mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets

Abstract

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 rapamy- IntroductionPlatelets are critical effector cells in hemostasis and inflammation in health and disease. [1][2][3] Previously unrecognized responses of human platelets continue to emerge. 1,2,4 One of the most unexpected is synthesis of new proteins from constitutively expressed but silenced messenger RNAs (mRNAs) by activated platelets. [4][5][6][7][8][9] Activation-dependent synthesis of proteins is contrary to conventional expectations because platelets are anucleate. Nevertheless, early 10,11 and more recent 4,5,8,9,[12][13][14] observations demonstrate that mature circulating human platelets have a significant complement of mRNAs generated by precursor megakaryocytes. Furthermore, platelets translate some of these constitutive transcripts in response to activating signals, 9 providing a previously unrecognized mechanism to alter the platelet proteome. 15 Activated human platelets also process pre-mRNAs to mature, translatable transcripts. 4 The diversity of regulatory mechanisms in the platelet repertoire suggests that posttranscriptional gene expression is an important feature of their biology [4][5][6][7][8][9] and is again contrary to previous dogma that they are functionally simple cells.B-cell lymphoma-3 (Bcl-3), a member of the Ik␤␣ family of factors, 16,17 is an index example of signal-dependent synthesis of a specific protein by human platelets. [5][6][7]9 Its translation from constitutively repressed mRNA, which is rapid and yields new protein within minutes, is triggered by cellular activation via thrombin receptors and is modulated by engagement of integrin ␣ IIb ␤ 3 . [5][6][7] Inhibition of synthesis of Bcl-3 by the therapeutic macrolide rapamycin provided evidence that its translation in activated platelets is controlled by mammalian target of rapamycin (mTOR). 5 This conserved phosphatidylinositol kinaserelated kinase is a key regulator of cell-cycle progression, growth, and nutrient balance. [18][19][20] It was, however, not known to control expression of...

PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids

“…However, plasma PAF-AH mRNA levels were markedly increased ( Figure 1B) in differentiated cells (ie, culture day 13; referred to as megakaryocytes) that no longer expressed CD34 (data not shown) but did express ␣ IIb integrins ( Figure 1A far right panel) and displayed other megakaryocyte-like features as previously described. [25][26][27] Protein for plasma PAF-AH protein was absent in CD34 ϩ cells and megakaryocyte precursors but was expressed by megakaryocytes ( Figure 1C). Specificity for the PAF-AH antibody was demonstrated by the marked reduction in megakaryocyte staining when the peptide immunogen was present ( Figure 1C far right panel).…”

“…[25][26][27] Undifferentiated CD34 ϩ cells did not possess the platelet-specific biomarker integrin ␣ IIb ( Figure 1A left panel) and expressed very low levels of mRNA for plasma PAF-AH ( Figure 1B and data not shown). mRNA for plasma PAF-AH increased slightly but remained low at culture day 7 ( Figure 1B) and a subset of the cells (referred to as megakaryocyte precursors) began to express ␣ IIb integrins ( Figure 1A middle panel).…”

“…After the fixation step, the cells were washed, permeabilized, and immunostained as previously described. [25][26][27][28][29][30] The cells were viewed by confocal microscopy using a 60ϫ/1.42 NA oil objective on an FV300 Olympus IX81 microscope (Melville, NY), with PMT values adjusted to no signal with IgG controls. The following antibodies, peptides, and recombinant proteins were used for these studies: anti-integrin ␣ IIb (sc-15328), anti-plasma PAF-AH (sc-16952), and corresponding blocking peptide from Santa Cruz Biotechnology (Santa Cruz, CA); anti-␤-tubulin (T5293) from Sigma-Aldrich (St Louis, MO); anti-PAF-AH1b2 (ab15875) from Abcam (Cambridge, MA); recombinant human PAF-AH1b2 (G22423F) from Genway (San Diego, CA); and goat anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546 from Invitrogen.…”

“…Cells at culture day 7 are referred to as megakaryocyte precursors. Cells at culture day 13, which lacked CD34 but expressed ␣ IIb integrins and other critical platelet biomarkers, [25][26][27] are referred to as megakaryocytes.…”

Leptin and mTOR: Partners in metabolism and inflammation

“…29,30 mTOR signaling pathway also have roles during inflammatory responses. In leukocytes, vascular cells and platelets, mTOR activation by inflammatory or adhesion signals translationally and/or transcriptionally regulates the rapid synthesis of key gene products [31][32][33][34][35][36] and may have implications to atherosclerosis and other inflammatory diseases. 35 mTOR pathway has also been implicated in inflammation-induced angiogenesis through the observations that IKKβ, activated by TNFα, could inhibit TSC1.…”