Abstract 17084: Aspirin Influences Megakaryocytic Gene Expression Leading to Up-Regulation of Multidrug Resistance Protein 4 in Human Platelets
Recently, we demonstrated that MRP4 over-expression, an organic anion cellular detoxifier, has a role for the reduced aspirin action in patients after by-pass surgery (JACC in press). In other cell lines Mrp4 induction is an adaptive response necessary to reduce intracellular organic anion toxicity or cholestasis. To understand if aspirin treatment induces MRP4 up-regulation in human platelets we studied its quantitative variations in human megakacaryoblastic cell line, Dami, in platelets from megakaryocytic culture and from two different populations, aspirin treated patients (Asp-patients) and healthy volunteers (HV), in-vitro and in-vivo treated with aspirin. MRP4 expression was evaluated by mRNA intracellular expression (Real-time, PCR analysis), protein expression (immunofluorescence-WesternBlot). Aspirin in-vitro treatment (50 microM for 48 h) enhances of about 70% and 93% induction in MRP4 mRNA expression in Dami cells (5.3±0.2 vs 3.1±0.5 ΔΔCt) and in platelets from megakaryocytic culture (0.081±0.028 vs 0.042 ±0.013 ΔΔCt) respectively. In human platelets we found an association between in-vivo Aspirin treatment and MRP4 mRNA expression; which was higher than HV if the patients were treated for more than 2 months (HV 0.049±0.005 n=50; <1months 0.052±0.010 n=19; >2months 0.070±0.007 n=50 ΔΔCt respectively). In addition, platelets from in-vivo aspirin treated HV (330 mg/die n.=5) present an enhancement of induction in MRP4 mRNA expression after 15 days while, after 7 days, it is similar to those obtained before the treatment (HV 0.042±0.013; 7 days 0.048±0.022; 15 days 0.085±0.029ΔΔCt). Immunofluorescence and immunoblot analysis confirm that aspirin enhances MRP4 expression after either in-vitro or in-vivo treatment. Thus, MRP4 over-expression was associated with aspirin treatment. This is the first evidence that aspirin treatment induces a protein up-regulation in human platelets.
- © 2011 by American Heart Association, Inc.