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 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.
Highlights
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
Treatment of platelets with thrombin resulted in phosphorylation of VASP at Ser157, the site preferred by PKA (Fig. 1A)
Increase of cGMP in thrombin- and collagen-stimulated platelets has been described in several publications, whereas cAMP has been shown to decrease in thrombin-stimulated platelets [25,26]
Summary
Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFB-IB Complex*□S. 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 NFB-IB␣-PKAc complex. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IB␣, disruption of an IB␣-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. In the initial steps of platelet activation, the platelet receptor glycoproteins (GP)31b and GPVI interact with extracellular matrix (ECM) proteins, causing platelets to tether and roll on the injured endothelium or subendothelial ECM [5] Stimulation of these receptors triggers intracellular signaling cascades that activate integrin ␣IIb3 and induce the release of secondary mediators like ADP and thromboxane A2 (TXA2), leading to full platelet activation and thrombus formation. This particular pathway for thrombin/collagen activation of PKA is described for the first time in platelets, and has characteristics of a novel feedback inhibitory mechanism, which would reduce the likelihood of platelet activation, under weak stimulus conditions
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