Methods Materials Culture media, trypsin, penicillin and streptomycin were from Invitrogen (Breda, the Netherlands). Bovine fibroblast growth factor (BFGF), thrombin, forskolin, IBMX, H-89, BAPTA-AM and dibutyryl-camp were from Sigma-Aldrich Chemie (Steinheim, Germany). 8-pCPT-2 -O-Me-cAMP (Me-cAMP) was from Biolog (Bremen, Germany). Anti-VWF polyclonal antibody was obtained from Dako A/S (Glostrup, Denmark). Chemiluminescence blotting substrate was from Roche Diagnostics (Mannheim, Germany). Anti-RalA monoclonal antibody was from BD Transduction Laboratories (Lexington, USA). Glutathion-Sepharose was purchased from Amersham Biosciences (Uppsala, Sweden). The Ral-derived peptide TATRal-c was synthesised using Fmoc (N-(9- fluorenyl)methoxycarbonyl) solid phase chemistry and correspond to the sequence: biotin- YARAAARQARAGARKMEDSKEKNGKKKRKSLAKRIRER, in which the underlined sequence represents the TAT sequence, the boxed amino acid represents a glycine linker and the sequence in plain characters represents the Ral carboxy-terminus. At the used concentration of 200 g/ml, transduction of the peptide into cells occurred within 1 minute and was stable for at least 30 minutes (data not shown). All chemicals used were of analytical grade. Cell culture Endothelial cells were isolated from umbilical veins and cultured in medium containing RPMI 1640 and M199 (1:1), 20% human serum, 100 units/ml penicillin, 100 g/ml streptomycin, 33 g/ml glutamin and 1 ng/ml bfgf as described previously 1. Stimulation of endothelial cells by thrombin, epinephrine, forskolin, dibutyryl-camp (db-camp) or 8-pCPT-2 -O-Me-cAMP (Me-cAMP) was performed in the following manner. Endothelial cells grown for 24 hours in 6 wells plates were washed two times with culture medium in which the human serum was replaced by 1% human albumin (serum-free (SF) medium). After washing, the cells were pre-incubated with SF medium for 1 hour. At the beginning of stimulation, the pre-incubation medium was replaced by SF medium containing 1 U/ml thrombin, 10 M epinephrine and 100 M IBMX, 10 M forskolin and 100 M IBMX, 1 mm db-camp, 100 M Me-cAMP or no stimulating agent. In experiments with the
TATRal-c peptide or H-89 endothelial cells were, additionally to the 1 hour pre-incubation step with SF medium, incubated for 5 or 10 minutes with SF medium containing 200 g/ml TATRal-c or 100 M H-89 respectively. The amount of VWF secreted in the medium upon stimulation was measured by ELISA. Ral-activation assay The amount of Ral that is activated upon stimulation was measured in a pull-down assay. The Ral binding domain (RalBD) of the putative Ral effector RalBP1 fused to a GST tag was expressed in IPTG-induced bacteria as described previously 2. Purified GST-RalBD (50-200 g/sample) was precoupled to 30 l/sample of glutathione Sepharose for 1 hour at 4 o C. The pre-coupled glutathione Sepharose was then washed 3 times with Ral buffer containing 15% (v/v) glycerol, 1% NP-40, 50 mm Tris (ph 7.5), 200 mm NaCl, 2.5 mm MgCl 2, 1 mm PMSF and 100 nm aprotinin. Subsequently, aliquots of 200 l/sample pre-coupled glutathione Sepharose beads in Ral buffer were prepared and stored on ice. Stimulated endothelial cells were lysed at indicated time points in 400 l Ral buffer. The activated, GTP-bound form of Ral was then isolated from endothelial cell lysates by incubation of 300 l cell lysate with the RalBD pre-coupled glutathione Sepharose for 1 hour at 4 o C. Finally, the Sepharose beads were washed 4 times with Ral buffer and analysed on a 12.5 % SDS-PAGE gel by western blotting with the anti-rala monoclonal antibody. Immunofluorescence HUVECs were grown on glass coverslips for 24 hours till confluency. After stimulation cells were fixed with 3.7% formaldehyde for 10 minutes and permeabilised with 0.02% saponin in PBS containing 1% BSA. VWF was visualised using the polyclonal anti-vwf antibody and a TexasRedlabelled secondary goat anti-rabbit antibody in PBS; 1% BSA; 0.02% saponin. Cells were embedded in Vectashield mounting medium and viewed by confocal microscopy using a Zeiss LSM510. Images were generated by making optical sections (Z-stacks with 400 nm intervals) and three-dimensional analysis using depth-coding software (application for Zeiss LSM510 version 2.3) that allowed for monitoring of all WPBs present in a single cell. Differences in WPB numbers were statistically analysed using a Student s t-test.
