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J. Biol. Chem., Vol. 283, Issue 18, 11995-12003, May 2, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/18/11995    most recent M800146200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Karim, Z. A. Articles by Whiteheart, S. W. PubMed PubMed Citation Articles by Karim, Z. A. Articles by Whiteheart, S. W. Social Bookmarking                      What's this?

Primary Platelet Signaling Cascades and Integrin-mediated Signaling Control ADP-ribosylation Factor (Arf) 6-GTP Levels during Platelet Activation and Aggregation^*

From the Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509

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 [GenBank] ) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 [GenBank] 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) /β, 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 PKC/β and 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 β3 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.

Received for publication, January 7, 2008 , and in revised form, February 27, 2008.

^* This work was supported by Grant HL56652 from the National Institutes of Health (to S. W. W.) and the Ohio Valley Affiliate of the American Heart Association (to W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, 741 South Limestone, Biomedical Biological Sciences Research Bldg., Lexington, KY 40536-0509. Tel.: 859-257-4882; Fax: 859-257-2283; E-mail: whitehe{at}uky.edu.

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