title: Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation.
abstract: Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) alpha/beta, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKCalpha/beta and delta inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in beta3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509, USA.
authors.forename: Zubair A
authors.initials: ZA
authors.lastname: Karim
authors.forename: Wangsun
authors.initials: W
authors.lastname: Choi
authors.forename: Sidney W
authors.initials: SW
authors.lastname: Whiteheart
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects; D003435:Crotalid Venoms / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D037181:Lectins, C-Type; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000072377:Syk Kinase; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D002118:Calcium; D003435:Crotalid Venoms; D006160:Guanosine Triphosphate; D039661:Integrin beta3; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D037181:Lectins, C-Type; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D013917:Thrombin; D010738:Type C Phospholipases; D019061:src-Family Kinases; C028890:convulxin; C000606896:SYK protein, human; C000606897:Syk protein, mouse; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse