Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells

Weibel-Palade bodies are endothelial cell-specific organelles, which contai n von Willebrand factor (vWF), P-selectin, and several other proteins. Rece ntly, we found that the small GTP-binding protein Ral is present in a subce llular fraction containing Weibel-Palade bodies. In the present study, we i nvestigated whether Ral is involved in the regulated exocytosis of Weibel-P alade bodies. Activation of endothelial cells by thrombin resulted in trans ient cycling of Ral from its inactive GDP-bound to its active GTP-bound sta te, which coincided with release of VWF. Ral activation and exocytosis of W eibel-Palade bodies were inhibited by incubation with trifluoperazine, an i nhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constit utively active and dominant-negative Ral variants in primary endothelial ce lls. Introduction of active Ral G23V resulted in the disappearance of Weibe l-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodie s in transfected cells. These results indicate that Ral is involved in regu lated exocytosis of Weibel-Palade bodies by endothelial cells.