Arf6 plays an early role in platelet activation by collagen and convulxin.
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 latter 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.
Choi
Wangsun
W
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536-0509, USA.
Karim
Zubair A
ZA
Whiteheart
Sidney W
SW
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D003435:Crotalid Venoms / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D037181:Lectins, C-Type; D010973:Platelet Adhesiveness / Q000187:drug effects* / Q000502:physiology; D010974:Platelet Aggregation / Q000187:drug effects* / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013928:Thromboxane A2 / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D003435:Crotalid Venoms; D037181:Lectins, C-Type; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; C028890:convulxin; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D003094:Collagen; C116687:ARF3 protein, human; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020764:cdc42 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein