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Thromb Haemost 1999; 82(03): 1177-1181
DOI: 10.1055/s-0037-1614349
Letters to the Editor
Schattauer GmbH

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

Hubert P. J. C. de Leeuw
1   From the Department of Blood Coagulation, CLB, Amsterdam, The Netherlands
,
Pauline M. Wijers-Koster
1   From the Department of Blood Coagulation, CLB, Amsterdam, The Netherlands
,
Jan A. van Mourik
1   From the Department of Blood Coagulation, CLB, Amsterdam, The Netherlands
,
Jan Voorberg
1   From the Department of Blood Coagulation, CLB, Amsterdam, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 31 December 1998

Accepted after revision 30 April 1999

Publication Date:
09 December 2017 (online)

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Summary

In endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Keywords

Weibel-Palade bodies - RalA - GTP - von Willebrand factor - endothelial cells
 

  • References

  • 1 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical cord veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-56.
    CrossrefPubMedGoogle Scholar
  • 2 Nachman R, Levine R, Jaffe EA. Synthesis of factor VIII antigen by cultured guinea pig megakaryocytes. J Clin Invest 1970; 60: 914-21.
    PubMedGoogle Scholar
  • 3 Sporn LA, Chavin SI, Marder VJ, Wagner DD. Biosynthesis of von Willebrand factor by human megakaryocytes. J Clin Invest 1985; 76: 1102-6.
    CrossrefPubMedGoogle Scholar
  • 4 Ruggeri ZM. Von Willebrand factor. J Clin Invest 1997; 99: 559-64.
    CrossrefPubMedGoogle Scholar
  • 5 Weibel ER, Palade GE. New cytoplasmic components in arterial endothelia. J Cell Biol 1964; 23: 101-12.
    CrossrefPubMedGoogle Scholar
  • 6 Wagner DD, Olmsted JB, Marder VJ. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol 1982; 95: 355-60.
    CrossrefPubMedGoogle Scholar
  • 7 Reinders JH, de Groot PG, Gonsalves MD, Zandbergen J, Loesberg C, Van Mourik JA. Isolation of a storage and secretory organelle containing von Willebrand protein from cultured human endothelial cells. Biochim Biophys Acta 1984; 80: 361-9.
    PubMedGoogle Scholar
  • 8 McEver RP, Beckstrad JH, Moore KL, Marshall-Carlson L, Bainton DF. GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest 1989; 84: 92-9.
    CrossrefPubMedGoogle Scholar
  • 9 Vischer UM, Wagner DD. CD63 is a component of Weibel-Palade bodies of human endothelial cells. Blood 1993; 82: 1184-91.
    PubMedGoogle Scholar
  • 10 Russell FD, Skepper JN, Davenport AP. Evidence using immunoelectron microscopy for regulated and constitutive pathways in the transport and release of endothelin. J Cardiovasc Pharm 1998; 31: 424-30.
    CrossrefPubMedGoogle Scholar
  • 11 Wolff B, Burns AR, Middleton J, Rot A. Endothelial Cell “Memory” of Inflammatory stimulation: Human venular endothelial cells store interleukin 8 in Weibel-Palade bodies. J Exp Med 1998; 188: 1757-62.
    CrossrefPubMedGoogle Scholar
  • 12 De Groot PhG, Gonsalves MD, Loesberg C, van Buul-Wortelboer MF, van Aken WG, van Mourik JA. Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation: Prostacyclin synthesis is independent of phospholipid methylation. J Biol Chem 1984; 259: 13329-33.
    PubMedGoogle Scholar
  • 13 Vischer UM, Wollheim CB. Epinephrine induces von Willebrand Factor release from cultured endothelial cells: involvement of cyclic AMP-dependent signalling in exocytosis. Thromb Haemost 1997; 77: 1182-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Vischer UM, Jornot L, Wollheim CB, Theler J-M. Reactive oxygen intermediates induce regulated secretion of von Willebrand factor from cultured human vascular endothelial cells. Blood 1995; 85: 3164-72.
    PubMedGoogle Scholar
