neefjes; Jan A. van Mourik; Jan Voorberg

From the Departments of Plasma Proteins and Blood Coagulation (H.P.J.C.d.L., T.R.d.W., P.M.W.-K., J.A.v.M., J.V.) and the Department of Experimental Immunohaematology (P.L.H.), CLB; the Division of Tumor Biology (M.F.-B., E.A.J.R., J.N.), The Netherlands Cancer Institute; and the Department of Vascular Medicine (J.A.v.M.), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Correspondence to Jan Voorberg, PhD, Department of Plasma Proteins, CLB, Plesmanlaan 125, 1066 CX Amsterdam, Netherlands. E-mail J_Voorberg{at}clb.nl

	Abstract

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Abstract—Weibel-Palade bodies are endothelial cell–specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.

Key Words: von Willebrand factor • Weibel-Palade bodies • Ral • GTP-binding protein • endothelial cells

	Introduction

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Amultimeric glycoprotein, von Willebrand factor (vWF) is involved in the adhesion of platelets to a damaged vessel wall.^{1 2} Synthesis of vWF is confined to endothelial cells and megakaryocytes. During its biosynthesis in endothelial cells, part of vWF is segregated from the bulk flow of proteins and stored in rod-shaped organelles, the Weibel-Palade bodies.^{3 4} A number of other components have been identified in Weibel-Palade bodies; these include P-selectin, CD63, endothelin, and interleukin-8.^{5 6 7 8 9} On stimulation with agonists such as thrombin and histamine, Weibel-Palade bodies release their contents into the blood.^{10 11 12} The mechanism of thrombin-induced exocytosis of Weibel-Palade bodies has been only partially elucidated.^{13 14 15} Thrombin induces the elevation of intracellular Ca²⁺ levels, which appears crucial for the release of vWF from Weibel-Palade bodies.^{15 16} Inhibition studies have shown that intracellular Ca²⁺ exerts its effect on regulated secretion of vWF via calmodulin.^{13 15}

Little attention has been directed at involvement of small GTPases in regulated secretion in endothelial cells. Small GTPases cycle between an active GTP-bound and inactive GDP-bound form. Guanine nucleotide exchange factors enhance the conversion from the inactive GDP-bound to the active GTP-bound form, whereas GTPase activating proteins promote the GTP hydrolysis of small GTPases. In many cells, small GTP-binding proteins have been implicated in regulated exocytosis, as exemplified by the pivotal role of Rab3A in the release of synaptic vesicles at the nerve terminal.¹⁷ Therefore, it seems likely that small GTP-binding proteins are also involved in the release of vWF through the regulated pathway in endothelial cells. Recently, we identified the small GTP-binding protein Ral in a subcellular fraction containing Weibel-Palade bodies, suggesting a role for this GTPase in regulated exocytosis of these organelles.¹⁸ Ral is a geranylgeranylated GTPase that is ubiquitously expressed.¹⁹ Activated GTP-bound Ral binds to RLIP76 (or Ral binding protein), an effector molecule that possesses GTPase activity for cdc42 and Rac, suggesting a link between the activation of Ral and rearrangement of the cytoskeleton.²⁰ Morphological studies have identified Ral on dense granules in platelets and on synaptic vesicles in nerve terminals, suggesting a role for Ral in regulated exocytosis.^{21 22} Interestingly, Ral has been proposed to interact with calmodulin in a Ca²⁺-dependent manner.²³ Binding to calmodulin enhances GTP binding to Ral 2- to 3-fold.²⁴ These observations may suggest a regulatory role for Ral in the calmodulin-mediated release of vWF from endothelial cells.

In the present study, we investigated the involvement of Ral in the secretion of Weibel-Palade bodies by endothelial cells. We show that activation of Ral correlates with thrombin-induced secretion of vWF from Weibel-Palade bodies. The expression of "constitutively active" Ral in endothelial cells results in the exocytosis of Weibel-Palade bodies, whereas the expression of a dominant-negative Ral variant did not show this effect. Together, these findings suggest that Ral is involved in regulated exocytosis of Weibel-Palade bodies from endothelial cells.

