Thrombin and collagen induce a feedback inhibitory signaling pathway in platelets involving dissociation of the catalytic subunit of protein kinase A from an NFkappaB-IkappaB complex.
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 NFkappaB-IkappaBalpha-PKAc complex. We demonstrate mRNA and protein expression for most of the NFkappaB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IkappaBalpha, and conversely, IkappaBalpha 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 IkappaBalpha, disruption of an IkappaBalpha-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBalpha phosphorylation, PKA-IkBalpha dissociation, and VASP phosphorylation, and potentiated integrin alphaIIbbeta3 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 NFkappaB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.
Gambaryan
Stepan
S
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Grombühlstrasse 12, D-97080 Wuerzburg, Germany. gambaryan@klin-biochem.uni-wuerzburg.de
Kobsar
Anna
A
Rukoyatkina
Natalia
N
Herterich
Sabine
S
Geiger
Joerg
J
Smolenski
Albert
A
Lohmann
Suzanne M
SM
Walter
Ulrich
U
D001792:Blood Platelets / Q000378:metabolism*; D020134:Catalytic Domain; D003094:Collagen / Q000737:chemistry*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D013917:Thrombin / Q000737:chemistry*
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D003094:Collagen; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D051550:I-kappa B Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin; D006152:Cyclic GMP