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

Abstract

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. 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 -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 Ca 2ϩ levels, which appears crucial for the release of vWF from WeibelPalade bodies. 15,16 Inhibition studies have shown that intracellular Ca 2ϩ exerts its effect on regulated secretion of vWF via calmodulin. 13,15 See coverLittle 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 Ca 2ϩ -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 ...

The Epac-Rap1 Signaling Pathway Controls cAMP-mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells

“…These findings show that both epinephrine-and thrombin-induced exocytoses of WPB coincide with the activation of the small GTPase Rap1. Remarkably, the kinetics of Rap1 activation in response to thrombin and epinephrine are very similar to that observed for the activation of the small GTPase RalA (12,34), suggesting that these two GTPases are activated in a coordinated fashion.…”

“…Previous studies have supported the concept that both signaling pathways converge at the level of the guanine exchange factor RalGDS (11). Subsequent activation of the small GTPase Ral promotes assembly of the exocyst complex and mediates phospholipase D 1 -facilitated fusion of WPBs with the plasma membrane (16,34). In this study, we show that Epac1, through the activation of the small GTPase Rap1, is crucial for epinephrine-but not for thrombininduced release of WPBs.…”

Exocytosis of Endothelial Lysosome-Related Organelles Hair-Triggers a Patchy Loss of Glycocalyx at the Onset of Sepsis

“…Both lysosomal and WPB compartments are calcium sensitive, and in endothelial cells this process can be blocked by NO, calcium chelators, 16 a 22-amino acid N-ethylmaleimide (NEM)-sensitive factor fused to a Tat-carrier peptide, 20 NEM itself, a dominant-negative Ral variant, 21 and a glycine-(Na-Et)lysine-proline-arginine (ITF 1697) peptide. 22,23 By pharmacologically manipulating exocytosis of these organelles and examining the integrity of glycocalyx in response to stressors we obtained the key test results for the proposed hypothesis.…”

“…20 NEM itself is also a potent inhibitor of exocytosis. de Leeuw et al 21 demonstrated that a small GTPbinding protein, Ral, is involved in exocytosis of WPBs and that expression of a dominant-negative Ral variant prevented it. Bertuglia et al 22 explored a lysine-proline motif encountered in several biologically active small peptides and synthesized a glycine-(Na-Et)lysine-proline-arginine (ITF 1697) peptide, with the biological half-life of 20 to 120 minutes, and demonstrated that it inhibits ischemia-reperfusioneinduced exocytosis of WPBs and protected pulmonary microcirculation by preventing increase in permeability, leukocyte and platelet adhesion, P-selectin, and vWF secretion.…”

Functional consequences of inhibiting exocytosis of Weibel-Palade bodies in acute renal ischemia

“…Matsushita et al (16) developed fusion polypeptides composed of a 22-amino acid N-ethyl-maleimidesensitive factor (a regulator of exocytosis) and a carrier peptide derived from the human immunodeficiency virus transactivating regulatory protein domain and demonstrated that inhibition of WPB exocytosis decreased leukocyte trafficking and peritonitis (7). de Leeuw et al (6) demonstrated that a small GTP-binding protein, Ral, is involved in exocytosis of WPB and that expression of a dominant-negative Ral variant prevented it. More recently, Bertuglia et al (4) explored a lysineproline motif encountered in several biologically active small peptides and synthesized a glycine-(N␣-Et)lysine-proline-arginine (ITF 1697) peptide, with the biological half-life of 20 -120 min, and demonstrated that it inhibits ischemia-reperfusion-induced exocytosis of WPB and protected pulmonary microcirculation by preventing increase in permeability, leukocyte and platelet adhesion, P-selectin, and von Willebrand factor secretion.…”