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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1315.)
© 2004 American Heart Association, Inc.

Thrombosis

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

Mariska G. Rondaij; Erica Sellink; Karina A. Gijzen; Jean Paul ten Klooster; Peter L. Hordijk; Jan A. van Mourik; Jan Voorberg

From the Departments of Plasma Proteins (M.G.R., E.S., K.A.G., J.A.v.M., J.V.) and Experimental Immunohematology (J.P.t.K., P.L.H.), Sanquin Research at CLB, Amsterdam, the Netherlands; and the Department of Vascular Medicine (J.A.v.M.), Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.

Correspondence to Dr J. Voorberg, Department of Plasma Proteins, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands. E-mail J.Voorberg{at}sanquin.nl

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.

Key Words: Weibel-Palade bodies • von Willebrand factor • Ral • cAMP • endothelial cells

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