	Methods

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Materials
Culture media, trypsin, penicillin, streptomycin, and fungizone were from GIBCO-BRL. Human serum was from healthy donors. Heparin (5000 IU/mL) was purchased from Leo Pharmaceutical Products. Bovine fibroblast growth factor and soybean trypsin inhibitor were from Sigma-Aldrich Chemie. Chemiluminescence blotting substrate was from Boehringer-Mannheim. Monoclonal antibody CLB-RAg 35 directed against vWF has been described previously.²⁵ Monoclonal antibody CLB-HEC 75 directed against CD31 has been characterized previously.²⁶ Peroxidase-conjugated polyclonal rabbit IgG against human vWF was obtained from Dakopatts A/S. Monoclonal anti-Ral antibody was from Transduction Laboratories. Hybridoma cell line 9E10 was from American Type Culture Collection. Protease inhibitor cocktail, Complete Mini, was from Boehringer-Mannheim. Vectashield was from Vecta Laboratories. All chemicals used were of analytical grade.

Cell Culture
Endothelial cells isolated from human umbilical veins (HUVECs) were cultured as described previously.¹⁸ Stimulation of endothelial cells by thrombin, Ca²⁺ ionophore A23187, and phorbol myristic acetate (PMA) was performed as follows. Endothelial cells were washed 3 times with PBS and cultured for 2 hours in medium 199 supplemented with 1% human serum albumin. At the onset of stimulation, the culture medium was replaced by medium containing thrombin, Ca²⁺ ionophore, PMA, or no agonist. To study the effect of the calmodulin inhibitor trifluoperazine (TFP) on thrombin-induced secretion, cells were preincubated for 30 minutes with 40 µmol/L TFP before stimulation by thrombin. The amount of vWF secreted from stimulated and nonstimulated cells was determined in triplicate for each individual time point.

Ral Activation Assay
The GTP-bound form of Ral was isolated from total cell lysates by incubating the cell lysate with glutathione S-transferase (GST)–Ral-binding domain (RalBD) coupled to glutathione Sepharose essentially as described previously.²⁷ Vector pGEX4T3-GST-RalBD was kindly provided by Dr J.L. Bos (Utrecht University, Utrecht, the Netherlands). GST-RalBD was purified from isopropyl ß-D-thiogalactopyranoside (IPTG)-induced bacteria as described previously.²⁷ HUVECs were cultured in 6-well dishes and grown to confluence. Stimulation of endothelial cells was performed as described in the previous paragraph. At indicated time periods, HUVECs were lysed in Ral buffer (15% [vol/vol] glycerol, 1% NP-40, 50 mmol/L Tris [pH 7.4], 200 mmol/L NaCl, 2.5 mmol/L MgCl₂, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.1 µmol/L Trasylol, Bayer). Cell lysates were incubated with 15 µg GST-RalBD precoupled to glutathione Sepharose for 60 minutes at 4°C. Beads were washed and analyzed by 12.5% SDS-PAGE and Western blotting with a monoclonal anti-Ral antibody. All RalBD experiments were performed at least 3 times and yielded similar findings.

Transient Expression of Ral and Rab3B Variants in HUVECs
Total cDNA of HUVECs and plasmid pGEM-T-Myc-Rab30 were used to construct epitope-tagged human Ral, Ral G23V, Ral S28N, Rab3B, Rab3B, T36N, and Rab3B Q81L.²⁸