  • 15 Palmer DS, Aye MT, Ganz PR, Halpenny M, Hashem S. Adenosine nucleotides and serotonin stimulate von Willebrand factor release from cultured human endothelial cells. Thromb Haemost 1994; 72: 132-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 Collins PW, Macay MG, Cahill MR, Newland AC. Von Willebrand factor release and P-selectin expression is stimulated by thrombin and trypsin but not IL-1 in cultured human endothelial cells. Thromb Haemost 1993; 70: 346-50.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Datta YH, Romano M, Jacobson BC, Golan DE, Serhan CN, Ewenstein BM. Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human umbilical vein endothelial cells. Circulation 1995; 92: 3304-11.
    CrossrefPubMedGoogle Scholar
  • 18 Paleolog EM, Crossman DC, McVey JH, Pearson JD. Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells. Blood 1990; 75: 688-95.
    CrossrefPubMedGoogle Scholar
  • 19 Pinsky DJ, Naka Y, Liao H, Oz MC, Wagner DD, Mayadas TN, Johnson RC, Hynes RO, Heath M, Lawson CA, Stern DM. Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies: A mechanism for neutrophil recruitment after cardiac preservation. J Clin Invest 1996; 97: 493-500.
    CrossrefPubMedGoogle Scholar
  • 20 Van den Eijnden-Schrauwen Y, Atsma DE, Lupu F, de Vries RE, Kooistra T, Emeis JJ. Involvement of calcium and G proteins in the acute release of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells. Arterioscler Throm Vasc Biol 1997; 17: 2177-87.
    CrossrefPubMedGoogle Scholar
  • 21 Frearson JA, Harrison P, Scrutton MC, Pearson J. Differential regulation of von Willebrand factor exocytosis and prostacyclin synthesis in electropermeabilized endothelial cell monolayers. Biochem J 1995; 309: 473-9.
    CrossrefPubMedGoogle Scholar
  • 22 Fayos BE, Wattenberg BW. Regulated exocytosis in vascular endothelial cells can be triggered by intracellular guanine nucleotides and require a hydrophobic, thiol-sensitive component. Studies of regulated von Willebrand Factor secretion from digitonin permeabilized endothelial cells. Endothelium 1997; 5: 339-52.
    CrossrefPubMedGoogle Scholar
  • 23 De Leeuw HPJC, Koster PM, Calafat J, Janssen H, Van Zonneveld A-J, Van Mourik JA, Voorberg J. Small GTP-binding proteins in human endothelial cells. Brit J Haematol 1998; 103: 15-9.
    CrossrefPubMedGoogle Scholar
  • 24 Stel HV, Sakariassen KS, Scholte BJ, Veerman ECI, van der Kwast ThH, de Groot PhG, Sixma JJ, van Mourik JA. Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: Relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium. Blood 1984; 63: 1408-15.
    PubMedGoogle Scholar
  • 25 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. Brit J Haematol 1994; 87: 332-42.
    CrossrefPubMedGoogle Scholar
  • 26 Ewenstein BM, Warhol MJ, Handin RI, Pober JS. Composition of the von Willebrand Factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells. J Cell Biol 1987; 104: 1423-33.
    CrossrefPubMedGoogle Scholar
  • 27 Borchiellini A, Fijnvandraat K, ten Cate JW, Pajkrt D, van Deventer SJ, Pasterkamp G, Meijer-Huizinga F, Zwart-Huinink L, Voorberg J, van Mourik JA. Quantitative analysis of von Willebrand factor propeptide release in vivo: effect of experimental endotoxemia and administration of 1-deamino-8-D-arginine vasopressin in humans. Blood 1996; 88: 2951-8.
    PubMedGoogle Scholar
  • 28 Laemlli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedGoogle Scholar
  • 29 O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975; 250: 4001-21.
    PubMedGoogle Scholar
  • 30 Lapetina EG, Reep BR. Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc Natl Acad Sci USA 1987; 84: 2261-5.
    CrossrefPubMedGoogle Scholar
