Expression and functionality of Toll-like receptor 3 in the megakaryocytic lineage.

BACKGROUND: In addition to their key role in hemostasis, platelets and megakaryocytes regulate immune and inflammatory responses, in part through their expression of Toll-like receptors (TLRs). Among the TLRs, TLR3 recognizes dsRNA associated with viral infection. Thrombocytopenia is a frequent complication of viral infection. However, the expression and functionality of TLR3 in megakaryocytes and platelets is not yet well understood. OBJECTIVE: To study the expression and functionality of TLR3 in the megakaryocytic lineage. METHODS AND RESULTS: RT-PCR, flow cytometric and immunofluorescence assays showed that TLR3 is expressed in CD34(+) cells, megakaryocytes, and platelets. Immunoblotting assays showed that stimulation of megakaryocytes with two synthetic agonists of TLR3, Poly(I:C) and Poly(A:U), activated the nuclear factor-κB (NF-κB), phosphoinositide 3-kinase (PI3K)/Akt, extracellular signal-related kinase (ERK)1/2 and p38 pathways. TLR3-megakaryocyte activation resulted in reduced platelet production in vitro and interferon-ß release through the PI3K-Akt and NF-κB signaling pathways. TLR3 ligands potentiated the aggregation mediated by classic platelet agonists. This effect was also observed for ATP release, but not for P-selectin or CD40L membrane exposure, indicating that TLR3 activation was not involved in α-granule release. In addition, TLR3 agonists induced activation of the NF-κB, PI3K-Akt and ERK1/2 pathways in platelets. Reductions in platelet production and platelet fibrinogen binding mediated by Poly(I:C) or Poly(A:U) were prevented by the presence of an inhibitor of the TLR3-dsRNA complex. CONCLUSIONS: Our findings indicate that functional TLR3 is expressed in CD34(+) cells, megakaryocytes, and platelets, and suggest a potential role for this receptor in the megakaryopoiesis/thrombopoiesis alterations that occur in viral infections.

Expression and functionality of type I interferon receptor in the megakaryocytic lineage.

BACKGROUND: Type I interferons (IFN-I) negatively regulate megakaryo/thrombopoiesis. However, expression of the IFN-I receptor (IFNAR) in the megakaryocytic lineage is poorly characterized. OBJECTIVES: To study the expression and functionality of IFNAR in the megakaryocytic lineage. METHODS AND RESULTS: Although IFNAR mRNA was found in every cell type studied, its protein expression showed differences between them. According to flow cytometry and immunofluorescence, IFNAR1 was observed in Meg-01, Dami, CD34+ cells and megakaryocytes, but not in proplatelets or platelets. Immunoblotting assays showed that IFNAR1 and IFNAR2 were highly expressed in all cell types, except in platelets where it was barely detectable. Regarding IFNAR1, 130- and 90-kDa bands were detected in Meg-01 and Dami, whereas 130- and 60-kDa bands were found in CD34+ cells and megakaryocytes. Activation of megakaryocytic IFNAR by IFN-ß induced pSTAT1/2 and upregulated the antiviral genes IRF7 and MXA. The latter response was completely suppressed by IFNAR blockade. In contrast, the low levels of IFNAR in platelets were not functional as pSTAT1/2, aggregation and P-selectin expression were not induced by IFN-I. In addition, megakaryocytes increased IFN-I transcript levels and produced IFN-ß upon stimulation with PolyI:C, a synthetic dsRNA that mimics viral infection. CONCLUSIONS: Early progenitors and mature megakaryocytes, but not platelets, express functional IFNAR and synthetize/release IFN-ß, revealing not only that megakaryo/thrombopoiesis regulation by IFN-I is associated with a specific interaction with its receptor, but also that megakaryocytes may play a role in the antiviral defense by being both IFN producers and responders.

Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha-granule proteins.

BACKGROUND: Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures. OBJECTIVES: To explore the effect of higher temperatures on platelet physiology. METHODS: Platelet responses including adhesion, spreading (fluorescence microscopy), α(IIb)ß(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied. RESULTS: Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion. CONCLUSIONS: Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.