Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events

Abstract

Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation. (Blood. 2011;118(7): 1903-1911) IntroductionThe biogenesis of platelets from megakaryocytes is a complex and intricate process that is incompletely understood. What is generally agreed on, however, is that platelets are assembled along intermediate pseudopodial-like extensions that derive from the cytoplasm of megakaryocytes. 1 These extensions are referred to as proplatelets. 1 Proplatelet formation has been modeled in vitro 2 and more recently observed in vivo. 3 Mechanisms that control proplatelet formation and platelet release are emerging, including the critical role of microtubules in driving the elongation of proplatelets. 4 Microtubules are also thought to be involved in sorting mitochondria as well as ␣ and dense granules into individual platelet buds. 5,6 Granules readily track along microtubular-rich shafts of proplatelets and distinct granule subpopulations are partitioned into platelets. 6 This suggests that platelet biogenesis is under discrete control.Protein synthesis also ramps up as proplatelets form. Adhesion molecules, secretome constituents, and other proteins are synthesized and packaged into discrete locations, including membranes and granules, so that platelets are loaded with requisite components before they are released into the bloodstream. 7 In addition to proteins, megakaryocytes send thousands of mRNAs and miRNAs into platelets. [8][9][10] The mRNAs are capped and polyadenylated on their 5Ј-and 3Ј-untranslated region (UTR), respectively, and code for protein when they are placed in in vitro tra...

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“…RNA from human CD34-differentiated or fetal liver-derived proplatelet-producing megakaryocytes was isolated and prepped for deep sequencing as previously described (43)(44)(45). In brief, RNA was prepared for sequencing according to Illumina's (DNA vision) TruSeq kit V2 for poly-A RNA.…”

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