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CC BY-NC-ND 4.0 · Thromb Haemost 2016; 116(02): 272-284
DOI: 10.1160/TH15-11-0891
Cellular Haemostasis and Platelets
Schattauer GmbH

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines

Joy R. Wright
1   Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
3   Department of Cardiovascular Sciences, University of Leicester and NIHR Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester, UK
,
Stefan Amisten
2   Division of Diabetes and Nutritional Sciences, Kings College, London, UK
,
Alison H. Goodall
3   Department of Cardiovascular Sciences, University of Leicester and NIHR Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester, UK
,
Martyn P. Mahaut-Smith
1   Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
› Author Affiliations Financial support: We acknowledge support of the British Heart Foundation (grant no PG/11/56) and the Leicester NIHR Biomedical Research Unit in Cardiovascular Disease, Glenfield Hospital, Leicester, UK.
Further Information

Publication History

Received: 19 November 2015

Accepted after major revision: 30 April 2016

Publication Date:
09 March 2018 (online)

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Summary

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288–11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These “channelome” datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Platelet physiology - megakaryocytes - molecular biology methods - receptors

 

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