HUVECs were transfected by electroporation using a Genepulser equipped with an RF module (Bio-Rad). Confluent HUVECs were trypsinized, and 2 million cells were resuspended in 350 µL HEPES-buffered media. Five-micrograms of CsCl-purified plasmid was added to the cell suspension and incubated for 5 minutes at room temperature. Electroporation was performed in 2-mm cuvettes at 240 V. After transfection, cells were seeded on coverslips and cultured for 48 hours. Cells were fixed with 3.7% formaldehyde for 10 minutes and permeabilized with 0.02% saponin in PBS supplemented with 1% BSA. Cells were then stained with monoclonal anti-myc antibody 9E10 and polyclonal anti-vWF antibody in PBS/0.02% saponin/1% BSA. Secondary antibodies used were fluorescein isothiocyanate (FITC)-labeled goat anti-mouse (CLB) and Texas red–labeled horse anti-rabbit (Vector Laboratories) antibodies. FITC-conjugated CLB-HEC/75 (CLB) was used for staining of CD31 in endothelial cells. Cells were embedded in Vectashield mounting medium and viewed by confocal microscopy by using a Leica TCS NT (Leica Microsystems). Results of 2 independent experiments are given. The number of Weibel-Palade bodies present in endothelial cells expressing Ral wild type, G23V, or S28N was determined. For each construct, 20 to 30 individual transfected cells were evaluated.

	Results

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Thrombin-Induced Secretion of vWF Coincides With Activation of Ral
Stimulation of endothelial cells by agents such as thrombin results in exocytosis of Weibel-Palade bodies.^{10 11} Recently, we have shown the presence of Ral in a subcellular fraction containing Weibel-Palade bodies.¹⁸ To investigate whether the secretion of Weibel-Palade bodies coincides with the activation of Ral, HUVECs were stimulated with thrombin for various periods of time (Figure 1). Exocytosis of Weibel-Palade bodies was determined by measuring the release of vWF in the medium. Secretion of vWF was initiated 30 seconds after the addition of 1 U/mL thrombin and gradually increased in time (Figure 1A). The specificity of RalBD of the Ral effector molecule RLIP76 for GTP-bound Ral has previously been used to monitor the activation of Ral in thrombin-stimulated platelets.²⁷ Similarly, we used GST-tagged RalBD to measure the activation of Ral in thrombin-stimulated endothelial cells (Figure 1B). A transient activation of Ral, which reached a maximum after 2 minutes of stimulation with thrombin, was observed. After 10 minutes, the amount of GTP-bound Ral had decreased significantly. No increase in activation of Ral could be detected in unstimulated cells, and the total amount of Ral was similar in all samples analyzed (Figure 1C). These results show that the activation of Ral coincides with the release of vWF in endothelial cells after stimulation by thrombin. Similar to thrombin, stimulation of endothelial cells by the Ca²⁺ ionophore A23187 and the phorbol ester PMA also resulted in the activation of Ral (please see supplementary Figures I and II, which can be accessed online at http://atvb.ahajournals.org).

View larger version (31K): [in this window] [in a new window]   Figure 1. Thrombin-induced secretion of vWF coincides with Ral activation in endothelial cells. Endothelial cells were cultured in 6-well plates and grown to confluence. Two hours before stimulation, culture medium was replaced by medium 199/1% (vol/vol) human serum albumin. Subsequently, cells were stimulated with thrombin (1 U/mL) for the indicated periods of time. Medium was collected, and cells were lysed in Ral-binding buffer. The concentration of vWF in culture medium was determined by ELISA. Activation of Ral was measured as described in Methods. A, Concentration of vWF in medium (open bar, control; black bar, stimulation with thrombin). B, Activation of Ral in endothelial cells stimulated with thrombin. C, Activation of Ral in nonstimulated endothelial cells.

