Aspirin influences megakaryocytic gene expression leading to up‐regulation of multidrug resistance protein‐4 in human platelets

Abstract

AimThe aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα).MethodsThe effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV).ResultsIn DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment.ConclusionsThe present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

“…33 It has been shown that MRP4 expression and aspirin are linked and that aspirin treatment enhances MRP4 expression by megacaryocytes. 34 In conclusion, our results provide evidence for a novel role of MRP4 in platelet cyclic nucleotide regulation. They also show that MRP4 is not the main transporter responsible for ADP and ATP accumulation in dense granules.…”

MiR‐21 role in aspirin‐dependent PPARα and multidrug resistance protein 4 upregulation

“…The transcriptional effect on megakaryocytes, responsible for platelet gene expression adaptome , was previously showed, in fact transcriptional change that can be transmitted to circulating platelets is evident for PPARα activation in human bone marrow megakaryocytes 6, 21…”

“…To investigate whether aspirin could regulate miR‐21 expression even after in vivo administration and to verify the correlation between MRP4 and miR‐21, we explored miR‐21 expression in platelets obtained from volunteers after aspirin treatment which have aspirin induced MRP4 over‐expression, as previously shown 6, 8…”

“…Platelet pellet was resuspended with Tyrode buffer (containing 134 mmol/L NaCl, 12 mmol/L NaHCO3, 2.9 mmol/L KCl, 5 mmol/L Glucose, 20 mmol/L Hepes, 1 mmol/L MgCl 2 , 0.34 mmol/L Na 2 HPO 4 , pH 7.3) with addition of EDTA (5 mmol/L), and then filtered on a 5 m syringe‐adaptable filter to remove contamination of white blood cell 6. After centrifugation at 800  g for 15 minutes a platelet pellet without contaminants was obtained, that was stored at −80°C.…”

“…The analysis of gene expression was performed according to Massimi et al 6. MRP4, PPARα, CXCL8, and ACTIN mRNA amounts were quantified by using ΔΔCt method using SDS software version 2.3 (Life Technologies, Warrington, UK)…”