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Blood, 15 April 2006, Vol. 107, No. 8, pp. 3145-3152.
Prepublished online as a Blood First Edition Paper on December 13, 2005; DOI 10.1182/blood-2005-09-3563.
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Submitted September 6, 2005
Accepted November 28, 2005
Arf6 plays an early role in platelet activation by collagen and convulxin
Wangsun Choi, Zubair A Karim, and Sidney W Whiteheart*
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
* Corresponding author; email: whitehe{at}pop.uky.edu.
Small GTPases play critical roles in hemostasis, though the roster of such molecules in platelets is not complete. In this study, we report the presence of Ras-related GTPases of the ADP Ribosylation Factor (Arf) family. Platelets contain Arf1 or 3 and Arf6 with the later being predominantly membrane-associated. Using effector domain pull-down assays, we show, counter to other GTPases, that Arf6-GTP is present in resting platelets and decreases rapidly upon activation with collagen or convulxin. This decrease does not completely rely on secondary agonists (ADP and thromboxane A2) or require integrin signaling. The decrease in free Arf6-GTP temporally precedes activation of Rho family GTPases (RhoA, Cdc42, and Rac1). Using a membrane-permeant, myristoylated peptide, which mimics the N-terminus of Arf6, we show that the Arf6-GTP decrease is essential for collagen- and convulxin-induced aggregation, platelet adherence and spreading on collagen-coated glass. Treatment with this peptide also affects the activation of Rho family GTPases, but has little effect on RalA and Rap1 or on agonist-induced calcium mobilization. These data show that Arf6 is a key element in activation through GPVI, and is required for activation of the Rho family GTPases and the subsequent cytoskeletal rearrangements needed for full platelet function.
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