Role of Calmodulin in Activation of Ral and Regulated Secretion of vWF
In endothelial cells, calmodulin has been implicated in the thrombin-induced exocytosis of Weibel-Palade bodies.^{12 15} Recently, a binding site for calmodulin on Ral has been detected, and calmodulin has been shown to enhance the binding of GTP to Ral.^{23 24} We investigated whether thrombin-induced activation of Ral is affected by antagonists of calmodulin. Endothelial cells were stimulated with thrombin in the presence and absence of the calmodulin inhibitor TFP. Two minutes after the addition of thrombin, TFP inhibited the secretion of vWF by 70%. A slightly lower inhibition was observed at 5 and 10 minutes after incubation with TFP (Figure 2A). In the same series of experiments, the effect of TFP on activation of Ral was determined. In the absence of TFP, the amount of GTP-bound Ral increased 6- to 7-fold after incubation with thrombin for 2 minutes (Figure 2B and 2C). Incubation with TFP resulted in a 3-fold increase in the amount of intracellular GTP-bound Ral at the same time point (Figure 2C). At 5 and 10 minutes after stimulation with thrombin, no significant effect of TFP on the activation of Ral was observed (Figure 2C). Our findings suggest that TFP partially inhibits the activation of Ral and release of vWF in endothelial cells stimulated with thrombin.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of calmodulin inhibitor TFP on thrombin-induced secretion of Weibel-Palade bodies and activation of Ral. Endothelial cells were cultured in 6-well plates and grown to confluence. Two hours before stimulation, culture medium was replaced by medium 199/1% human serum albumin. Then medium was replaced, and TFP was added to a final concentration of 50 µmol/L. In control cells, no TFP was added. After 30 minutes, cells were stimulated with thrombin (IIa, 1 U/mL) for indicated periods of time in the presence/absence of TFP and analyzed as described in Methods. A, Concentration of vWF in culture medium: nonstimulated endothelial cells (open bar) and cells stimulated with thrombin in the absence (black bar) or presence (gray bar) of TFP. B, Activation of Ral in nonstimulated endothelial cells (left) and cells stimulated with thrombin in the absence (middle) or presence (right) of TFP. C, Densitometric analysis of activation of Ral in the presence (gray bar) and absence (black bar) of TFP. On the y-axis, fold stimulation of Ral activation compared with nonstimulated cells is given. On the x-axis, time is depicted in minutes. Data from 3 independent experiments have been used to quantify activation of Ral in the presence and absence of TFP.

Overexpression of Variant Ral and Rab3b in Endothelial Cells
In the previous paragraphs, we have shown that Ral activation coincides with the thrombin-induced release of vWF. To study the functional role of Ral in the exocytosis of Weibel-Palade bodies, we expressed wild-type Ral, constitutively active (GTP-bound) Ral G23V, or dominant-negative (GDP-bound) Ral S28N in primary human endothelial cells by electroporation. Expression of myc-tagged Ral G23V revealed that in the majority of transfected cells, the number of Weibel-Palade bodies was greatly reduced (Figure 3A to 3C). In some of the transfected cells, a residual number of Weibel-Palade bodies could be detected (Table). Quantification of a large number of transfected cells revealed that 48% of cells expressing Ral G23V contained a limited number (<5) of Weibel-Palade bodies (Table). A similar phenotype was observed in cells expressing wild-type Ral (Table). In 48% of cells expressing wild-type Ral, the number of Weibel-Palade bodies is strongly decreased compared with the number of nontransfected cells. In cells transfected with dominant-negative mutant Ral S28N, normal amounts of Weibel-Palade bodies were observed. (Figure 3D through 3F). Quantitative analysis revealed that in only 8% of cells transfected with Ral S28N, reduced numbers of Weibel-Palade bodies were present. Similarly, 4% of nontransfected primary endothelial cells also contained reduced numbers of Weibel-Palade bodies. Overall, these results indicate that overexpression of Ral in the GTP-bound form reduces the number of Weibel-Palade bodies in endothelial cells. To provide evidence that cells expressing variant Ral are indeed endothelial cells, we performed colocalization studies with a monoclonal antibody directed against CD31, a transmembrane adhesion molecule that is abundantly expressed at intracellular junctions of endothelial cells.²⁹ CD31 was observed at the junctions of nontransfected cells and cells expressing Ral G23V (data not shown). To further establish the specificity of the Ral G23V–mediated release of vWF from Weibel-Palade bodies, we expressed dominant-negative Rab3B (Rab3B T36N) and constitutively active Rab3B (Rab3B Q81L) in endothelial cells. Previously, Rab3B has been implicated in regulated exocytosis in neuroendocrine cells.³⁰ However, introduction of Rab3B T36N or Rab3B Q81L in human endothelial cells did not result in a decrease in the number of Weibel-Palade bodies (Figure 3G through 3L). These findings indicate that the expression of Rab3B does not induce exocytosis of Weibel-Palade bodies.

