Abstract
Abstract SCI-44
Platelets circulate in abundance in a quiescent state, yet are readily activated in physiologic control of blood loss and support for vascular integrity, with excessive or prolonged responsiveness underlying a range of cardiovascular events. Platelet activation is initiated by soluble agonists, including thrombin, platelet-activating factor, and ADP, and is accompanied by adhesion to injured vascular walls or to other circulating inflammatory cells. Activated platelets produce thromboxane A2, ATP and ADP, growth factors, and cytokines such as CD40 ligand, and they shed highly thrombotic microparticles. Anucleate platelets cannot generate new RNA to alter their proteome, but they store factors for prompt release after stimulation. Despite the handicap of lacking a nucleus to direct RNA transcript formation, platelets do, in fact, produce a number of new proteins after appropriate stimulation. The questions, then, are: how do platelets respond to inflammatory signaling in the absence of transcription factors that integrate so much of inflammatory signaling? How are new proteins produced in the absence of stored mRNA? To what events do new platelet proteins contribute? The answers to these questions derive both from stimulated translation of stored mRNA in unactivated platelets and from the presence of unspliced heteronuclear RNA transcripts of select genes, accompanied by stimulated posttranscriptional splicing that removes introns from stored heteronuclear RNA to generate functional mRNA, followed by stimulated translation to produce new protein. These mechanisms are retained in murine platelets, suggesting a continued role for the unique process of posttranscriptional protein production by activated platelets. Thrombin stimulates mTOR and S6 kinase signaling to stimulate spliceosome activity, while platelets respond to lipopolysaccharide through their TLR4 receptor, ultimately to stimulate the AKT and JNK kinases and then spliceosome activation. Human and murine platelets store interleukin-1β heteronuclear RNA, and, upon activation, both produce new pro-IL-1β, but also process it via caspase-1 activity to active cytokine. Activated platelets release functional IL-1β, in association with shed microparticles, to active endothelial cells and naïve platelets themselves. Platelets express signaling type 1 receptors for IL-1, and recombinant IL-1α and IL-1β each activate IL-1β RNA processing and cytokine production. Accordingly, specific blockade of IL-1 signaling with IL-1Ra, in clinical use as Anakinra, suppressed platelet stimulation by soluble IL-1, and suppressed microparticles shed from activated platelets. IL-1 signaling additionally affects platelets' structure and their interaction with immobile surfaces. Remarkably, IL-1Ra also abolished platelet stimulation by lipopolysaccharide, showing that platelets amplify TLR4 signaling by the IL-1 signaling axis. IL-1β accumulates in association with platelets within thrombi formed after FeCl3 damage to murine carotid arteries, not by captured or infiltrating mononuclear cells. The process of posttranscriptional RNA splicing and translation uniquely generates the paradigmatic inflammatory cytokine IL-1β in thrombi, and because this process can be tightly targeted by inhibition of posttranslational splicing, the contribution of platelets to vascular remodeling during and after thrombosis may be specifically addressed.
Disclosures:
No relevant conflicts of interest to declare.
Platelet-activating factor and oxidized phosphatidylcholines do not suppress endotoxin-induced pro-inflammatory signaling among human myeloid and endothelial cells