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J. Biol. Chem. 285 (24): 18352-18363

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NF{kappa}B-I{kappa}B Complex*Formula

Stepan Gambaryan{ddagger}§12, Anna Kobsar{ddagger}1, Natalia Rukoyatkina{ddagger}§, Sabine Herterich{ddagger}, Joerg Geiger{ddagger}, Albert Smolenski, Suzanne M. Lohmann{ddagger}, , and Ulrich Walter{ddagger}

From the {ddagger}Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Grombühlstrasse 12, D-97080 Wuerzburg, Germany,
the §Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr 44, 194223 St. Petersburg, Russia, and
the UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland

ABSTRACT Back to Top

Abstract: Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NF{kappa}B-I{kappa}B{alpha}-PKAc complex. We demonstrate mRNA and protein expression for most of the NF{kappa}B family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with I{kappa}B{alpha}, and conversely, I{kappa}B{alpha} was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of I{kappa}B{alpha}, disruption of an I{kappa}B{alpha}-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkB{alpha} phosphorylation, PKA-IkB{alpha} dissociation, and VASP phosphorylation, and potentiated integrin {alpha}IIbβ3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NF{kappa}B-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.

Key Words: Cyclic AMP (cAMP) • NF{kappa}B • Platelet • Protein Kinase A (PKA) • Protein Phosphorylation • NF{kappa}B • PKA • VASP • cAMP • Platelet

Received for publication October 21, 2009. Revision received March 30, 2010.

FOOTNOTES Back to Top

1 Both authors contributed equally to this work.

2 To whom correspondence should be addressed. Tel.: 49-931-32-93617; Fax: 49-931-3293630; E-mail: gambaryan{at}klin-biochem.uni-wuerzburg.de.

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