View larger version (42K): [in this window] [in a new window]   Figure 3. Wild-type Ral and constitutively active Ral induce exocytosis of Weibel-Palade bodies in endothelial cells. Primary endothelial cells were transfected with Ral and Rab3B variants and cultured on coverslips for 48 hours. Cells were fixed with 3.7% formaldehyde, and immunofluorescence was performed as described in Methods. Exogenous Ral and Rab3B were visualized by using monoclonal antibody 9E10 directed to myc-tagged and FITC-labeled secondary goat anti-mouse antibody (A, D, G, and J). vWF was visualized by using polyclonal antibody directed to vWF and a Texas red–labeled secondary horse anti-rabbit antibody (B, E, H, and K). A through C, Cells transfected with pCDNA3.1-Myc-Ral G23V. D through F, Cells transfected with pCDNA3.1-Myc-Ral S28N. G through I, Cells transfected with pCDNA3.1-Myc-Rab3B T36N. J through L, Cells transfected with pCDNA3.1-Myc-Rab3B Q81L.

View this table: [in this window] [in a new window]   Table 1. Quantitative Analysis of Number of Weibel-Palade Bodies in Transfected and Nontransfected Human Endothelial Cells

	Discussion

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Stimulation of endothelial cells with agents such as thrombin results in the release of high molecular weight multimers of vWF and translocation of P-selectin to the plasma membrane. Both these events are an immediate consequence of the thrombin-induced exocytosis of endothelial cell–specific storage organelles, the Weibel-Palade bodies. In the present study, we provide evidence that the small GTP-binding protein Ral is transiently activated after stimulation of endothelial cells by thrombin. A previous study has shown that activation of Ral also occurs on the stimulation of human platelets with thrombin.²⁷ In platelets, the activation of Ral reached a maximum 1 minute after stimulation with thrombin, whereas in endothelial cells, maximal levels were reached after 2 minutes (Figure 1).²⁷ Furthermore, the amount of activated Ral decreased rapidly (between 5 and 10 minutes) in endothelial cells, whereas in platelets, significant levels of activated Ral were still present 10 minutes after stimulation. Similar to what has been observed for platelets, elevation of intracellular Ca²⁺ levels resulted in a rapid activation of Ral. In the present study, we show that thrombin-induced activation of Ral is inhibited by the calmodulin antagonist TFP. This observation indicates that Ral functions downstream from calmodulin in endothelial cells. Also, the release of vWF is decreased in the presence of TFP (Figure 2). These findings lend additional support to a close correlation between the activation of Ral and regulated secretion of vWF.

Functional involvement of Ral in the regulated exocytosis of Weibel-Palade bodies by endothelial cells is suggested by the substantial decrease in the number of Weibel-Palade bodies in endothelial cells overexpressing wild-type and constitutively active Ral. In some transfected cells, a residual number of Weibel-Palade bodies can still be detected (Table). This may relate to variability in the expression levels of Ral variants among individual primary endothelial cells. A large variability in the number of Weibel-Palade bodies is also observed in nontransfected primary human endothelial cells (Table). A reduced number of Weibel-Palade bodies in a particular cell does not always result from exocytosis but may also be caused by cell-to-cell variability within primary cultures. Therefore, we determined the number of Weibel-Palade bodies in a large number of transfected primary endothelial cells. Our results suggest that constitutively active Ral (G23V) and wild-type Ral can induce the exocytosis of Weibel-Palade bodies. The number of Weibel-Palade bodies in endothelial cells transfected with dominant-negative Ral (S28N) is similar to control nontransfected cells. We also studied whether dominant-negative Ral interfered with the stimulus-induced exocytosis of Weibel-Palade bodies. Stimulation by thrombin, Ca²⁺ ionophore A23187, and PMA did result in a decrease in the number of Weibel-Palade bodies in cells expressing Ral S28N that was similar to that observed in nontransfected cells (data not shown). These findings indicate that under our experimental conditions, dominant-negative Ral is unable to block the stimulus-induced release of vWF from the Weibel-Palade bodies. Most likely, the concentration of Ral S28N in transfected cells is too low to completely block the cycling of endogenous Ral. Alternatively, Ral-independent signaling pathways that are capable of inducing regulated exocytosis of Weibel-Palade bodies may exist.