References 1. Brinkman HJ, Mertens K, Holthuis J, Zwart-Huinink LA, Grijm K, Van Mourik JA. The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes. Br J Haematol 1994;87:332-342. 2. Wolthuis RM, Franke B, van Triest M, Bauer B, Cool RH, Camonis JH, Akkerman JW, Bos JL. Activation of the small GTPase Ral in platelets. Mol Cell Biol 1998;18:2486-2491.
Figure I. Dibutyryl-cAMP induced VWF secretion and Ral activation. 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 the camp analogue dibutyryl-camp (db-camp; 1 mm) or SF medium alone (control). VWF secretion in response to incubation with epinephrine, db-camp or SF medium alone (control) was determined. VWF released by control cells after 20 minutes incubation with SF medium is taken as 100%. Results are given as the average of 4 independent experiments and error bars represent SEM. Ral activation in response to stimulation with epinephrine, thrombin or SF medium alone was measured using a Ral-GTP specific pull-down assay. Lower panels show total-ral levels as loading control. Figure II. TATRas-derived control peptides did not inhibit 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 M + 100 M IBMX) or SF medium alone (control) in the absence or presence of 200 g/ml of either TATHRas-c or TATKRas-c. VWF released by control cells after 30 minutes incubation with SF medium is taken as 100%. The result shows the average of 2 independent experiments. Error bars represent SEM. *P<0.05 by Student s t-test. Figure III. The TATRal-c peptide inhibits regulated exocytosis of WPBs. HUVECs were grown on gelatine coated glass coverslips to confluency. Cells were incubated for 1 hour with SF medium prior to stimulation with thrombin (1 U/ml), epinephrine (10 M + 100 M IBMX), forskolin (10 M + 100 M IBMX) or SF medium alone (control) in absence or presence of 200 g/ml of TATRal-c. After stimulation cells were fixed with 3.7% formaldehyde and immunofluorescence was performed as described in Materials and Methods. (A) Quantitative analysis of the number of WPBs in cells stimulated with different agonists for VWF secretion in absence or presence of TATRal-c. * P<0.001 by Student s t-test. (B) Representative, 3D-reconstructed confocal microscopy images of HUVECs stimulated with different agonists for VWF secretion in absence (a, b, c, d) or presence (e, f, g, h) of
the TATRal-c peptide. Relative depth is expressed by pseudo-colour coding in which red-green-blue represents basolateral-central-apical regions of the cell. Figure IV. Epac activation results in exocytosis of WPBs from HUVECs. HUVECs were cultured in 6-wells plates to confluency. Cells were incubated for 1 hour with SF medium prior to stimulation with epinephrine (10 M + 100 M IBMX), SF medium alone (control) or the Epac-specific camp analogue 8-pCPT-2 -O-Me-cAMP (Me-cAMP; 100 M). **P<0.005 by Student s t-test. Quantitative analysis of WPB-numbers in HUVECs of 3 independent experiments stimulated with epinephrine, Me-cAMP or SF medium alone.