Small GTP-Binding Protein Ral Is Involved in cAMP-Mediated Release of von Willebrand Factor From Endothelial Cells

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Abstract
Objective— von Willebrand factor (vWF) is synthesized by endothelial cells and stored in specialized vesicles called Weibel-Palade bodies (WPBs). Recently, we have shown that the small GTP-binding protein Ral is involved in thrombin-induced exocytosis of WPBs. In addition to Ca2+-elevating secretagogues such as histamine and thrombin, release of WPB is also observed after administration of cAMP-raising substances such as epinephrine and vasopressin. In the present study, we investigated whether Ral is also involved in cAMP-mediated vWF release. Methods and Results— Activation of Ral was observed 15 to 20 minutes after stimulation of endothelial cells with epinephrine, forskolin, or dibutyryl-cAMP. A cell-permeable peptide comprising the carboxy-terminal part of the Ral protein reduced both thrombin-induced and epinephrine-induced vWF secretion supporting a crucial role for Ral in this process. Furthermore, inhibition of protein kinase A by H-89 resulted in a marked reduction of vWF release and greatly diminished levels of GTP-Ral on stimulation with epinephrine. Activation of Ral was independent of the activation of Epac, a cAMP-regulated exchange factor for the small GTPases Rap1 and Rap2. Conclusions— These results suggest that protein kinase A-dependent activation of Ral regulates cAMP-mediated exocytosis of WPB in endothelial cells. Epinephrine, a cAMP-raising agonist of WPB exocytosis, activates the small GTPase Ral in a PKA-dependent manner. Furthermore, a cell-permeable, Ral-derived peptide inhibited epinephrine-induced and thrombin-induced vWF secretion. These results suggest that Ral is a crucial component of cAMP-dependent and Ca2+-dependent signaling pathways that mediate WPB exocytosis.

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