ALBERT

Description: Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

Description: The anatomy of platelets is unique and deceptively simple with a key feature being that platelets circulate without a nucleus. This has led to a common misconception that platelets lack the machinery to produce new proteins. However, over 40 studies from 1966 to 1997 collectively demonstrated and/or inferred that platelets synthesize protein. Recent investigations have not only confirmed these earlier reports but show that platelets possess a sophisticated translational apparatus consisting of template mRNAs, ribosomes, and translation initiation factors, such as eukaryotic initiation factor(eIF)-4E and eIF-2a. Current estimates indicate that platelets retain 4,000–6,000 transcripts and platelet-derived mRNAs are capped and polyadenylated at the 5′ - and 3′-untranslated regions, respectively. Data from our group also demonstrates that platelets contain a pool of precursor mRNAs (premRNAs), uridine-rich small nuclear RNAs, and essential splicing machinery. In response to activation, platelets splice specific pre-mRNAs into mature mRNAs. Published results from our group and others demonstrate that platelets use their splicing and translational machinery to synthesize new proteins. B-cell lymphoma 3 (Bcl-3), cyclooxygenase-1 (COX-1), integrin αIIbβ3, interleukin-1β (IL-1β), Na+-dependent transporter SVCT2, plasminogen activator inhibitor-1 (PAI-1), and tissue factor (TF) are among the most recently characterized proteins reported to be synthesized by platelets. The synthesis of these proteins is highly regulated and involves numerous checkpoints of control. Synthesis of Bcl-3, for example, is controlled by the mammalian Target of Rapamycin (mTOR), one of the most abundantly expressed proteins in platelets. In contrast, platelets use premRNA splicing pathways to regulate the production of pro-IL-1β protein. Processing of pro-IL-1β into its mature form is dependent on an innate cytosolic molecular complex referred to as the inflammasome. The inflammasome has been described in nucleated cells and consists of nucleotide-binding oligomerization domain (NOD)-like receptors, such as NALP-3, as well as caspase-1. Although not previously-described, recent studies from our group demonstrate that platelets possess a functional inflammasome that regulates pro- IL-1β maturation. Identifying and characterizing gene expression pathways in platelets will be an exciting area of investigation throughout the next decade. Our group is actively searching for new targets and, in addition to the inflammasome, we found that platelets harbor reverse transcriptase (RT) activity. Inhibition of RT activity modulates protein synthesis and differentiation responses in platelets. Gene expression pathways including the inflammasome and RT provide platelets with previously-unrecognized mechanisms for controlling thrombosis and inflammation and may be targets for future therapeutic interventions.

Description: New activities of human platelets continue to emerge. One unexpected response is new synthesis of proteins from previously transcribed RNAs in response to activating signals. We previously reported that activated human platelets synthesize B-cell lymphoma-3 (Bcl-3) under translational control by mammalian target of rapamycin (mTOR). Characterization of the ontogeny and distribution of the mTOR signaling pathway in CD34+ stem cell–derived megakaryocytes now demonstrates that they transfer this regulatory system to developing proplatelets. We also found that Bcl-3 is required for condensation of fibrin by activated platelets, demonstrating functional significance for mTOR-regulated synthesis of the protein. Inhibition of mTOR by rapamycin blocks clot retraction by human platelets. Platelets from wild-type mice synthesize Bcl-3 in response to activation, as do human platelets, and platelets from mice with targeted deletion of Bcl-3 have defective retraction of fibrin in platelet-fibrin clots mimicking treatment of human platelets with rapamycin. In contrast, overexpression of Bcl-3 in a surrogate cell line enhanced clot retraction. These studies identify new features of post-transcriptional gene regulation and signal-dependant protein synthesis in activated platelets that may contribute to thrombus and wound remodeling and suggest that posttranscriptional pathways are targets for molecular intervention in thrombotic disorders.