RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets.
Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively.
Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, 45701, United States of America.
Huzoor
H
Akbar
Division of Experimental Hematology and Cancer Biology, Children's Hospital Medical Center, University of Cincinnati, Cincinnati, OH, 45229, United States of America.
Xin
X
Duan
Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, 45701, United States of America.
Saima
S
Saleem
Division of Experimental Hematology and Cancer Biology, Children's Hospital Medical Center, University of Cincinnati, Cincinnati, OH, 45229, United States of America.
Ashley K
AK
Davis
Division of Experimental Hematology and Cancer Biology, Children's Hospital Medical Center, University of Cincinnati, Cincinnati, OH, 45229, United States of America.
Yi
Y
Zheng
Thromb Res. 2001 Sep 1;103(5):399-409
11553372
J Thromb Haemost. 2010 Oct;8(10):2283-93
20670370
J Biol Chem. 2013 Nov 22;288(47):34230-8
24106269
J Clin Invest. 1977 Jan;59(1):149-58
187622
Chem Biol. 2012 Feb 24;19(2):228-42
22365606
PLoS One. 2009 Nov 26;4(11):e8045
19956681
Biochem Biophys Res Commun. 2001 Sep 14;287(1):71-6
11549255
J Transl Med. 2010 Dec 06;8:128
21134286
J Cell Biol. 1999 Feb 22;144(4):745-54
10037795
Biochem J. 2005 Mar 15;386(Pt 3):401-16
15588255
PLoS One. 2011;6(11):e26238
22073153
J Biol Chem. 2014 Sep 5;289(36):24874-84
25056956
Proc Natl Acad Sci U S A. 2004 May 18;101(20):7618-23
15128949
J Thromb Haemost. 2012 Jun;10 (6):1133-41
22489915
Exp Mol Med. 2005 Dec 31;37(6):575-87
16391519
Blood. 1999 May 15;93(10 ):3408-17
10233893
Arterioscler Thromb Vasc Biol. 2013 Jul;33(7):1544-51
23640496
Methods Mol Biol. 2012;827:195-212
22144277
Am J Physiol Cell Physiol. 2013 Sep;305(5):C519-28
23784547
Physiol Rev. 2007 Jan;87(1):245-313
17237347
J Biol Chem. 2005 Nov 25;280(47):39474-84
16195235
Arterioscler Thromb Vasc Biol. 2011 Feb;31(2):423-34
21071703
J Biol Chem. 2013 Nov 22;288(47):34217-29
24106270
Blood. 1998 Jun 1;91(11):4206-15
9596668
Arterioscler Thromb Vasc Biol. 2004 Nov;24(11):1988-96
15374851
Biochem Biophys Res Commun. 2009 Jul 31;385(3):439-44
19470376
Br J Pharmacol. 2013 Jan;168(1):212-24
22881838
Methods Mol Biol. 2011;750:307-19
21618100
Mol Cell. 2000 Oct;6(4):899-907
11090627
Nat Immunol. 2001 Mar;2(3):211-5
11224519
J Clin Invest. 1977 May;59(5):959-65
192766
Science. 2010 Jan 15;327(5963):340-3
20075254
J Exp Med. 2013 Oct 21;210(11):2371-85
24101377
Biochem Biophys Res Commun. 2001 Feb 2;280(4):970-5
11162620
J Thromb Haemost. 2007 Aug;5(8):1747-55
17663742
Blood Cells Mol Dis. 2006 Mar-Apr;36(2):166-70
16469512
PLoS One. 2011;6(7):e22117
21789221
Nat Med. 2005 Aug;11(8):886-91
16025125
Blood. 2007 Jun 15;109(12):5260-9
17332246
Antioxid Redox Signal. 2004 Aug;6(4):691-8
15242549
Free Radic Biol Med. 2008 Jul 15;45(2):158-66
18452718
Thromb Haemost. 2014 May 5;111(5):817-23
24402688
Blood. 2009 Jan 22;113(4):893-901
18957688
Arterioscler Thromb Vasc Biol. 2016 May;36(5):846-54
26988594
Chem Biol. 2012 Jun 22;19(6):699-710
22726684
Methods Enzymol. 2006;406:554-65
16472687
Blood. 2012 Jan 26;119(4):1054-63
22045984
Blood. 2005 Oct 15;106(8):2757-60
15976180
Blood. 2009 Jul 9;114(2):415-24
19429871
Curr Opin Cell Biol. 2012 Oct;24(5):600-6
22980731
Circ Res. 2006 Dec 8;99(12):1293-304
17158345