It should be noted that our analysis does not allow for the direct monitoring of exocytosis of Weibel-Palade bodies. The amount of Weibel-Palade bodies in transfected cells is evaluated 48 hours after transfection. We cannot exclude that the observed effects of Ral on Weibel-Palade body content are not directly related to exocytosis. For instance, Ral may be involved in the formation of Weibel-Palade bodies from the trans-Golgi network, thereby reducing the number of these granules in cells transfected with active Ral G23V. A possible role for Ral in granule biogenesis has recently been proposed. Several studies have suggested that a complex of Ral with phospholipase D and Arf mediates vesicle budding from the Golgi apparatus.^{31 32}

In a previous study, we have reported that Ral associates with Weibel-Palade bodies in endothelial cells.¹⁸ In the present study, the expression of Ral variants did not reveal colocalization of Ral with vWF in Weibel-Palade bodies. Several reasons may be forwarded for this apparent discrepancy. First, Ral may only transiently associate with Weibel-Palade bodies. Finally, only a limited part of the intracellular amount of Ral may participate in the exocytosis of Weibel-Palade bodies. The intense membrane stain observed for Ral may interfere with the detection of small amounts of exogenous Ral that associate with Weibel-Palade bodies. Cell fractionation studies revealed that Ral is not exclusively present in subcellular fractions that contain Weibel-Palade bodies. A significant amount of Ral was detected in other subcellular fractions derived from endothelial cells (data not shown).

Several studies have suggested a role for Ral in cytoskeleton dynamics.¹⁹ Ral interacts in a GTP-dependent manner with filamin-inducing filopodia.³³ Furthermore, Ral interacts with RLIP76, a Ral effector protein with GTPase protein activity for cdc42.²⁰ Inspection of Figure 3A through 3C reveals that cells expressing Ral G23V appear larger than the surrounding endothelial cells. The observed morphological changes may result from an altered organization of the cytoskeleton in endothelial cells harboring constitutively active Ral G23V. At present, it is unclear whether the observed changes in organization of the cytoskeleton are related to the absence of Weibel-Palade bodies in cells expressing Ral G23V. Recently, it has been shown that thrombin-induced release of vWF can be potentiated by specific inhibition of Rho, a small GTPase involved in cytoskeletal rearrangements, such as stress fiber formation.³⁴ This interesting observation suggests that the cytoskeleton may modulate the agonist-induced release of Weibel-Palade bodies. It is possible that Ral-induced changes in the organization of the cytoskeleton may promote fusion of Weibel-Palade bodies with the plasma membrane.

Recently, Ral has also been implicated in endocytosis.³⁵ Interestingly, constitutively active and dominant-negative forms of Ral inhibited endocytosis. These observations suggest that GTP hydrolysis of active Ral is required for endocytosis. Potentially, Ral may be involved in the rapid endocytosis of integral membrane proteins such as P-selectin and CD63, which are also contained within the Weibel-Palade bodies, after fusion of these organelles with the plasma membrane. Taken together, it appears that Ral may have multiple functional roles within endothelial cells. Future studies will aim at defining individual steps in the biogenesis and release of Weibel-Palade bodies that are controlled by this small GTP-binding protein.

	Acknowledgments

 
This study was financially supported by a grant from the Netherlands Heart Foundation (No. 93.086).

Received August 30, 2000; accepted February 2, 2001.

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