  • 31 Huber LA, Ullrich O, Takai Y, Lütcke A, Dupree P, Olkkonen V, Virta H, De Hoop MJ, Alexandrov K, Peter M, Zerial M, Simons K. Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis. Proc Natl Acad Sci USA 1994; 91: 7874-8.
    CrossrefPubMedGoogle Scholar
  • 32 Bielinski DF, Pyun HY, Linko-Stentz K, Macara IG, Fine RE. Ral and Rab3a are major GTP-binding proteins of axonal rapid transport and synaptic vesicles and do not redistribute following depolarization stimulated synaptosomal exocytosis. Biochim Biophys Acta 1993; 1151: 246-56.
    CrossrefPubMedGoogle Scholar
  • 33 Volknandt W, Pevsner J, Elferink LA, Scheller RH. Association of three small GTP-binding proteins with cholinergic synaptic vesicles. FEBS lett 1993; 317: 53-6.
    CrossrefPubMedGoogle Scholar
  • 34 Mark BL, Jilkina O, Bhullar RP. Association of RalGTP-binding proteins with human platelet dense granules. Biochem Biophys Res Comm 1996; 225: 40-6.
    CrossrefPubMedGoogle Scholar
  • 35 Bos JL. All in the family? – New insights and questions regarding interconnectivity of Ras, Rap, and, Ral. EMBO J 1998; 17: 6776-82.
    CrossrefPubMedGoogle Scholar
  • 36 Hofer F, Fields S, Schneider C, Martin GS. Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. Proc Natl Acad Sci USA 1994; 91: 11089-93.
    CrossrefPubMedGoogle Scholar
  • 37 Kikuchi A, Demo SD, Ye ZH, Chen YW, Williams LT. RalGDS family members interact with the effector loop of ras p21. Mol Cell Biol 1994; 14: 7483-91.
    CrossrefPubMedGoogle Scholar
  • 38 Spaargaren M, Bischoff JR. Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap. Proc Natl Acad Sci USA 1994; 91: 12609-13.
    CrossrefPubMedGoogle Scholar
  • 39 Jiang H, Luo JO, Urano T, Frankel P, Lu Z, Foster D, Feig LA. Involvement of Ral GTPase in v-Src-induced phospholipase D activation. Nature 1995; 378: 409-12.
    CrossrefPubMedGoogle Scholar
  • 40 Luo JO, Liu X, Hammond SM, Colley WC, Feig LA, Frohman MA, Morris AJ, Foster DA. RalA interacts directly with the Arf-responsive PIP2-dependent phospholipase-D1. Biochem Biophys Res Comm 1997; 235: 854-9.
    CrossrefPubMedGoogle Scholar
  • 41 Luo JO, Liu X, Frankel P, Rotunda T, Ramos M, Flom J, Jiang H, Feig LA, Morris AJ, Kahn RA, Foster DA. Functional association between Arf and RalA in active phospholipase D complex. Proc Natl Acad Sci USA 1998; 95: 3632-7.
    CrossrefPubMedGoogle Scholar
  • 42 Bhullar RP, Seneviratne HD. Characterization of human platelet GTPase activating protein for the Ral GTP-binding protein. Biochem Biophys Acta 1996; 1311: 181-8.
    CrossrefPubMedGoogle Scholar
  • 43 Jullien-Flores V, Dorseuil O, Romero F, Letourner F, Saragosti S, Berger R, Tavitian A, Gacon G, Camonis JH. Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity. J Biol Chem 1995; 270: 22473-7.
    CrossrefPubMedGoogle Scholar
  • 44 Cantor SB, Urano T, Feig LA. Identification and characterization of Ral-Binding Protein 1, a potential downstream target of Ral GTPases. Mol Cell Biol 1995; 15: 4578-84.
    CrossrefPubMedGoogle Scholar
  • 45 Tapon N, Hall A. Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Current Opinion Cell Biology 1997; 9: 86-92.
    CrossrefPubMedGoogle Scholar
  • 46 Wolthuis RMF, Franke B, van Triest M, Bauer B, Cool RH, Camonis JH, Akkerman JWN, Bos JL. Activation of the small GTPase Ral in platelets. Mol Cell Biol 1998; 18: 2486-91.
    CrossrefPubMedGoogle Scholar
  • 47 Ling Wang KL, Khan MT, Roufogalis BD. Identification and characterization of a calmodulin-binding domain in RalA, A Ras-related GTP-binding protein purified from human erythrocyte membrane. J Biol Chem 1997; 272: 16002-9.
    CrossrefPubMedGoogle Scholar
  • 48 Birch KA, Pober JS, Zavoico GB, Means AR, Ewenstein BM. Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells. J Cell Biol 1992; 118: 1501-10.
    CrossrefPubMedGoogle Scholar