Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function

Department of Cardiovascular Science, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester, UK.
Blood (Impact Factor: 10.45). 11/2010; 116(22):4646-56. DOI: 10.1182/blood-2010-04-280925
Source: PubMed


Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

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    • "LRRFIP1 has been identified as one of the cancer-associated genes (Sjoblom et al., 2006) which promotes cell invasion and metastasis (Arakawa et al., 2010, Ohtsuka et al., 2011, Ariake et al., 2012). In addition, LRRFIP1 isoforms also interact with other proteins, such as Flightless-I (Fli-I), an actin-binding protein (Liu and Yin, 1998, Wilson et al., 1998, Fong and de Couet, 1999), Dishevelled (Dvl), a key molecule in the Wnt signalling cascade (Goodall et al., 2010, Ohtsuka et al., 2011), Toll-like receptor 3 (TLR3), an innate sensor of viral infection (Bagashev et al., 2010) and Drebrin 1 (DBN1), which is involved in the formation of dendritic spines and the stabilisation of tight junctions (Majoul et al., 2007, Goodall et al., 2010). With regards to the mouse isoforms, three different proteins have been reported, two of which, Fli-I leucine-rich repeat associated protein 1 (Flap-1) and Lrrfip1, have been studied. "
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    ABSTRACT: Lrrfip1 is an up-regulated protein after cerebral ischaemia whose precise role in the brain both in healthy and ischaemic conditions is unclear. Different Lrrfip1 isoforms with distinct roles have been reported in human and mouse species. The present study aimed to analyse the Lrrfip1 transcriptional variants expressed in rat cortex, to characterise their expression patterns and subcellular location after ischaemia, and to define their putative role in the brain. Five transcripts were identified and three of them (Lrrfip1, CRA_g and CRA_a' (Flap-1)) were analysed by qPCR. All the transcripts were up-regulated and showed differential expression patterns after in-vivo and in-vitro ischaemia models. The main isoform, Lrrfip1, was found to be up-regulated from the acute to the late phases of ischaemia in the cytoplasm of neurons and astrocytes of the peri-infarct area. This study demonstrates that Lrrfip1 activates β-catenin, Akt, and mTOR proteins in astrocytes and positively regulates the expression of the glutamate transporter GLT-1. Our findings point to Lrrfip1 as a key brain protein that regulates pro-survival pathways and proteins and encourages further studies to elucidate its role in cerebral ischaemia as a potential target to prevent brain damage and promote functional recovery after stroke.
    Neuroscience 03/2014; 268. DOI:10.1016/j.neuroscience.2014.02.051 · 3.36 Impact Factor
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    • "Lrrfip1 has been implicated as a regulator of platelet function [18]. Lrrfip1 also affects the cell cycle, which has been mainly documented in cancer cell proliferation, migration, invasion, and metastasis [19]. "
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    ABSTRACT: The proliferation and remodeling of vascular smooth muscle cells (VSMCs) is an important pathological event in atherosclerosis and restenosis. Here we report that microRNA-132 (miR-132) blocks vascular smooth muscle cells (VSMC) proliferation by inhibiting the expression of LRRFIP1 [leucine-rich repeat (in Flightless 1) interacting protein-1]. MicroRNA microarray revealed that miR-132 was upregulated in the rat carotid artery after catheter injury, which was further confirmed by quantitative real-time RT-PCR. Transfection of a miR-132 mimic significantly inhibited the proliferation of VSMCs, whereas transfection of a miR-132 antagomir increased it. miR-132 mimic inhibited VSMC migration and induced apoptosis. miR-132 mimic increased the protein amounts of both p27 and smooth muscle (SM) α-actin, whereas it decreased SM α-actin and Bcl2. Bioinformatics showed that LRRFIP1 is a target candidate of miR-132. miR-132 down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of Lrrfip1 in a sequence-specific manner. LRRFIP1 induced VSMC proliferation and increased phosphorylation of ERK. Immunohistochemical analysis revealed that Lrrfip1 was clearly expressed along with the basal laminar area of smooth muscle, and its expression pattern was disrupted 7 days after arterial injury. LRRFIP1 mRNA was decreased 14 days after injury. Delivery of miR-132 to rat carotid artery reduced LRRFIP1 expression and attenuated neointimal proliferation in carotid artery injury models. Our results suggest that miR-132 is a novel regulator of VSMC proliferation that represses neointimal formation by inhibiting LRRFIP1 expression.
    Atherosclerosis 08/2013; 229(2):348-55. DOI:10.1016/j.atherosclerosis.2013.05.009 · 3.99 Impact Factor
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    • "The average log2-normalized expression of each long total RNA transcript across the 4 samples was ranked by transcript abundance and compared to published platelet transcript profiles obtained on Affymetrix GeneChip [32] and Illumina BeadChip microarray platforms [15,17]. A Spearman’s correlation coefficient was computed for the genes that are represented on all platforms. "
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    ABSTRACT: BackgroundHuman blood platelets are essential to maintaining normal hemostasis, and platelet dysfunction often causes bleeding or thrombosis. Estimates of genome-wide platelet RNA expression using microarrays have provided insights to the platelet transcriptome but were limited by the number of known transcripts. The goal of this effort was to deep-sequence RNA from leukocyte-depleted platelets to capture the complex profile of all expressed transcripts.ResultsFrom each of four healthy individuals we generated long RNA (≥40 nucleotides) profiles from total and ribosomal-RNA depleted RNA preparations, as well as short RNA (<40 nucleotides) profiles. Analysis of ~1 billion reads revealed that coding and non-coding platelet transcripts span a very wide dynamic range (≥16 PCR cycles beyond β-actin), a result we validated through qRT-PCR on many dozens of platelet messenger RNAs. Surprisingly, ribosomal-RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts. Of the known protein-coding loci, ~9,500 are present in human platelets. We observed a strong correlation between mRNAs identified by RNA-seq and microarray for well-expressed mRNAs, but RNASeq identified many more transcripts of lower abundance and permitted discovery of novel transcripts.ConclusionsOur analyses revealed diverse classes of non-coding RNAs, including: pervasive antisense transcripts to protein-coding loci; numerous, previously unreported and abundant microRNAs; retrotransposons; and thousands of novel un-annotated long and short intronic transcripts, an intriguing finding considering the anucleate nature of platelets. The data are available through a local mirror of the UCSC genome browser and can be accessed at:
    BMC Genomics 01/2013; 14(1):1. DOI:10.1186/1471-2164-14-1 · 3.99 Impact Factor
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