Laura Hille1, Maximilian Lenz2, Andreas Vlachos2,3, Björn Grüning4, Lutz Hein5,6, Franz-Josef Neumann7, Thomas G Nührenberg1,5,7, Dietmar Trenk1. 1. Department of Cardiology and Angiology II, Clinical Pharmacology, University Heart Center Freiburg-Bad Krozingen, Bad Krozingen, Germany. 2. Department of Neuroanatomy, Institute of Anatomy and Cell Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany. 3. Center for Basics in Neuromodulation, University of Freiburg, Freiburg, Germany. 4. Bioinformatics Group, Department of Computer Science, University of Freiburg, Freiburg, Germany. 5. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany. 6. BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany. 7. Department of Cardiology and Angiology II, University Heart Center Freiburg-Bad Krozingen, Bad Krozingen, Germany.
Abstract
BACKGROUND: Reticulated platelets (RP) are the youngest circulating platelets in blood. An increased amount of this subpopulation is associated with higher cardiovascular risk and mortality. OBJECTIVES: It is unknown to what extent intrinsic properties of RP contribute to their hyperreactive features. This study is the first providing a multifactorial approach based on ultrastructural, transcriptional, and functional analysis of RP compared to non-RP sorted by flow cytometry. METHODS: Reticulated platelets and non-RP were sorted after platelet staining with SYTO 13. Employing transmission electron microscopy, 1089 micrographs were analyzed for platelet size, amounts of intracellular structures, and anatomical surrogates indicating activation. Long and small RNA-sequencing (RNA-seq) were performed for analyzing differential gene expression. Functional analysis of P-selectin-an upregulated mRNA in RP-was performed in healthy subjects and patients on P2Y12 -inhibitors. RESULTS: Electron micrographs uncovered distinct ultrastructural differences in RP versus non-RP. Cross sections were 1.9-fold larger in RP (P < .0001). Amounts of α-granules, dense granules, open canalicular system-openings, and mitochondria were increased in RP, which persisted after adjustment for platelet size. Long RNA-seq showed 1212 upregulated transcripts that are predominantly associated to platelet shape change, aggregation, and activation; 1264 mRNAs were downregulated in RP. Small RNA-seq did not reveal any differentially expressed transcripts. Functional analysis displayed higher P-selectin expression as compared to non-RP upon ADP- or TRAP-stimulation. CONCLUSIONS: Our results demonstrate that altered intrinsic structural and molecular properties contribute to the hyperreactivity of RP. These properties and an increased amount of RP may account for the association with cardiovascular risk.
BACKGROUND: Reticulated platelets (RP) are the youngest circulating platelets in blood. An increased amount of this subpopulation is associated with higher cardiovascular risk and mortality. OBJECTIVES: It is unknown to what extent intrinsic properties of RP contribute to their hyperreactive features. This study is the first providing a multifactorial approach based on ultrastructural, transcriptional, and functional analysis of RP compared to non-RP sorted by flow cytometry. METHODS: Reticulated platelets and non-RP were sorted after platelet staining with SYTO 13. Employing transmission electron microscopy, 1089 micrographs were analyzed for platelet size, amounts of intracellular structures, and anatomical surrogates indicating activation. Long and small RNA-sequencing (RNA-seq) were performed for analyzing differential gene expression. Functional analysis of P-selectin-an upregulated mRNA in RP-was performed in healthy subjects and patients on P2Y12 -inhibitors. RESULTS: Electron micrographs uncovered distinct ultrastructural differences in RP versus non-RP. Cross sections were 1.9-fold larger in RP (P < .0001). Amounts of α-granules, dense granules, open canalicular system-openings, and mitochondria were increased in RP, which persisted after adjustment for platelet size. Long RNA-seq showed 1212 upregulated transcripts that are predominantly associated to platelet shape change, aggregation, and activation; 1264 mRNAs were downregulated in RP. Small RNA-seq did not reveal any differentially expressed transcripts. Functional analysis displayed higher P-selectin expression as compared to non-RP upon ADP- or TRAP-stimulation. CONCLUSIONS: Our results demonstrate that altered intrinsic structural and molecular properties contribute to the hyperreactivity of RP. These properties and an increased amount of RP may account for the association with cardiovascular risk.
Authors: Harriet E Allan; Melissa A Hayman; Simone Marcone; Melissa V Chan; Matthew L Edin; Tania Maffucci; Abhishek Joshi; Laura Menke; Marilena Crescente; Manuel Mayr; Darryl C Zeldin; Paul C Armstrong; Timothy D Warner Journal: J Thromb Haemost Date: 2021-08-27 Impact factor: 16.036
Authors: Thomas G Nührenberg; Jasmin Stöckle; Federico Marini; Mark Zurek; Björn A Grüning; Vladimir Benes; Lutz Hein; Franz-Josef Neumann; Christian Stratz; Marco Cederqvist; Willibald Hochholzer Journal: PLoS One Date: 2022-01-27 Impact factor: 3.240